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Registered report: Diverse somatic mutation patterns and pathway alterations in human cancers

  1. Vidhu Sharma
  2. Lisa Young
  3. Anne B Allison
  4. Kate Owen
  5. Reproducibility Project: Cancer Biology Is a corresponding author
  1. Applied Biological Materials, Canada
  2. Piedmond Virginia Community College, United States
  3. University of Virginia, United States
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Cite as: eLife 2016;5:e11566 doi: 10.7554/eLife.11566

Abstract

The Reproducibility Project: Cancer Biology seeks to address growing concerns about reproducibility in scientific research by conducting replications of selected experiments from a number of high-profile papers in the field of cancer biology. The papers, which were published between 2010 and 2012, were selected on the basis of citations and Altmetric scores (Errington et al., 2014). This Registered Report describes the proposed replication plan of key experiments from "Diverse somatic mutation patterns and pathway alterations in human cancers" by Kan and colleagues published in Nature in 2010 (Kan et al., 2010). The experiments to be replicated are those reported in Figures 3D-F and 4C-F. Kan and colleagues utilized mismatch repair detection (MRD) technology to identify somatic mutations in primary human tumor samples and identified a previously uncharacterized arginine 243 to histidine (R243H) mutation in the G-protein α subunit GNAO1 in breast carcinoma tissue. In Figures 3D-F, Kan and colleagues demonstrated that stable expression of mutant GNAO1R243D conferred a significant growth advantage in human mammary epithelial cells, confirming the oncogenic potential of this mutation. Similarly, expression of variants with somatic mutations in MAP2K4, a JNK pathway kinase (shown in Figures 4C-E) resulted in a significant increase in anchorage-independent growth. Interestingly, these mutants exhibited reduced kinase activity compared to wild type MAP2K4, indicating these mutations impose a dominant-negative influence to promote growth (Figure 4F). The Reproducibility Project: Cancer Biology is a collaboration between the Center for Open Science and Science Exchange and the results of the replications will be published in eLife.

https://doi.org/10.7554/eLife.11566.001

Introduction

Human cancer is driven by the acquisition of mutations in cells of somatic origin. Somatic mutations comprise several distinct classes of DNA sequence changes, including single-nucleotide substitutions, small insertions and deletions (indels), copy number alterations, and structural rearrangements (Weir et al., 2007; Chin and Gray, 2008; Stratton et al., 2009; Pleasance et al., 2010). Somatic mutations can be further characterized based on their oncogenic ability: genetic variations that are directly involved in cancer development are termed “driver” mutations, whereas mutations that do not confer any obvious advantage are referred to as “passenger” mutations (Davies et al., 2005). In all cases, genetic changes in somatic cells arise as a result of defective DNA repair mechanisms and/or imprecise DNA replication, and can develop spontaneously, be acquired over the lifetime of an individual, or by direct exposure to mutagens, such as tobacco smoke and ionizing UV radiation (Pfeifer, 2010; Pleasance et al., 2010; Helleday et al., 2014). Over the past 10 years, technologies for the detection of wide-spread genetic alterations have been developed and used to analyze cancer genomes (Stratton et al., 2009; Watson et al., 2013). Its is clear that cancer cell genomes often harbor substantial somatic mutation burdens, thus the ability to generate a comprehensive genetic cancer profile has the potential to significantly improve patient diagnosis and treatment.

The combination of PCR and Sanger sequencing to identify mutations in tumor genomes has proven to be a powerful approach in the study of cancer genomics (Collins et al., 2003). However, this technology is constrained by limited throughput and cost (Chin et al., 2011). Here, Kan and colleagues utilized mismatch repair detection (MRD) technology as a low-cost, high throughput alternative to identify somatic mutations in a large number of primary human tumor samples (Peters et al., 2007). Using this technique, Kan and colleagues identified an uncharacterized somatic mutation in GNAO1 from breast carcinoma tissue (Kan et al., 2010). GNAO1 encodes the Gαo subunit of heterotrimeric guanine-binding proteins (G proteins) (Jastrzebska, 2013). G proteins function as molecular switches that alternate between “on” (GTP-bound) and “off” (GDP-bound) states to control signal transduction in eukaryotes (Gilman, 1987; Birnbaumer, 2007b; 2007a). While previous studies have reported oncogenic mutations in the Gα subunits of other G proteins, including GNAS, GNAI2 and GNAQ (Landis et al., 1989; Lyons et al., 1990; Forbes et al., 2008; Van Raamsdonk et al., 2009), the arginine 243 to histidine (R243H) conversion identified in GNAO1 does not correspond to any previously described mutations within G proteins (Garcia-Marcos et al., 2011). In Figure 3D–F, the oncogenic potential of this mutation was tested. Human mammary epithelial cells (HMECs) stably expressing equivalent levels of wild type GNAO1 or GNAO1R243H were suspended in agar before assessment for colony formation. This key experiment reported that the R243H mutation promotes a two-fold increase in anchorage-independent growth compared to cells expressing wild type GNAO1, and will be replicated in Protocol 1. Subsequent work on GNAO1 has characterized the molecular basis underlying the oncogenic properties of the R243H mutation. Importantly, these studies have determined that the R243H mutation renders Gαo constitutively active via Src-STAT3 signaling (Garcia-Marcos et al., 2011; Leyme et al., 2014).

Kan and colleagues also identified a number of somatic mutations in mitogen activated protein kinase kinase 4 (MAP2K4) (Kan et al., 2010). MAP2K4 is a component of a triple kinase cascade that involves the successive activation of downstream MAP kinases, culminating in the activation of c-Jun NH2-terminal kinases (JNK) and p38 (Derijard et al., 1995; Chang and Karin, 2001; Johnson and Lapadat, 2002). Both the JNK and p38 signaling pathways mediate cellular responses to cytokine signals, stress and other extracellular stimuli (Johnson and Lapadat, 2002). While mutations in MAP2K4 have been reported here (Kan et al., 2010) and elsewhere (Teng et al., 1997; Parsons et al., 2005; Greenman et al., 2007; Forbes et al., 2008), the role of MAP2K4 in cancer has remained complex and contradictory. Some studies have suggested MAP2K4 functions as a pro-oncogenic molecule in breast and pancreatic tumors (Wang et al., 2004), melanoma (Finegan and Tournier, 2010), and in prostate cancer tumors (Lotan et al., 2007; Pavese et al., 2014), whereas other early reports identified MAP2K4 as a putative tumor suppressor gene due to its frequent inactivation in human cancer cell lines and tumor tissues, including pancreatic, breast, ovarian, and colon cancer cells and tissues (Su et al., 1998; 2002; Nakayama et al., 2006; Ahn et al., 2011).

In Figure 4C–E, the functional relevance of six select MAP2K4 mutants (5 located in the kinase domain, 1 outside the kinase domain) were tested in vitro (Kan et al., 2010). NIH3T3 fibroblasts stably expressing equivalent levels of either WT or mutant MAP2K4 were assessed for their ability to promote anchorage-independent growth. Importantly, all six MAP2K4 variants resulted in significantly enhanced agar colony formation compared to cells expressing wild type MAP2K4. A majority of the MAP2K4 mutants resulted in reduced activity to either JNK or myelin basic protein (MBP) when tested in an in vitro kinase assay suggesting that reduced MAP2K4 signaling plays a dominant-negative role in the control of cell growth. A related study examined the invasiveness of cells where endogenous MAP2K4 was depleted and various MAP2K4 mutants were added back, including four of the mutants tested by Kan and colleagues (Ahn et al., 2011). The effect on invasion was directly proportional to the kinase activities of the mutants. The mutations that resulted in loss-of-function kinase activity (including R154W, S251N, and N234I examined by Kan and colleagues) resulted in increased invasion, while mutations with gain-of-function kinase activity, or comparable kinase activity to wild-type (including A279T examined by Kan and colleagues), did not (Ahn et al., 2011). More recent studies have confirmed these findings, showing that MAP2K4 genetic inactivation is prevalent in high grade serous and endometrioid carcinomas, breast cancer, and pancreatic cancer (Davis et al., 2011; Yeasmin et al., 2011b; Yeasmin et al., 2011a; Curtis et al., 2012; Huang et al., 2013). Furthermore, genetic polymorphisms that increase MAP2K4 promoter activity are associated with reduced risk of prostate, lung, and sporadic colorectal cancers (Wei et al., 2009; Liu et al., 2010; Shao et al., 2012). A recent study by Haeusgen and colleagues (Haeusgen et al., 2014) suggests that the balance between MAP2K4 and a novel MAP2K4 splice variant may be important in regulating appropriate cell growth. The key experiments described in Figures 4C–F will be replicated in Protocol 2.

Materials and methods

Unless otherwise noted, all protocol information was derived from the original paper, references from the original paper, or information obtained directly from the authors. An asterisk (*) indicates data or information provided by the Reproducibility Project: Cancer Biology core team. A hashtag (#) indicates information provided by the replicating lab.

Protocol 1: Generation of N-terminally Flag-tagged MAP2K4 and GNAO1 wild-type and mutant vectors

This protocol generates N-terminally flag-tagged wild type or mutant GNAO1 and wild type or mutant MAP2K4 vectors. These vectors will be used in Protocols 2 and 4.

Sampling

  • This experiment will be performed once in order to generate vectors.

Materials and reagents

ReagentTypeManufacturerCatalog #Comments
pRetroX-IRES-ZsGreen1 VectorPlasmidClontech632520Original product number not specified;
replaces pRetro-IRES-GFP-Vector
MAP2K4WT Myc-DDK tagged –includes FLAG tag1PlasmidOrigeneRC206051Original product number not specified
GNAO1WT Myc-DDK tagged (Variant 1) – includes FLAG tag1PlasmidOrigeneRC217958Original product number not specified
Agilent - QuikChange Lightning Multi Site-Directed Mutagenesis KitKitAgilent210516Original product number not specified
  1. 1DDK is equivalent to FLAG which is a registered trademark of Sigma Aldrich

Procedure

  1. 1. Generate GNAO1 and MAP2K4 mutant constructs:

    1. Perform site-directed mutations on cDNA ORFs using #Agilent QuikChange Kit according to manufacturer’s protocol.

      1. Point mutations:

        1. GNAO1: arginine 243 to histidine (R243H)

        2. MAP2K4: arginine 228 to lysine (R228K)

        3. MAP2K4: alanine 279 to threonine (A279T)

  2. Clone inserts (includes FLAG tag) into #pRetroX-IRES-ZsGreen1 vector backbone according to manufacturer’s protocols.

    1. Specific molecular cloning steps and reagents used will be recorded and reported later.

    2. #Perform PCR cloning using primers that encompass the ORF and FLAG-tag insert from the original cDNA

  3. Sequence vectors to confirm identity as well as mutational status, and run on gel to confirm integrity. [additional QC]

    1. #Use the following sequencing primers:

      1. GNAO1R243H Forward: GCCCTTTTTGAGTTTGGATC

      2. GNAOR243H Reverse: GTAAAGCATGTGCACCGAGG

      3. MAP2K4R228K Forward: GCCCTTTTTGAGTTTGGATC

      4. MAP2K4R228K Reverse: GTAAAGCATGTGCACCGAGG

      5. MAP2K4A279T Forward: GCCCTTTTTGAGTTTGGATC

      6. MAP2K4A279T Reverse: GTAAAGCATGTGCACCGAGG

Deliverables

  • Data to be collected

    • Sequencing information and gel verification of vectors

  • Sample delivered for further analysis:

    • Plasmids for use in Protocols 2 and 4:

      • pRetroX-IRES-ZsGreen1

      • pRetroX-FLAG-GNAO1WT-IRES-ZsGreen1

      • pRetroX-FLAG-GNAO1R243H-IRES-ZsGreen1

      • pRetroX-FLAG-MAP2K4WT-IRES-ZsGreen1

      • pRetroX-FLAG-MAP2K4R228K-IRES-ZsGreen1

      • pRetroX-FLAG-MAP2K4A279T-IRES-ZsGreen1

Confirmatory analysis plan

  • None applicable.

Known differences from the original study

The vector backbone pRetroX-IRES-ZsGreen1 will be used instead of pRetroX-IRES-FLAG because the latter is no longer available. The replicating lab will use a cDNA with an ORF tagged with myc-DDK (the same as FLAG) for downstream protocols. Not all mutants used in the original study will be replicated. We will not generate MAP2K4 mutations G85R, R154W, N234I or S251N. All known differences are listed in the materials and reagents section above with the originally used item listed in the comments section. All differences have the same capabilities as the original and are not expected to alter the experimental design.

Provisions for quality control

Sequencing and gel analysis of plasmids will be reported. All of the raw data, including the analysis files, will be uploaded to the project page on the OSF (https://osf.io/jpeqg/) and made publically available.

Protocol 2: Generation of human mammary epithelial cells stably expressing wild-type or GNAO1R243H

This protocol describes the generation of HMECs stably expressing WT or mutant GNAO1R243H protein. Expression of GNAO1 will be confirmed by Western blot that will be a replication of Figure 3F. These cells will subsequently be used in Protocol 3.

Sampling

  • This experiment to be conducted one time to confirm stable expression of GNAO1WT or GNAO1R243H protein.

  • The experiment has 4 cohorts:

    • Cohort 1: Uninfected HMECs [additional negative control]

    • Cohort 2: HMECs transduced with pRetroX-IRES-ZsGreen1 -empty vector [additional negative control]

    • Cohort 3: HMECs transduced with pRetroX-FLAG-GNAO1WT-IRES-ZsGreen1

    • Cohort 4: HMECs transduced with pRetroX-FLAG-GNAO1R243H-IRES-ZsGreen1

  • Western blotting will be performed for the following proteins:

    • FLAG

    • β-ACTIN

Materials and reagents

ReagentTypeManufacturerCatalog #Comments
pRetroX-IRES-ZsGreen1 vectorPlasmidProduced in Protocol 1
pRetroX- FLAG-GNAO1WT
-IRES-ZsGreen1 vector
PlasmidProduced in Protocol 1
pRetroX-FLAG-GNAO1R243H
-IRES-ZsGreen1 vector
PlasmidProduced in Protocol 1
HMECsCell lineATCCPCS-600-010Original product number not specified;
Replaces Life Technology brand used
in original study
HMEC mediumCell cultureATCCPCS-600-03Original product number not specified;
Replaces Life Technology brand used
in original study
HMEC supplementCell cultureATCCPCS-600-040Original product number not specified;
Replaces Life Technology brand used
in original study
Bovine pituitary extractCell cultureLife Technologies13028014
Penicillin/StreptomycinCell cultureApplied Biological MaterialsG255Original not specified
Phoenix amphoteric cellsCell lineATCCATCC CRL-3213Replaces Orbigen brand
used in original study
DMEMCell cultureSigma11965-092Original not specified
Fetal bovine serum (FBS)Cell cultureLife Technologies12483-020Original not specified
L-glutamineCell cultureLife Technologies35050-061Original not specified
GlucoseCell cultureLife TechnologiesA2494001Original not specified
Lipofectamine 2000Transfection ReagentLife Technologies11668027
Opti-MEMTransfection ReagentSigma-Aldrich31985070Original not specified
PBSBufferGIBCO10010023Original not specified
0.45 µm syringe filterLabwareMilliporeSLHV033RBOriginal not specified
Trypsin EDTABufferABMTM050Original not specified
FBSBufferGIBCO12483Original not specified
SDSChemicalLeft to the discretion of the replicating lab
2-mercaptoethanolChemical
GlycerolChemical
bromophenol blueChemical
Tris-HClChemical
Bradford AssayDetection assaySigmaB6916-500 MLOriginal not specified
12% SDS-PAGE gelWestern Blot ReagentInvitrogenEC60252BOXOriginal 4–20%
OptiProtein MarkerWestern Blot ReagentApplied Biological MaterialsG252Original not specified
PVDF membraneWestern Blot ReagentBiorad162-0015Original Nitrocellulose
Skim milk powderWestern Blot ReagentFisher Scientific361021617Original not specified
1X TBS solutionBufferFisher ScientificBP2471-100Original not specified
Anti-FLAG M2 antibodyAntibodySigmaF1804
Anti-ß-ACTIN antibodyAntibodyAbcamAb8227Original not specified
Anti-mouse HRP-conjugated
secondary antibody
AntibodyAbcamAb6728Original not specified
ECL Reagent A and BWestern Blot ReagentApplied Biological MaterialsG075Replaces Thermo
Fisher brand.
X-ray FilmWestern Blot ReagentKodakXBT-1Original not specified

Procedure

Notes:

  • HMECs are grown in complete HMEC medium: HMEC medium supplemented with HMEC supplement, #0.05 mg/mL bovine pituitary extract, 100 U/mL penicillin and 100 mg/ml streptomycin cultured at 37°C and 5% CO2.

  • Phoenix cells are grown in complete DMEM medium: DMEM supplemented with 10% (v/v) FBS, 2 mM L-glutamine and 4.5 g/L glucose, 100 U/mL penicillin and 100 mg/ml streptomycin cultured at 37°C and 5% CO2.

  • All cells will be sent for mycoplasma testing and STR profiling.

  1. #Transfect Phoenix cells with the appropriate retroviral constructs using Lipofectamine 2000 according to manufacturer’s instructions.

    1. On the day before transfection, transfer Phoenix cells to fresh medium in 6 well plates and maintain at 37°C and 5% CO2.

    2. On the day of transfection, dilute 2.5 µg plasmid DNA in 500 µl Opti-MEM medium and mix gently.

      1. pRetroX-FLAG-GNAO1WT-IRES-ZsGreen1

      2. pRetroX-FLAG-GNAO1R243H-IRES-ZsGreen1

      3. pRetroX-IRES-ZsGreen1 (empty vector)

    3. Incubate for 30 min at room temperature.

    4. Add DNA-Opti-MEM mixture to 500 µl Lipofectamine 2000.

    5. Add DNA-Lipofectamine LTX complex to wells containing Phoenix cells and mix gently

    6. Incubate cells for 18–48 hr.

      1. Change media after 4–6 hr to complete media containing serum.

    7. Harvest virus-containing supernatants 48 hr post transfection and re-feed cells with DMEM. Incubate at 37°C in a humidified 5% CO2 incubator. Note: Multiple rounds of collection may be required for concentrating stock.

      1. This initial collected media can be stored briefly at 4°C.

    8. After an additional 12–24 hr of culture, collect viral supernatants again and pool with first collection.

    9. #Concentrate viral stock.

      1. Centrifuge the viral supernatant at 3000 rpm for 15 min to remove any cell debris.

      2. Filter the supernatant through a 0.45 µm syringe filter.

      3. Ultracentrifuge at 22,000 rpm for 2 hr at 4°C to produce concentrated viral stocks.

      4. Aliquot virus into screw-cap centrifuge tubes and store at -70°C.

    10. #Titre retrovirus

      1. One day before harvesting viral supernatant, plate 1.2 × 105 HMECs per well of a 6 well dish.

      2. On the day of viral supernatant harvesting, count the number of cells in one well to determine cell number at time of infection.

      3. Add a range of volumes between 2 to 5 µl of concentrated viral supernatant to the wells. Incubate for 72 hr.

      4. Remove culture medium, wash the wells once with 2 ml PBS.

      5. Add 0.5 ml of 0.25% trypsin EDTA

      6. Incubate 5 min at 37°C.

      7. Add 0.5 ml DMEM-10 or 15 (10–15% FBS).

      8. Pipette up and down with 1 ml pipette and transfer cells to a FACS tube.

      9. Determine the percentage of GFP-positive cells by FACS analysis.

      10. Calculate the number of transfection units (TU/ml):

        1. Divide the % GFP-positive cells by 100.

        2. Multiply that by the number of cells at the time of infection

        3. Divide that number by the volume of the virus added (ml)

        4. This will yield the number of viral particles per ml.

    11. Use resulting virus to transduce HMECs in Step 3.

  2. #One day prior to transduction, seed HMECs in 15 cm plates so they will be 70–90% confluent on the day of transfection.

  3. #Transduce HMECs with the appropriate viruses (Optimal MOI will be determined prior to transduction).

    1. Infect HMECs on a 24-well plate with lentivirus.

      1. Cohort 1: Uninfected HMECs [additional negative control]

      2. Cohort 2: HMECs transduced with pRETRO-IRES-ZsGreen1-empty vector [additional negative control]

      3. Cohort 3: HMECs transduced with pRETRO-FLAG-GNAO1WT-IRES- ZsGreen1

      4. Cohort 4: HMECs transduced with pRETRO-FLAG-GNAO1 R243H-IRES- ZsGreen1

    2. After 72 hr, check cells under fluorescence microscope to calculate infection rate.

  4. #Sterile sort the top 10% of the transduced HMECs by #flow cytometry based on GFP expression.

    1. Trypsinize the cells and resuspend in PBS with 0.5% FBS (FACS buffer)

    2. Pass the cells through the cell strainer to make a single cell suspension.

    3. Sort cells for GFP signal (top 10% selected) on FACS sorter (Influx 100 μm-18 psi)

      1. 100,000 cells are collected per tube.

  5. #Perform Western blots on top 10% GFP positive HMECs to confirm expression of GNAO1:

    1. Spin down the cells for 5 min at maximum speed using an eppendorf tube centrifuge. Aspirate and discard the supernatant.

    2. Add 100 µl to 200 µl of protein lysis buffer depending on the size of the cell pellet.

      1. #Protein lysis buffer: 4% SDS, 10% 2-mercaptoethanol, 20% glycerol, 0.004% bromophenol blue, 0.125 M Tris HCl pH 6.8

      2. Quantify protein concentration using a #Bradford assay according to manufacturer’s instructions.

    3. Load equal amounts of total protein in 25 µl sample on a #12% SDS-PAGE gel.

      1. Boil for 7 min before loading

      2. Load one lane with 8 µl protein marker ladder.

      3. Run at 150V for 10–15 min.

      4. When samples reach separation gel, turn to 100V and run for approximately 1.5 hr. Record running time.

    4. Wet transfer to #PVDF membrane #at 95V for 70 min.

    5. #Block membrane with 3% skim milk in Tris-buffered saline (TBS) on shaker for 30 min

    6. Incubate with the following primary antibodies for 1 hr at 37°C:

      1. Mouse Anti-FLAG M2 (1:500 dilution)

      2. #Mouse Anti-ß-ACTIN (#1:1000 dilution)

    7. Wash membrane 3 times in 1X TBS for 5 min each on shaker.

    8. Incubate with anti-mouse HRP conjugated secondary antibody (#1:1000) for 1 hr on shaker at room temperature.

    9. Remove membrane from secondary antibody and wash three times in 1X TBS for 5 min each.

    10. Prepare ECL solution and incubate membrane.

    11. Expose membrane to X-ray film, develop and scan.

Deliverables

  • Data to be collected:

    • Data for viral titration

    • Flow cytometry data (for viral titration and sorting of transduced cells)

    • Protein determination assay data.

    • Figure 3F: Full scans of all films for each western blot with ladder.

  • Sample delivered for further analysis:

    • HMECs transduced with:

      • pRetroX-IRES-ZsGreen1 (empty vector)

      • pRetroX-FLAG-GNAO1WT-IRES-ZsGreen1

      • pRetroX-FLAG-GNAO1R243H-IRES-ZsGreen1

Confirmatory analysis plan

  • None applicable.

Known differences from the original study

All known differences are listed in the materials and reagents section above with the originally used item listed in the comments section. All differences have the same capabilities as the original and are not expected to alter the experimental design.

Provisions for quality control

The cell line used in this experiment will undergo STR profiling to confirm its identity and will be sent for mycoplasma testing to ensure there is no contamination. GNAO1 expression will be confirmed in the top 10% GFP positive HMECs with western blots. All of the raw data, including the analysis files, will be uploaded to the project page on the OSF (https://osf.io/jpeqg/) and made publically available.

Protocol 3: Anchorage-independent colony formation assay of HMECs transduced with wild-type or mutant GNAO1

This experiment tests the effect of WT or mutant GNAO1 expression on anchorage-independent colony formation of HMECs. It is a replication of the experiments reported in Figure 3D–E.

Sampling

  • Experiment to be repeated a total of 3 times for a power of 99%.

    • See Power Calculations section for details.

  • Experiment has 4 cohorts:

    • Cohort 1: Uninfected HMECs [additional negative control]

    • Cohort 2: HMECs transduced with pRetroX-IRES-ZsGreen1-empty vector [additional negative control]

    • Cohort 3: HMECs transduced with pRetroX-FLAG-GNAO1WT-IRES-ZsGreen1

    • Cohort 4: HMECs transduced with pRetroX-FLAG-GNAO1R243H-IRES-ZsGreen1

  • Each cohort will have anchorage independent colony formation quantified.

Materials and reagents

ReagentTypeManufacturerCatalog #Comments
HMECsCell lineATCCPCS-600-010Original product number not specified;
Replaces Life Technology brand used
in original study
HMECs transduced with
pRetroX-IRES-ZsGreen1-empty vector
Cell lineProduced in Protocol 2
HMECs transduced with
pRetroX-FLAG-GNAO1WT-IRES-ZsGreen1
Cell lineProduced in Protocol 2
HMECs transduced with
pRetroX-FLAG-GNAO1 R243H-IRES-ZsGreen1
Cell lineProduced in Protocol 2
HMEC mediumCell cultureATCCPCS-600-03Replaces Life Technology brand
used in original study
HMEC supplementCell cultureATCCPCS-600-040Original product number not specified;
Replaces Life Technology brand used
in original study
Bovine pituitary extractCell cultureLife Technologies13028014Originl not specified
Penicillin/streptomycinCell cultureABMG255Original not specified
6 well platesLabwareFisher ScientificBiolite 12556004Original not specified
Low melting temperature agarCell cultureBioworld40100048-2Original not specified
Crystal violetDyeLeft to the discretion of the replicating labNot originally used
Methanol (MeOH)Chemical
Acetic acidChemical
ImageJSoftwareNIHReplaces Oxford Optronix GelCount
imager and software

Procedure

Notes:

  • Transduced HMECs are generated in Protocol 2.

  1. Grow 3 flasks of transduced and untransduced control HMECs in complete HMEC medium: HMEC medium supplemented with HMEC supplement, #0.5 mg/mL bovine pituitary extract, 100 U/mL penicillin and 100 mg/mL streptomycin cultured at 37°C and 5% CO2 (these will be the biological replicates).

  2. Plate a lower layer of #1 ml 0.5% agar per well in twelve wells of 6-well plates.

    1. Let solidify.

  3. Suspend 3 wells each of 3 × 104 HMECs in 1 ml full media containing 0.35% agar containing either:

    1. untransduced HMECs

    2. HMECs transduced with pRetroX-IRES-ZsGreen1 (empty vector)

    3. HMECs transduced with pRetroX-FLAG-GNAO1WT-IRES-ZsGreen1

    4. HMECs transduced with pRetroX-FLAG-GNAO1R243H-IRES-ZsGreen1

  4. Plate #1 ml suspended cells on top of the lower layer of 0.5% agar in 6-well plates.

  5. Incubate the plates for 3 weeks at 37°C and 5% CO2.

    1. #Refresh growth media on top layer every 2–3 days.

  6. Assess the presence of colonies.

    1. Stain wells with crystal violet.

      1. Remove media from wells.

      2. Fix with 500 µl of 10% MeOH/10% acetic acid for 10 min.

      3. Remove and stain with 500 µl 0.01% crystal violet for 1 hr.

      4. Remove stain and wash wells.

    2. Image entire well with high-resolution camera.

      1. Include calibration scale in image.

    3. Quantify the number of colonies greater than 200 µm in diameter using ImageJ software.

      1. Set threshold using calibration scale taken during image acquisition.

Deliverables

  • Data to be collected:

    • Figure 3D: Images of colonies.

    • Raw numbers for quantification of colonies for each sample.

    • Figure 3E: Graph of mean number of colonies for each cohort.

Confirmatory analysis plan

  • Statistical Analysis of the Replication Data:

  • Note: At the time of analysis we will perform the Shapiro-Wilk test and generate a quantile-quantile plot to assess the normality of the data. We will also perform Levene’s test to assess homoscedasticity. If the data appears skewed we will perform the appropriate transformation in order to proceed with the proposed statistical analysis. If this is not possible we will perform the equivalent non-parametric test.

    • Unpaired two-tailed t-test of the mean number of colonies in HMECs expressing exogenous GNAO1WT or GNAO1R243H.

  • Meta-analysis of original and replication attempt effect sizes:

    • This replication attempt will perform the statistical analysis listed above, compute the effects sizes, compare them against the reported effect size in the original paper and use a meta-analytic approach to combine the original and replication effects, which will be presented as a forest plot

Known differences from the original study

The original study counted cell colonies using GelCount to image, count, and analyze colonies, while the replication attempt will stain with crystal violet to enhance detection of cell colonies, image wells with a high-resolution camera, and use ImageJ software to count and analyze colonies. Since the software and approach used by the original and replication attempt are different, there will likely be some differences in sensitivity and error rates. All known differences are listed in the materials and reagents section above with the originally used item listed in the comments section. All differences have the same capabilities as the original and are not expected to alter the experimental design.

Provisions for quality control

The cell line used in this experiment will undergo STR profiling to confirm its identity and will be sent for mycoplasma testing to ensure there is no contamination. All of the raw data, including the analysis files, will be uploaded to the project page on the OSF (https://osf.io/jpeqg/) and made publically available.

Protocol 4: Generation of NIH3T3 cells stably expressing wild-type or mutant MAP2K4

This protocol describes the generation of NIH3T3 cells stably expressing wild-type or mutant MAP2K4 proteins. This protocol also describes verification of expression of MAP2K4 by western blot that will be a replication of Figure 4E. These cells will subsequently be used in Protocols 4 and 5.

Sampling

  • This experiment will be conducted one time to confirm stable expression of exogenous MAP2K4.

  • Experiment has 5 cohorts:

    • Cohort 1: Uninfected NIH3T3 cells [additional negative control]

    • Cohort 2: transduced with pRetroX-IRES-ZsGreen1 (empty vector)

    • Cohort 3: transduced with pRetroX-FLAG-MAP2K4WT-IRES-ZsGreen1

    • Cohort 4: transduced with pRetroX-FLAG-MAP2K4R228K-IRES-ZsGreen1

    • Cohort 5: transduced with pRetroX-FLAG-MAP2K4A279T-IRES-ZsGreen1

  • To confirm MAP2K4 expression, Western blotting will be performed for the following proteins:

    • FLAG

    • β-ACTIN [Additional loading control]

Materials and reagents

ReagentTypeManufacturerCatalog #Comments
NIH3T3 cellsCell lineATCCCRL-1658
DMEM mediumCell cultureSigma11965-092Original not specified
FBSCell cultureLife Technologies12483-020Original not specified
L-glutamineCell cultureLife Technologies35050-061Original not specified
Penicillin/StreptomycinCell cultureApplied Biological MaterialsG255Original not specified
pRetroX-IRES-ZsGreen1 vectorPlasmidProduced in Protocol 1
pRetroX-FLAG-MAP2K4WT-
IRES-ZsGreen1 vector
PlasmidProduced in Protocol 1
pRetroX-FLAG-MAP2K4R228K-
IRES-ZsGreen1 vector
PlasmidProduced in Protocol 1
pRetroX-FLAG-MAP2K4A279T-
IRES-ZsGreen1 vector
PlasmidProduced in Protocol 1
Phoenix amphoteric cellsCell lineATCCATCC CRL-3213Replaces Orbigen
brand used in original study
Lipofectamine 2000Transfection ReagentLife Technologies11668027
Opti-MEMTransfection ReagentSigma-Aldrich31985070Original not specified
PBSBufferGIBCO10010023Original not specified
0.45 µm syringe filterLabwareMilliporeSLHV033RBOriginal not specified
Trypsin EDTABufferABMTM050Original not specified
FBSBufferGIBCO12483Original not specified
SDSChemical
2-mercaptoethanolChemical
GlycerolChemical
bromophenol blueChemical
Tris-HClChemical
Bradford AssayDetection assaySigmaB6916-500 MLOriginal not specified
12% SDS-PAGE gelWestern Blot ReagentInvitrogenEC60252BOXOriginal 4–20%
OptiProtein MarkerWestern Blot ReagentApplied Biological MaterialsG252Original not specified
PVDF membraneWestern Blot ReagentBiorad162-0015Original Nitrocellulose
1X TBS solutionBufferFisher ScientificBP2471-100Original not specified
Anti-FLAG M2 antibodyAntibodySigmaF1804
Anti-ß-ACTIN antibodyAntibodyAbcamAb8227Original not specified
Anti-mouse HRP-conjugated
secondary antibody
AntibodyAbcamAb6728Original not specified
ECL Reagent A and BWestern Blot ReagentApplied Biological MaterialsG075Replaces Thermo Fisher brand.
X-ray FilmWestern Blot ReagentKodakXBT-1Original not specified

Procedure

Notes:

  • NIH3T3 cells are grown in complete DMEM medium: DMEM medium supplemented with 10% (v/v) FBS, 2 mM L-glutamine, 100 U/mL penicillin and 100 mg/mL streptomycin cultured at 37°C and 5% CO2.

  • Phoenix cells grown in complete DMEM medium: DMEM supplemented with 10% (v/v) FBS, 2 mM L-glutamine, 100 U/mL penicillin and 100 mg/mL streptomycin cultured at 37°C and 5% CO2.

  • All cells will be sent for mycoplasma testing and STR profiling.

  1. Transfect Phoenix cells with the appropriate constructs as in Protocol 2 step 1.

  2. Transduce NIH3T3 cells with the appropriate viruses as in Protocol 2 steps 2 and

  3. Sterile sort the top 10% of the transduced NIH3T3 cells by flow cytometry based on GFP expression as in Protocol 2 Step 4.

  4. Perform western blot on sorted cells to confirm expression of MAP2K4 as in Protocol 2 Step 5.

Deliverables

  • Data to be collected:

    • Data for viral titration

    • Flow cytometry data (for viral titration and sorting of transduced cells)

    • Protein determination assay data.

    • Figure 4E: Full scans of all films for each western with ladder.

  • Sample delivered for further analysis:

    • NIH3T3 cells transduced with:

      • pRetroX-IRES-ZsGreen1 (empty vector)

      • pRetroX-FLAG-MAP2K4WT-IRES-ZsGreen1

      • pRetroX-FLAG-MAP2K4R228K-IRES-ZsGreen1

      • pRetroX-FLAG-MAP2K4A279T-IRES-ZsGreen1

Confirmatory analysis plan

  • None applicable.

Known differences from the original study

Not all mutants used in the original study will be replicated. We will not generate MAP2K4 mutations G85R, R154W, N234I or S251N. All known differences are listed in the materials and reagents section above with the originally used item listed in the comments section. All differences have the same capabilities as the original and are not expected to alter the experimental design.

Provisions for quality control

The cell lines used in this experiment will undergo STR profiling to confirm their identity and will be sent for mycoplasma testing to ensure there is no contamination. MAP2K4 expression will be confirmed in the top 10% GFP positive HMECs with Western blots. All of the raw data, including the analysis files, will be uploaded to the project page on the OSF (https://osf.io/jpeqg/) and made publically available.

Protocol 5: Anchorage-independent colony formation assay of NIH3T3 cells transduced with wild-type or mutant MAP2K4

This experiment tests the effect of WT or mutant MAP2K4 expression on anchorage-independent colony formation of NIH3T3 cells. It is a replication of the experiments reported in Figure 4C and 4D.

Sampling

  • Experiment to be repeated a total of 3 times for a minimum power of 99%.

    • See Power Calculations section for details.

  • Experiment has 5 (generated in Protocol 4) cohorts:

    • Cohort 1: Uninfected NIH3T3 cells [additional negative control]

    • Cohort 2: NIH3T3 cells transduced with with pRetroX-IRES-ZsGreen1-empty vector

    • Cohort 3: NIH3T3 cells transduced with with pRetroX-FLAG-MAP2K4WT-IRES-ZsGreen1

    • Cohort 4: NIH3T3 cells transduced with with pRetroX-FLAG-MAP2K4R228K-IRES-ZsGreen1

    • Cohort 5: NIH3T3 cells transduced with with pRetroX-FLAG-MAP2K4A279T-IRES-ZsGreen1

  • Each cohort will have anchorage independent colony formation quantified.

Materials and reagents

ReagentTypeManufacturerCatalog #Comments
NIH3T3 cells transduced with
pRetroX-IRES-ZsGreen1-empty vector
Cell lineProduced in Protocol 4
NIH3T3 cells transduced with
pRetroX-FLAG-MAP2K4WT-IRES-ZsGreen1
Cell lineProduced in Protocol 4
NIH3T3 cells transduced with
pRetroX-FLAG-MAP2K4R228K-IRES-ZsGreen1
Cell lineProduced in Protocol 4
NIH3T3 cells transduced with
pRetroX-FLAG-MAP2K4A279T-IRES-ZsGreen1
Cell lineProduced in Protocol 4
DMEM mediumCell cultureSigma11965-092Original not specified
FBSCell cultureLife Technologies12483-020Original not specified
L-glutamineCell cultureLife Technologies35050-061Original not specified
Penicillin/streptomycinCell cultureApplied Biological MaterialsG255Original not specified
6 well platesLabwareFisher ScientificBiolite 12556004Original not specified
Low melting temperature agarCell cultureBioworld40100048-2Original not specified
Crystal violetDyeLeft to the discretion of the replicating lab
Not originally used
Methanol (MeOH)Chemical
Acetic acidChemical
ImageJSoftwareNIHReplaces Oxford Optronix GelCount
imager and software

Procedure

Note:

  • All cells will be sent for mycoplasma testing and STR profiling.

  1. Grow 3 flasks each of transduced NIH3T3 cells generated in Protocol 4 in complete DMEM medium: DMEM medium supplemented with 10% (v/v) FBS, 2 mM L-glutamine, 100 U/mL penicillin and 100 mg/mL streptomycin cultured at 37°C and 5% CO2. (these will be the biological replicates)

  2. Plate a lower layer of #1 ml 0.5% agar per well in 16 wells of 6-well plates.

    1. Let solidify.

  3. Suspend 3 plates each of 1 × 104 cells/plate in 1 ml full media containing 0.35% agar containing either:

    • uninfected NIH3T3 cells [additional negative control]

    • NIH3T3 cells transduced with pRetroX-IRES-ZsGreen1-empty vector

    • NIH3T3 cells transduced with pRetroX-FLAG-MAP2K4WT-IRES-ZsGreen1

    • NIH3T3 cells transduced with pRetroX-FLAG-MAP2K4R228K-IRES-ZsGreen1

    • NIH3T3 cells transduced with pRetroX-FLAG-MAP2K4A279T-IRES-ZsGreen1

  4. Plate #1 ml suspended cells on top of the lower layer of 0.5% agar in 6-well plates.

  5. Incubate the plates for 3 weeks at 37°C and 5% CO2.

    1. #Refresh growth media from top layer every 2–3 days.

  6. Assess the presence of colonies.

    1. Stain wells with crystal violet.

      1. Remove media from wells.

      2. Fix with 500 µl of 10% MeOH/10% acetic acid for 10 min.

      3. Remove and stain with 500 µl 0.01% crystal violet for 1 hr.

      4. Remove stain and wash wells.

    2. Image entire well with a high-resolution camera.

      1. Include calibration scale in image.

    3. Quantify the number of colonies greater than 100 µm in diameter using ImageJ software.

      1. Set threshold using scale taken during image acquisition.

Deliverables

  • Data to be collected:

    • Figure 4C: Images of colonies.

    • Raw numbers for quantification of colonies for each sample.

    • Figure 4D: Graph of mean number of colonies for each cohort.

Confirmatory analysis plan

  • Statistical Analysis of the Replication Data:

  • Note: At the time of analysis we will perform the Shapiro-Wilk test and generate a quantile-quantile plot to assess the normality of the data. We will also perform Levene’s test to assess homoscedasticity. If the data appears skewed we will perform the appropriate transformation in order to proceed with the proposed statistical analysis. If this is not possible we will perform the equivalent non-parametric test.

    • One-way ANOVA of the mean number of colonies in NIH3T3 cells expressing exogenous MAP2K4WT, MAP2K4R228K, or MAP2K4A279T followed by planned comparisons using Fisher’s LSD:

      • MAP2K4WT vs MAP2K4R228K

      • MAP2K4WT vs MAP2K4A279T

  • Meta-analysis of original and replication attempt effect sizes:

    • Compute the effect sizes of each comparison, compare them against the effect size in the original paper and use a random effects meta-analytic approach to combine the original and replication effects, which will be presented as a forest plot.

Known differences from the original study

Not all mutants used in the original study will be replicated. We will not generate MAP2K4 mutations G85R, R154W, N234I or S251N. The original study counted cell colonies using GelCount to image, count, and analyze colonies, while the replication attempt will stain with crystal violet to enhance detection of cell colonies, image wells with a high-resolution camera, and use ImageJ software to count and analyze colonies. Since the software and approach used by the original and replication attempt are different, there will likely be some differences in sensitivity and error rates. All known differences are listed in the materials and reagents section above with the originally used item listed in the comments section. All differences have the same capabilities as the original and are not expected to alter the experimental design.

Provisions for quality control

The cell line used in this experiment will undergo STR profiling to confirm its identity and will be sent for mycoplasma testing to ensure there is no contamination. All of the raw data, including the analysis files, will be uploaded to the project page on the OSF (https://osf.io/jpeqg/) and made publically available.

Protocol 6: Assessing the kinase activity of wild-type or mutant MAP2K4

This experiment tests the in vitro kinase activity of WT or mutant MAP2K4 immunoprecipitated from NIH3T3 cells. It is a replication of the experiment reported in Figure 4F.

Sampling

  • Experiment to be repeated a total of 4 times.

    • The original data is qualitative, thus to determine an appropriate number of replicates to initially perform, sample sizes were determined based on a range of potential variance.

      • See Power Calculations section for details.

  • Experiment has 5 cohorts:

    • Cohort 1: Uninfected NIH3T3 cells [additional negative control]

    • Cohort 2: NIH3T3 cells transduced with pRetroX-IRES-ZsGreen1 (empty vector)

    • Cohort 3: NIH3T3 cells transduced with pRetroX-FLAG-MAP2K4WT-IRES-ZsGreen1

    • Cohort 4: NIH3T3 cells transduced pRetroX-FLAG-MAP2K4R228K-IRES-ZsGreen1

    • Cohort 5: NIH3T3 cells transduced with pRetroX-FLAG-MAP2K4A279T-IRES-ZsGreen1

  • A kinase assay is performed for each cohort using the following substrates:

    • Myelin basic protein (MBP)

    • Inactive MAPK9/JNK2

Materials and reagents

ReagentTypeManufacturerCatalog #Comments
NIH3T3 cells transduced with
pRetroX-IRES-ZsGreen1-empty vector
Cell lineProduced in Protocol 4
NIH3T3 cells transduced with
pRetroX-FLAG-MAP2K4WT-
IRES-ZsGreen1
Cell lineProduced in Protocol 4
NIH3T3 cells transduced with
pRetroX-FLAG-MAP2K4R228K-
IRES-ZsGreen1
Cell lineProduced in Protocol 4
NIH3T3 cells transduced with
pRetroX-FLAG-MAP2K4A279T-
IRES-ZsGreen1
Cell lineProduced in Protocol 4
DMEM mediumCell cultureSigma11965-092Original not specified
FBSCell cultureLife Technologies12483-020Original not specified
L-glutamineCell cultureLife Technologies35050-061Original not specified
EZview FLAG-M2-antibody-coupled
affinity gel
ChromatographySigmaA2220
Penicillin/streptomycinCell cultureApplied Biological MaterialsG255Original not specified
Cell Lysis BufferBufferCell Signaling Technology9803Original product number
not specified
Phenylmethanesulfonyl
Fluoride (PMSF)
Protease inhibitorCell Signaling Technology8853Original not specified
Myelin basic proteinProteinSignalchemM42-51NReplaces Millipore AB15542
Inactive MAPK9/JNK2ProteinInvitrogenPV3621Listed as MAP2K7 in
original paper.
[γ-32P]ATPChemicalPerkin ElmerBLU002H250UC
Kinase Reaction BufferBufferCell Signaling Technology9802Original product number
not specified
Anti-FLAG M2 Magnetic BeadsKinase assay reagentSigma-AldrichM8823Original not specified
12% SDS-PAGEWestern Blot ReagentInvitrogenEC60252BOXOriginal 4–20%
Bradford AssayDetection assaySigmaB6916-500 MLOriginal not specified
OptiProtein MarkerWestern Blot ReagentApplied Biological MaterialsG252Original not specified
PVDF membraneWestern Blot ReagentBiorad162-0015Original Nitrocellulose
Skim milk powderWestern Blot ReagentFisher Scientific361021617Original not specified
1X TBS solutionBufferFisher ScientificBP2471-100Original not specified
Anti-FLAG M2 antibodyAntibodySigmaF1804
Anti-ß-ACTIN antibodyAntibodyAbcamAb8227Original not specified
Anti-mouse HRP-conjugated
secondary antibody
AntibodyAbcamAb6728Original not specified
ECL Reagent A and BWestern Blot ReagentApplied Biological MaterialsG075Replaces Thermo
Fisher brand.
X-ray FilmWestern Blot ReagentKodakXBT-1Original not specified

Procedure

Note:

  • Transduced NIH3T3 cells are generated in Protocol 4.

  • All cells will be sent for mycoplasma testing and STR profiling.

  1. Grow 4 flasks of NIH3T3 in complete DMEM medium: DMEM medium supplemented with 10% (v/v) FBS, 2 mM L-glutamine, 100 U/mL penicillin and 100 µg/mL streptomycin cultured at 37°C and 5% CO2. These are the biological replicates.

  2. Generate cell lysates:

    1. Plate cells for kinase assay so they will be 70–90% confluent on the day of harvest.

    2. Replace with serum free media (0% FBS) and serum starve cells for 24 hr.

    3. Wash cells with PBS and lyse with Cell Lysis Buffer (with 1 mM PMSF added just before use).

    4. Clarify lysates.

    5. Quantify protein concentration using a Bradford Assay according to manufacturer’s instructions.

    6. Adjust samples to equalize for total amount of protein and concentration.

  3. Perform immunoprecipitation:

    1. Incubate clarified lysates with anti-FLAG-M2 conjugated beads overnight at 4°C.

    2. Spin beads down at 10,000xg for 30 s, remove supernatant and wash three times with Cell lysis buffer (with 1 mM PMSF added just before use).

    3. Remove sample for input analysis and divide sample equally between two microcentrifuge tubes.

    4. Spin beads down and remove supernatant.

  4. Kinase Assay

    1. Add 25 µl of Kinase Reaction Buffer supplemented with 10 µM ATP and 2 µCi [γ-32P]ATP with either:

      1. Myelin basic protein (MBP) (#1:2000)

      2. Inactive MAPK9/JNK2 (#1:2000).

    2. Incubate samples for 30 min at 30°C.

    3. Stop kinase reactions by adding SDS sample buffer.

    4. Resolve kinase reactions on #15% SDS-PAGE gel with protein ladder.

    5. #Fix gel for 15 min in 5% methanol, 7% acetic acid and dry at 60°C for 30 min.

    6. Expose gel to X-ray film and scan images.

  5. Perform western blot on input sample for MAP2K4 expression as in Protocol 2 Step 5.

  6. For each replicate normalize each protein (MBP and MAPK9/JNK2) to MAP2K4 input levels and then normalize each sample to MAP2K4WT.

Deliverables

  • Data to be collected:

    • Protein determination assay data.

    • Figure 4F: Full images of autoradiographs for each kinase assay substrate with ladder.

    • Figure 4F: Scans of full films for western blot of MAP2K4 input with ladder.

Confirmatory analysis plan

  • Statistical Analysis of the Replication Data:

  • Note: At the time of analysis we will perform the Shapiro-Wilk test and generate a quantile-quantile plot to assess the normality of the data. We will also perform Levene’s test to assess homoscedasticity. If the data appears skewed we will perform the appropriate transformation in order to proceed with the proposed statistical analysis. If this is not possible we will perform the equivalent non-parametric test.

    • Bonferroni corrected one-sample t-tests of normalized pMBP levels from the following MAP2K4 variants compared to 1 (MAP2K4WT):

      • MAP2K4R228K

      • MAP2K4A279T

    • Bonferroni corrected one-sample t-tests of normalized pMAPK9/pJNK levels from the following MAP2K4 variants compared to 1 (MAP2K4WT):

      • MAP2K4R228K

      • MAP2K4A279T

  • Meta-analysis of effect sizes:

    • Since some of the band intensities in the original paper were unable to be quantified the replication study will record and make accessible all autoradiographs collected. This will allow for a subjective comparison of the original images and the replication images. Additionally, the replication will quantify the results in an additional exploratory measure. This cannot be compared to the original reported results, but will be presented to understand the utility of analyzing the data in a quantitative manner.

Known differences from the original study

Not all mutants used in the original study will be replicated. We will not generate MAP2K4 mutations G85R, R154W, N234I or S251N. All known differences are listed in the materials and reagents section above with the originally used item listed in the comments section. All differences have the same capabilities as the original and are not expected to alter the experimental design.

Provisions for quality control

The cell line used in this experiment will undergo STR profiling to confirm its identity and will be sent for mycoplasma testing to ensure there is no contamination. All of the raw data, including the analysis files, will be uploaded to the project page on the OSF (https://osf.io/jpeqg/) and made publically available.

Power calculations

For additional details on power calculations, please see analysis scripts and associated files on the Open Science Framework:

https://osf.io/bxr2d/

Protocol 1:

  • Not applicable

Protocol 2:

  • Not applicable

Protocol 3:

Summary of original data

  • Note; values are from data shared by authors, which was reported in Figure 3E.

VectorMean # of colonies >200 μm diameterStdevN
WT113.510.6072
R243H201.516.2632

Test family

  • Two-tailed t test, difference between two independent means, alpha error = 0.050

Power calculations

Group 1Group 2Effect size dA priori powerGroup 1 sample sizeGroup 2 sample size
WTR243H6.4095487.2%1,22121
  1. 1 3 samples per group will be used making the achieved power of 99.9%.

  2. 2 The calculation was also performed with the non-parametric Wilcoxon-Mann-Whitney test, which gives an achieved power of 99.9% with a sample size of 3 per group.

Protocol 4:

  • Not applicable

Protocol 5:

Summary of original data

  • Note: values are from data shared by authors, which was reported in Figure 4D:

VectorMean # of colonies >100 μm diameterStdevN
WT162.82842
R228K362.82842
A279T94.57.77822

Test family

  • ANOVA: Fixed effects, omnibus, one-way, alpha error = 0.05

Power calculations

  • Performed with G*Power software, version 3.1.7 (Faul et al., 2007).

  • ANOVA F test statistic and partial η2 performed with R software, version 3.1.2 (Team, 2014).

GroupsF test statisticPartial η2Effect size fA priori powerTotal sample size
NIH3T3 cells transduced with
WT or MAP2K4 mutants
F(2,3) = 130.520.988649.328099.9%61
(3 groups)
  1. 19 total samples (3 per group) will be used as a minimum sample size making the power 99.9%.

Test family

  • Two-tailed t test, difference between two independent means, Fisher’s LSD: alpha error = 0.05

Power calculations

Group 1Group 2Effect size dA priori powerGroup 1 sample sizeGroup 2 sample size
WTR228K7.0710799.9%1,22121
WTA279T13.413499.9%1,22121
  1. 1 3 samples per group will be used making the power 99.9%.

  2. 2 The calculation was also performed with the non-parametric Wilcoxon-Mann-Whitney test, which gives an achieved power of 99.9% with a sample size of 3 per group.

Protocol 6

Summary of original data

  • Note: data estimated from the image reported in Figure 4F.

    • The original data presented is qualitative (images of Western blots). We used ImageJ version 1.50a (Schneider et al., 2012) to perform densitometric analysis of the presented bands to quantify the original effect size where possible. The data presented in Figure 4F for Input MAP2K4 were unable to be quantified for all bands and were thus excluded from the normalization. Additionally, the WT values provide under-estimates of the actual values since the WT bands were saturated and unable to be quantified.

VariantNormalized pJNK band intensity to WTNormalized pMBP band intensity to WT
WT11
R228K0.2997360.057556
A279T0.6133780.096804
  • The original data does not indicate the error associated with multiple biological replicates. To identify a suitable sample size, power calculations were performed using different levels of relative variance.

Test family

  • t-test: Means: Difference from constant (one sample case): Bonferroni’s correction: alpha error = 0.0125.

Power calculations

SubstrateVariantConstant (WT)Effect size dA priori powerSample size per group
P-JNKR228K1116.81399.9%3
A279T131.515799.9%3
P-MBPR228K1818.71399.9%3
A279T1466.50799.9%3
  • 15% variance

SubstrateVariantConstant (WT)Effect size dA priori powerSample size per group
P-JNKR228K115.575199.9%3
A279T14.2021092.2%4
P-MBPR228K1109.16299.9%3
A279T162.200999.9%3
  • 28% variance

SubstrateVariantConstant (WT)Effect size dA priori powerSample size per group
P-JNKR228K18.3438092.6%3
A279T12.2511288.7%6
P-MBPR228K158.479599.9%3
A279T133.321999.9%3
  • 40% variance

SubstrateVariantConstant (WT)Effect size dA priori powerSample size per group
P-JNKR228K15.8406699.5%4
A279T11.5757982.5%8
P-MBPR228K140.935799.9%3
A279T123.325499.9%3
  • Based on these ranges of variance, which use a conservative effect size estimate since the original data were unable to be quantified, we will run the experiment four times.

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    eLife, 3, 10.7554/eLife.04333.
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Decision letter

  1. Tony Hunter
    Reviewing Editor; Salk Institute, United States

In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.

Thank you for submitting your work entitled "Registered report: Diverse somatic mutation patterns and pathway alterations in human cancers" for consideration by eLife. Your submission has been evaluated by a Senior Editor, who would like you to revise the article as follows prior to peer review:

1) In the case of MAP2K4 there has been independent validation by others that the mutants lack kinase activity, and can drive transformation. In this regard, the authors have missed an important paper (Ahn et al. MCB 31:4270), where the authors examined the properties of 11 MAP2K4 cancer mutants for kinase activity and their ability to affect cell migration and invasion in culture, and alter autochthonous mutant KRas driven NSCLC tumor growth in vivo, reaching the conclusion that MAP2K4 acts as a tumor suppressor. We suggest that the authors add discussion of the Ahn et al. paper and review the literature on MAP2K4 mutations both prior to and subsequent to the Kan et al. paper in question, comprehensively, to ensure that there aren't any other omissions.

2) A technical issue relates to the authors' proposed use of inactive MAP2K7/JNK2 as a substrate for the in vitro MAP2K4 kinase assay. MAP2K7 (aka MKK7) is not the same as JNK2, and it is not a substrate for MAP2K4. This notation came from the original Kan et al. paper, but I suspect they mean MAPK9/JNK2, which is what the Invitrogen website lists under the PV3621 catalogue number they give. Could you check and correct this accordingly?

[Editors' note: further revisions were requested prior to acceptance, as described below.]

Thank you for submitting your work entitled "Registered report: Diverse somatic mutation patterns and pathway alterations in human cancers" for consideration by eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by Tony Hunter as the Senior Editor and Reviewing Editor. Two of the three reviewers, Somasekar Seshagiri and John Brognard, have agreed to share their names.

The reviewers have discussed the reviews with one another and the Reviewing editor has drafted this decision to help you prepare a revised submission.

Summary:

As part of the Reproducibility Project: Cancer Biology, the authors will set out to replicate experimental data on mutant forms of GNAO1 and MAP2K4 that were published in Kan et al. (Nature 2010). The authors note that the functional relevance of the mutant forms of GNAO1 and MAP2K4 in cancer has been established by subsequent studies and propose to replicate the colony formation assay and kinase assay (MAP2K4) experiments reported in Kan et al. (Nature 2010). The authors describe the reagent generation, experimental work and analysis in great detail. The reviewers agree that the proposed plan is appropriate and closely adheres to published methods, but we have the following requests for revisions before publication of the Registered Report.

Essential revisions:

In the Registered Report, the authors use six protocols to replicate the experiments. For protocol 3, 5 and 6 power calculations and confirmatory analysis plans are described. In the confirmatory analysis plan of the protocol five One-way Anova is followed by planned comparisons using Fisher's LSD and in the corresponding power calculations for the t-tests a 0.05 α error is used. Fisher's LSD, however, does not control the family wise error rate (see Hayter, 1986) and it is useful only for the calculation of the effect size d. A Bonferroni correction should be used (α = 0.025. This issue needs to be addressed.

Reference: Anthony J Hayter. The maximum familywise error rate of Fisher's least significant difference test. Journal of the American Statistical Association, 81(396): 1000-1004, 1986. doi: 10.1080/01621459.1986.10478364

https://doi.org/10.7554/eLife.11566.002

Author response

1) In the case of MAP2K4 there has been independent validation by others that the mutants lack kinase activity, and can drive transformation. In this regard, the authors have missed an important paper (Ahn et al. MCB 31:4270), where the authors examined the properties of 11 MAP2K4 cancer mutants for kinase activity and their ability to affect cell migration and invasion in culture, and alter autochthonous mutant KRas driven NSCLC tumor growth in vivo, reaching the conclusion that MAP2K4 acts as a tumor suppressor. We suggest that the authors add discussion of the Ahn et al.

paper and review the literature on MAP2K4 mutations both prior to and subsequent to the Kan et al. paper in question, comprehensively, to ensure that there aren't any other omissions.

We have incorporated a discussion of this study (Ahn et al. 2011) into the Introduction. We have also added other citations to expand the state of knowledge about the function of MAP2K4 mutations.

2) A technical issue relates to the authors' proposed use of inactive MAP2K7/JNK2 as a substrate for the in vitro MAP2K4 kinase assay. MAP2K7 (aka MKK7) is not the same as JNK2, and it is not a substrate for MAP2K4. This notation came from the original Kan et al. paper, but I suspect they mean MAPK9/JNK2, which is what the Invitrogen website lists under the PV3621 catalogue number they give. Could you check and correct this accordingly?

We have made this change. The Invitrogen PV3621 MAPK9 is listed correctly listed in the reagents list.

[Editors' note: further revisions were requested prior to acceptance, as described below.]

Essential revisions: In the Registered Report, the authors use six protocols to replicate the experiments. For protocol 3, 5 and 6 power calculations and confirmatory analysis plans are described. In the confirmatory analysis plan of the protocol five One-way Anova is followed by planned comparisons using Fisher's LSD and in the corresponding power calculations for the t-tests a 0.05 α error is used. Fisher's LSD, however, does not control the family wise error rate (see Hayter, 1986) and it is useful only for the calculation of the effect size d. A Bonferroni correction should be used (α

= 0.025. This issue needs to be addressed. Reference: Anthony J Hayter. The maximum familywise error rate of Fisher's least significant difference test. Journal of the American Statistical Association, 81(396): 1000-1004, 1986. doi: 10.1080/01621459.1986.10478364

We agree with the reviewers’ comment on the use of a correction, such as Bonferroni or the modification of LSD by Hayter as ways to control for the MFWER; however as Hayter describes in his 1986 paper, this applies in situations where the ANOVA is unbalanced or with a balanced design with four or more populations. Since the proposed analysis is balanced with three population groups, the LSD is sufficiently conservative and powerful to account for the multiple comparisons in this specific situation. This is further explained by Levin et al., 1994 and discussed in Maxwell and Delaney, 2004 (Chapter 5) and Cohen, 2001 (Chapter 12).

References:

Levin, J.R., Serline, R.C., & Seaman M.A. (1994). A controlled, powerful multiple-comparison strategy for several situations. Psychological Bulletin, 115, 153-159.

Maxwell, S.E. & Delaney, H.D. (2004). Designing experiments and analyzing data: a model comparison perspecitive. Lawrence Erlbaum Associates, Mahwah, N.J., 2nd edition.

Cohen, B.H. (2001). Explaining psychological statistics. John Wiley and Sons, New York, 2nd edition.

https://doi.org/10.7554/eLife.11566.003

Article and author information

Author details

  1. Vidhu Sharma

    Applied Biological Materials, Richmond, Canada
    Contribution
    VS, Drafting or revising the article
    Competing interests
    VS the experiments presented in this manuscript will be conducted at Applied Biological Materials, which is a Science Exchange lab.
  2. Lisa Young

    Applied Biological Materials, Richmond, Canada
    Contribution
    LY, Drafting or revising the article
    Competing interests
    LY the experiments presented in this manuscript will be conducted at Applied Biological Materials, which is a Science Exchange lab.
  3. Anne B Allison

    Piedmond Virginia Community College, Charlottesville, United States
    Contribution
    ABA, Drafting or revising the article
    Competing interests
    No competing interests declared.
  4. Kate Owen

    University of Virginia, Charlottesville, United States
    Contribution
    KO, Drafting or revising the article
    Competing interests
    No competing interests declared.
  5. Reproducibility Project: Cancer Biology

    Contribution
    RP:CB, Conception and design, Drafting or revising the article
    For correspondence
    nicole@scienceexchange.com
    Competing interests
    RP:CB employed by and holds shares in Science Exchange Inc.
    1. Elizabeth Iorns, Science Exchange, Palo Alto, United States
    2. William Gunn, Mendeley, London, United Kingdom
    3. Fraser Tan, Science Exchange, Palo Alto, United States
    4. Joelle Lomax, Science Exchange, Palo Alto, United States
    5. Nicole Perfito, Science Exchange, Palo Alot, United States
    6. Timothy Errington, Center for Open Science, Charlottesville, United States

Funding

Laura and John Arnold Foundation

  • The Reproducibility Project: Cancer Biology Core Team

The Reproducibility Project: Cancer Biology is funded by the Laura and John Arnold Foundation, provided to the Center for Open Science in collaboration with Science Exchange. The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

The Reproducibility Project: Cancer Biology core team would like to thank the original authors, in particular Bijay Jaiswal, for generously sharing critical information to ensure the fidelity and quality of this replication attempt. We are grateful to Courtney Soderberg at the Center for Open Science for assistance with statistical analyses. We would also like to thank the following companies for generously donating reagents to the Reproducibility Project: Cancer Biology; American Type Culture Collection (ATCC), Applied Biological Materials, BioLegend, Charles River Laboratories, Corning Incorporated, DDC Medical, EMD Millipore, Harlan Laboratories, LI-COR Biosciences, Mirus Bio, Novus Biologicals, Sigma-Aldrich, and System Biosciences (SBI).

Reviewing Editor

  1. Tony Hunter, Reviewing Editor, Salk Institute, United States

Publication history

  1. Received: September 15, 2015
  2. Accepted: January 8, 2016
  3. Version of Record published: February 19, 2016 (version 1)

Copyright

© 2016, Sharma et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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