Registered Report: COT drives resistance to RAF inhibition through MAP kinase pathway reactivation

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 “COT drives resistance to RAF inhibition through MAPK pathway reactivation” by Johannessen and colleagues, published in Nature in 2010 (Johannessen et al., 2010). The key experiments to be replicated are those reported in Figures 3B, 3D-E, 3I, and 4E-F. In Figures 3B, D-E, RPMI-7951 and OUMS023 cells were reported to exhibit robust ERK/MEK activity concomitant with reduced growth sensitivity in the presence of the BRAF inhibitor PLX4720. MAP3K8 (COT/TPL2) directly regulated MEK/ERK phosphorylation, as the treatment of RPMI-7951 cells with a MAP3K8 kinase inhibitor resulted in a dose-dependent suppression of MEK/ERK activity (Figure 3I). In contrast, MAP3K8-deficient A375 cells remained sensitive to BRAF inhibition, exhibiting reduced growth and MEK/ERK activity during inhibitor treatment. To determine if RAF and MEK inhibitors together can overcome single-agent resistance, MAP3K8-expressing A375 cells treated with PLX4720 along with MEK inhibitors significantly inhibited both cell viability and ERK activation compared to treatment with PLX4720 alone, as reported in Figures 4E-F. The Reproducibility Project: Cancer Biology is collaboration between the Center for Open Science and Science Exchange and the results of the replications will be published in eLife. DOI: http://dx.doi.org/10.7554/eLife.11414.001


Introduction
Activation of the canonical mitogen activated protein kinase (MAPK) pathway occurs in response to the binding of growth factors, hormones, or neurotransmitters to receptor tyrosine kinase receptors located at the cell surface (Dhomen and Marais, 2009;Lopez-Bergami et al., 2008). In untransformed cells, receptor ligation induces the sequential activation of the small GTPase RAS, followed by RAF, MEK and ERK, which relays proliferative signals generated at the cell periphery into the nucleus to control cellular survival, differentiation and growth (Inamdar et al., 2010;Panka et al., 2006). Not surprisingly, dysregulation of MAPK signaling is common in many human cancers including melanoma. Mutations in the RAF and RAS genes (Davies et al., 2002;Mercer and Pritchard, 2003) that trigger constitutive activation of the MAPK pathway can result in uncontrolled cell proliferation, invasion, metastasis, survival and angiogenesis (Panka et al., 2006;Sharma et al., 2006;. BRAF is one of three members of the RAF family, which includes ARAF, BRAF, and CRAF (or RAF-1) (Dhomen and Marais, 2009). In melanoma, BRAF represents the most commonly mutated gene in the MAPK signaling cascade where 90% of tumors carry a valine to glutamic acid transition at codon 600 (V600E) that renders BRAF constitutively active and hyperactivates the MAPK cascade (Davies et al., 2002;Dhomen and Marais, 2009;Michaloglou et al., 2008). While preclinical and clinical studies have shown that targeting BRAF (V600E) melanomas with the use of RAF-selective inhibitors results in initial tumor regression (Fedorenko et al., 2015;Flaherty et al., 2010;Shtivelman et al., 2014), responses to RAF inhibitors are transient, with acquired resistance triggering disease progression (Shtivelman et al., 2014). Although progress has been made in the development of drugs that target RAF, the clinical outcome regarding long-term usage and the mechanisms of acquired resistance warrants further evaluation. In their study, Johannessen and colleagues sought to identify kinases involved in mediating resistance to the RAF kinase inhibitor PLX4720 (Johannessen et al., 2010).
Using a kinase open reading frame (ORF) collection and a high throughput screening methodology, Johannessen and colleagues identified MAP3K8 (the gene encoding cancer osaka thyroid (COT)/TPL2), as a driver of resistance to BRAF inhibition with PLX4720 (Johannessen et al., 2010). Johannessen and colleagues first examined basal MAP3K8 expression in multiple cell lines harboring the V600E mutation (Johannessen et al., 2010). As shown in Figure 3B and reported by others, RPMI-7951 and OUMS-23 cells were found to express high intrinsic levels of MAP3K8 compared to A375 cells where MAP3K8 was undetectable (Johannessen et al., 2010;Paraiso et al., 2012). RPMI-7951 and OUMS-23 cells also exhibited robust, undiminished ERK and MEK activity concomitant with reduced growth sensitivity in the presence of PLX4720 (Figure 3D-E; Johannessen et al., 2010). This is supported by additional findings demonstrating that RPMI-7981 cells treated with the closely related BRAF inhibitor PLX4032/vemurafenib (a successor of PLX4720) also remain refractory to inhibitor treatment as assessed by annexin V staining (Paraiso et al., 2012) and MTS assay (Park et al., 2013). However, others have reported RPMI-7981 cells as exhibiting modest sensitivity to PLX4720 (Schayowitz et al., 2012). In the latter case, ERK activity was reduced by 50% after incubation with inhibitor, although these differences in sensitivity may reflect the significantly shorter time course and experimental design used by Park and colleagues. Finally, in Figure 3I, Johannessen and colleagues determined that MAP3K8 kinase activity is required to regulate MEK/ERK activation in RPMI-7951 cells, findings that further confirm MAP3K8 is an essential upstream activator of the MEK-ERK signaling cascade (George and Salmeron, 2009;Johannessen et al., 2010). The key experiments outlined in Figures 3B,D,E, and 3I will be replicated in protocols 1, 2, 3, and 4.
Resistance to targeted agents, such as BRAF inhibitors, is a frequent cause of therapy failure, as noted above. Importantly, chronic BRAF inhibition can lead to cross-resistance to several BRAFselective inhibitors, indicating that resistance is not likely to be overcome by switching to a new RAF inhibitor (Corcoran et al., 2010;Villanueva et al., 2011). It has been suggested previously that combination treatment with MEK and BRAF inhibitors may be useful in preventing the emergence of resistance or in overcoming resistance to single agent therapies targeting either molecule alone (Corcoran et al., 2010). To examine whether the combined use of RAF and MEK inhibitors bypass MAP3K8-driven resistance, Johannessen and colleagues ectopically expressed MAP3K8 in A375 melanoma cells before treatment with BRAF inhibitor (PLX4720) alone or in combination with the MEK inhibitors CI-1040 or AZD6244. As shown in Figures 4E and 4F, both viability and ERK activation was dramatically reduced in MAP3K8-expressing cells treated with either of the combination therapies, similar to cells ectopically expressing MEK1, which remained sensitive to PLX4720 (Johannessen et al., 2010). Similar results were obtained in RPMI-7951 cells expressing high basal levels of MAP3K8 treated with PLX4032 and a second MEK inhibitor AS703026 (Park et al., 2013). Interestingly, overexpression of constitutively active MEK (MEK1 DD ) resulted in increased sensitivity to BRAF inhibition combined with AZD6244, but not CI-1040 (Johannessen et al., 2010). These findings confirm that MAP3K8 is able to reactivate MAPK signaling despite BRAF inhibition and that targeting RAF and MEK in combination may be an effective anti-melanoma treatment strategy. These experiments will be replicated in Protocols 5 and 6.

Protocol 1: MAPK pathway analysis in cells expressing elevated MAP3K8
This experiment assesses the effect the RAF inhibitor, PLX4720, has on the MAPK pathway, in cells expressing elevated MAP3K8, as analyzed via Western blot. It utilizes RPMI-7951 and OUMS-23 cells, which express a high level of MAP3K8 and A375 cells, which have undetectable levels. This protocol replicates the experiments reported in Figures 3B and 3E.

Sampling
Experiment to be repeated a total of 4 times for a minimum power of 80%. The original data is qualitative, thus to determine an appropriate number of replicates to initially perform, sample sizes based on a range of potential variance was determined.
. Cells will be sent for mycoplasma testing and STR profiling.
1. Plate 500,000 A375 cells, 750,000 RPMI-7951, and 750,000 OUMS-23 cells in 6-well plates and incubate for 24-36 hr to achieve log phase growth. 2. 24-36 hr after seeding treat cells with 0.1, 1, and 10 mM PLX4720 or DMSO. Incubate for 24 hr. a. Add drug directly to each well using a 1000X stock (in DMSO). i. Final DMSO concentration kept to 0.1%. 3. Wash cells with 1-2 ml ice-cold PBS and lyse in 1% NP-40 lysis buffer supplemented with 2X protease inhibitors and 1X phosphatase inhibitor cocktails I and II. a. Add~100-200 ml 1% NP-40 lysis buffer to ensure that protein concentration is between 2-3 mg/ml. b. b. Scrape each plate with a rubber cell scraper, collect lysates, and clarify by centrifugation at max speed ( 8. Apply appropriate HRP-linked secondary antibodies for 1 hr at RT with constant agitation, and then detect signal using chemiluminescence following manufacturer's instructions. a. Note: If a Li-COR Odyssey imaging system is available for use, IR Dye-labeled secondary antibodies and a low fluorescence membrane will be used instead, and images will be acquired following manufacturer's instructions. 9. Analyze bands with image analysis software and normalize to loading controls. a. pERK1/2 (T202/Y204) normalized to MEK1/2 (total). b. pMEK1/2 (S217/221) normalized to ERK1/2 (total). c. MAP3K8 normalized to Actin. 10. Repeat steps 1-9 independently three additional times.

Deliverables:
. Data to be collected: Full image western blot films of all immunoblots including ladder. (Compare to Figures  3B and 3E) Raw data of band analysis and normalized bands for each sample.

Confirmatory analysis plan
. Statistical Analysis of the Replication Data: Two-way MANOVA of normalized pERK1/2 and pMEK1/2 levels of A375, RPMI-7951, and OUMS-23 cells with the following planned comparisons using the Bonferroni correction: Planned contrast of normalized pMEK1/2 levels from OUMS-23 cells treated with vehicle compared to cells treated with PLX4720 (all doses).
. Meta-analysis of original and replication attempt effect sizes: The replication data (mean and 95% confidence interval) will be plotted with the original reported data value plotted as a single point on the same plot for comparison.

Known differences from the original study
The replication will not include the other BRAF (V600E) cell lines reported in the original paper. The original NP40 cell lysis buffer was composed of: 150 mM NaCl, 50 mM Tris pH 7.5, 2 mM EDTA pH 8, 25 mM NaF, and 1% NP-40. The replication will use a commercial formula, which has the following composition: 250 mM NaCl, 50 mM Tris pH 7.4, 5 mM EDTA, 50 mM NaF, 1 mM Na 3 VO 4 , and 1% NP-40. The western blots will use Actin, instead of Vinculin, which was reported in Figure 3B. 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/lmhjg/) and made publically available.
Protocol 2: Determine the range of detection of the replicating lab's plate reader This is a general protocol that determines the range of detection of the plate reader in order to calculate the required number of A375, RPMI-7951, and OUMS-23 cells to yield 90-95% confluency in 5 days for Protocols 3 and 5.

Sampling
This experiment is performed a total of once with three cell lines (A375, RPMI-7951, and OUMS-23 cells). Each cell line has 5 conditions to be performed with six technical replicates per experiment: .

Procedure
Note: . A375 cells maintained in RPMI medium supplemented with 10% FBS and 1% penicillin/streptomycin/L-glutamine at 37˚C in a humidified atmosphere at 5% CO 2 .
. Cells will be sent for mycoplasma testing and STR profiling.
1. Plate 800 -1600 A375 cells, 2600 -3400 RPMI-7951 cells, and 2600 -3400 OUMS-23 cells in 96 well plates with 100 ml of medium. Incubate for 5 days. a. Plate media alone (no cells) in columns 1 and 12. b. Plate cells in remaining wells (columns 2-11). c. Exclude plating in the first and last row to avoid edge effects and evaporation. 2. 5 days later estimate confluency and determine cell viability with the WST1 viability assay according to manufacturer's instructions. Briefly described: a. Add 11 ml/well reagent WST-1 (1:10 dilution). b. Incubate cells for 20-30 min. c. Shake thoroughly for 1 min on a shaker. d. Measure the absorbance against a background control as blank using a microplate reader at 420-480 nm. (If reference wavelength is to be determined, a filter >600 nm is recommended) e. Exclude rows A-H due to edge effects/evaporation, thus making each seeding six technical replicates. f. Calculate viability after background subtraction. g. Use starting cell numbers that give~90-95% confluency in 5 days and is in the linear range of the viability assay. i. Original report used 1500 A375 cells, 3000 RPMI-7951 cells, and 3000 OUMS-23 cells.
Deliverables . Data to be collected: Raw data and background subtracted absorbance at 420-480 nm.

Confirmatory analysis plan
. n/a 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. This All of the raw data, including the analysis files, will be uploaded to the project page on the OSF (https://osf.io/lmhjg/) and made publically available.

Protocol 3: PLX4720 growth inhibitory analysis in cells expressing elevated MAP3K8
This experiment assesses the effect the RAF inhibitor, PLX4720, has on cellular viability, in cells expressing elevated MAP3K8. It utilizes RPMI-7951 and OUMS-23 cells, which express a high level of MAP3K8 and A375 cells, which have undetectable levels. This protocol replicates the experiment reported in Figure 3D.

Sampling
Experiment to be repeated a total of 3 times for a minimum power of 80%. The original data is from a single biological replicate, thus to determine an appropriate number of replicates to initially perform, sample sizes based on a range of potential variance was determined.
. See Power Calculations section for details.

Procedure
Note: . A375 cells maintained in RPMI medium supplemented with 10% FBS and 1% penicillin/streptomycin/L-glutamine at 37˚C in a humidified atmosphere at 5% CO 2 .
. Cells will be sent for mycoplasma testing and STR profiling.
1. Plate number of A375, RPMI-7951, and OUMS-23 cells as determined in Protocol 2 in a 96 well plate with 90 ml of medium per well. Incubate for 24 hr. a. Plate media alone (no cells) in columns 1 and 12. b. Plate cells in remaining wells (columns 2-11). c. Exclude plating in the first and last row to avoid edge effects and evaporation. d. One plate is needed for each cell line.
a. Dilute stock of PLX4720 at 1000X final concentration of serial dilution stocks in DMSO (100 mM to 0.01 mM). b. Dilute 1000X serial dilution stocks 1:100 in complete growth medium to yield a 10X stock (1 mM to 10 -4 mM) that is added directly to the 90 ml of cell/medium. i. Final DMSO concentration kept to 0.1%. 3. Determine cell viability with the WST1 viability assay according to manufacturer's instructions.
Briefly described: a. Add 11 ml/well reagent WST-1 (1:10 dilution). b. Incubate cells for 20-30 min. c. Shake thoroughly for 1 min on a shaker. d. Measure the absorbance against a background control as blank using a microplate reader at 420-480 nm. (If reference wavelength is to be determined, a filter >600 nm is recommended) e. Exclude rows A-H due to edge effects/evaporation, thus making each cohort six technical replicates, except DMSO (vehicle), which has 12. f. Calculate viability as a percentage of control (DMSO (vehicle) cells) after background subtraction. g. Determine GI 50 value by fitting data using a nonlinear regression curve fit with a sigmoid dose-response curve (four-parameter log-logistic function). 4. Repeat steps 1-3 independently two additional times.
Deliverables . Data to be collected: Raw data and background subtracted absorbance at 420-480 nm.
GI 50 values of each biological replicate. Graph of average GI 50 values for each condition. (Compare to Figure 3D.)

Confirmatory analysis plan
. Statistical Analysis of the Replication Data: One way ANOVA of GI 50 values from A375, RPMI-7951, and OUMS-23 cells with the following planned comparisons using Fisher's LSD test.
. Meta-analysis of original and replication attempt effect sizes: The replication data (mean and 95% confidence interval) will be plotted with the original reported data value plotted as a single point on the same plot for comparison.

Known differences from the original study
The replication will not include the other BRAF (V600E) cell lines reported in the original paper. 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. The seeding density of each cell line was determined in Protocol 2. All of the raw data, including the analysis files, will be uploaded to the project page on the OSF (https://osf.io/lmhjg/) and made publically available.

Protocol 4: MAPK pathway analysis after MAP3K8 inhibition in cells expressing elevated MAP3K8
This experiment assesses the effect a MAP3K8 kinase inhibitor has on the MAPK pathway, in cells expressing elevated MAP3K8, as analyzed via Western blot. It utilizes RPMI-7951 cells, which express a high level of MAP3K8. This protocol replicates the experiment reported in Figure 3I.

Sampling
Experiment to be repeated a total of 8 times for a minimum power of 80%. The original data is qualitative, thus to determine an appropriate number of replicates to initially perform, sample sizes based on a range of potential variance was determined.
. See Power Calculations section for details.
Experiment has five conditions: .

Procedure
Note: . RPMI-7951 cells maintained in MEM medium supplemented with 10% FBS and 1% penicillin/ streptomycin at 37˚C in a humidified atmosphere at 5% CO 2 .
. Cells will be sent for mycoplasma testing and STR profiling. 9. Apply appropriate HRP-linked secondary antibodies for 1 hr at RT with constant agitation, and then detect signal using chemiluminescence following manufacturer's instructions. a. Note: If a Li-COR Odyssey imaging system is available for use, IR Dye-labeled secondary antibodies and a low fluorescence membrane will be used instead, and images will be acquired following manufacturer's instructions. 10. Analyze bands with image analysis software, normalize to loading controls, and normalize each dose of MAP3K8 inhibitor to Vehicle (DMSO). a. pERK1/2 (T202/Y204) normalized to MEK1/2 (total). b. pMEK1/2 (S217/221) normalized to ERK1/2 (total). 11. Repeat steps 1-10 independently seven additional times.

Deliverables
. Data to be collected: Full image western blot films of all immunoblots including ladder. (Compare to Figure 3I) Raw data of band analysis and normalized bands for each sample.

Confirmatory analysis plan
. Statistical Analysis of the Replication Data: One-way MANOVA of normalized pERK1/2 and pMEK1/2 levels of RPMI-7951 cells treated with MAP3K8 inhibitor with the following analysis using the Bonferroni correction: & One-way ANOVA of pERK1/2 levels of RPMI-7951 cells treated with MAP3K8 inhibitor.
. One-sample t-test of pERK1/2 levels of 20 mM treated cells compared to 1 (vehicle treated cells).
One-way ANOVA of pMEK1/2 levels of RPMI-7951 cells treated with MAP3K8 inhibitor.
. One-sample t-test of pMEK1/2 levels of 20 mM treated cells compared to 1 (vehicle treated cells).
. Meta-analysis of original and replication attempt effect sizes: The replication data (mean and 95% confidence interval) will be plotted with the original reported data value plotted as a single point on the same plot for comparison.

Known differences from the original study
The original NP40 cell lysis buffer was composed of: 150 mM NaCl, 50 mM Tris pH 7.5, 2 mM EDTA pH 8, 25 mM NaF, and 1% NP-40. The replication will use a commercial formula, which has the following composition: 250 mM NaCl, 50 mM Tris pH 7.4, 5 mM EDTA, 50 mM NaF, 1 mM Na 3 VO 4 , and 1% NP-40. 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/lmhjg/) and made publically available.

Protocol 5: Viability analysis following combinatorial MAPK pathway inhibition in cells expressing elevated MAP3K8
This experiment assesses the effect the RAF inhibitor, PLX4720, along with the MEK inhibitors, CI-1040 or AZD6244, has on cellular viability, in cells expressing MAP3K8. It utilizes A375 cells expressing MAP3K8, via ectopic expression of MAP3K8. This protocol replicates the experiment reported in Figure 4E.

Sampling
Generation of A375 cells expressing MEK1, MEK1 DD , and MAP3K8 to be performed once. Experiment (steps 4-6) to be repeated a total of 4 times for a minimum power of 80%. The original data is from a single biological replicate, thus to determine an appropriate number of replicates to initially perform, sample sizes based on a range of potential variance was determined.
. See Power Calculations section for details. Experiment has 3 cohorts: . . A375 cells maintained in RPMI medium supplemented with 10% FBS and 1% penicillin/streptomycin/L-glutamine at 37˚C in a humidified atmosphere at 5% CO 2 .
. 293T cells maintained in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin at 37˚C in a humidified atmosphere at 5% CO 2 .
. Cells will be sent for mycoplasma testing and STR profiling.
ii. Seed two wells per viral concentration for each virus (total wells = 30). b. Add polybrene (4-10 mg/ml final concentration) to plates, swirl to mix, then infect cells with varying concentrations of virus (1:5, 1:10, 1:12, 1:15, and 1:20) in duplicate (i.e. two wells per viral concentration). i. Thaw virus overnight at 4˚C, or in a 37˚C water bath, but without letting the virus get above 4˚C. ii. Add virus to the wells and swirl to mix. iii. Spin 6-well plates at 2250 RPM for 30 min at 37˚C. iv. Incubate cells overnight with virus, then change medium the following morning. c. Incubate for 24 hr, then remove medium and replace with growth medium with or without 10 mg/ml blasticidin. d. After 5-7 days of selection, count the cells in all wells and divide the counts with blasticidin by the non-selected control for each viral dilution. e. Use the lowest viral dilution that yields an 85-95% ratio of selected/non-selected cells (originally observed to be in the 1:8 -1:15 range).

Infect A375 cells with viral supernatant:
a. Seed 100,000 -125,000 A375 cells per well in 6 well plates in 2 ml medium. Incubate for 24 hr in normal growth conditions. i. Seed three wells per virus. (total wells = 9). b. Add polybrene (4-10 mg/ml final concentration) to plates, swirl to mix, then infect cells with dilution determined by titration protocol (step 4 above). i. Thaw virus overnight at 4˚C, or in a 37˚C water bath, but without letting the virus get above 4˚C. ii. Add virus to the wells and swirl to mix.
1. Alternatively, make a 3X virus/polybrene mixture, such that the addition of 1 ml of 3X virus to 2 ml of cells/medium yields an appropriate dilution of virus and polybrene. iii. Spin 6-well plates at 2250 RPM for 30 min at 37˚C. iv. Incubate cells overnight with virus, then change medium the following morning. c. Incubate for 24 hr, then remove medium and replace with growth medium with or without 10 mg/ml blasticidin. i. Two wells without selection and one well with blasticidin for each virus. d. After 5-7 days of selection, count cells in one well of no-blasticidin and the blasticidintreated well to calculate infection efficiency. i. Divide the counts with blasticidin by the non-selected control.

Deliverables:
. Data to be collected: Raw counts and titration percentages (step 2 above). Raw counts and infection efficiency (step 3 above). Raw data and background subtracted absorbance at 420-480 nm. Relative viability of A375 cells expressing MEK1, MEK1 DD , and MAP3K8 as a percentage of DMSO treatment for each cell line. (Compare to Figure 4E.) . Sample for additional protocol: MEK1, MEK1 DD , and MAP3K8 lentivirus for further use (Protocol 6).
. Meta-analysis of original and replication attempt effect sizes: The replication data (mean and 95% confidence interval) will be plotted with the original reported data value plotted as a single point on the same plot for comparison.
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. The seeding density of the A375 cell line was determined in Protocol 2. Infection efficiency will be determined for each replicate. The expression of the kinase of interest will be assessed using antibodies against the V5 tag as well as MAP3K8 and MEK1 as described in Protocol 6. All of the raw data, including the analysis files, will be uploaded to the project page on the OSF (https://osf.io/lmhjg/) and made publically available.

Protocol 6 MAPK pathway analysis following combinatorial MAPK pathway inhibition in cells expressing elevated MAP3K8
This experiment assesses the effect the RAF inhibitor, PLX4720, along with the MEK inhibitors, CI-1040 or AZD6244, has on the MAPK pathway, as analyzed via Western blot. It utilizes A375 cells expressing MAP3K8, via ectopic expression of MAP3K8. This protocol replicates the experiment reported in Figure 4F.

Sampling
Experiment to be repeated a total of 3 times for a minimum power of 80%. The original data is qualitative, thus to determine an appropriate number of replicates to initially perform, sample sizes based on a range of potential variance was determined.
. See Power Calculations section for details.

Procedure
Note: . A375 cells maintained in RPMI medium supplemented with 10% FBS and 1% penicillin/streptomycin at 37˚C in a humidified atmosphere at 5% CO 2 .
. Cells will be sent for mycoplasma testing and STR profiling.
1. Infect A375 cells with viral supernatant: a. Seed 100,000 -125,000 A375 cells per well in 6 well plates in 2 ml medium. Incubate for 24 hr in normal growth conditions. i. Seed six wells per virus. (total wells = 18). b. Add polybrene (4-10 mg/ml final concentration) to plates, swirl to mix, then infect cells following Protocol 5, step 5 using dilution determined by titration protocol (Protocol 5, step 4). c. Incubate for 24 hr, then remove medium and replace with growth medium with or without 10 mg/ml blasticidin. i. Five wells without selection and one well with blasticidin for each virus. d. After 5-7 days of selection, count cells in one well of no-blasticidin and the blasticidintreated well to calculate infection efficiency. i. Divide the counts with blasticidin by the non-selected control. 2. 72 hr after infection (96 hr after seeding) treat cells with DMSO or 1 mM PLX4720 with or without 1 mM AZD6244, 1 mM CI-1040, or DMSO. Incubate for 24 hr. a. Add drugs directly to each well using a 1000X stock (in DMSO). i. Final DMSO concentration kept to 0.2%. 3. Wash cells with 1-2 ml ice-cold PBS and lyse in 1% NP-40 lysis buffer (150 mM NaCl, 50 mM Tris pH 7.5, 2 mM EDTA pH 8, 25 mM NaF, and 1% NP-40) supplemented with 2X protease inhibitors and 1X phosphatase inhibitor cocktails I and II. a. Add~100-200 ml 1% NP-40 lysis buffer to ensure that protein concentration is between 2-3 mg/ml. b. Scrape each plate with a rubber cell scraper, collect lysates, and clarify by centrifugation at max speed ( 8. Apply appropriate HRP-linked secondary antibodies for 1 hr at RT with constant agitation, and then detect signal using chemiluminescence following manufacturer's instructions. a. Note: If a Li-COR Odyssey imaging system is available for use, IR Dye-labeled secondary antibodies and a low fluorescence membrane will be used instead, and images will be acquired following manufacturer's instructions. 9. Analyze bands with image analysis software and normalize to loading controls. a. pERK1/2 (T202/Y204) normalized to MEK1/2 (total). b. V5 (tag on exogenous proteins) normalized to Vinculin. 10. Repeat steps 1-9 independently two additional times.

Deliverables:
. Data to be collected: Raw counts and infection efficiency (step 1 above). Full image western blot films of all immunoblots including ladder. (Compare to Figures 2A and 4F.) Raw data of band analysis and normalized bands for each sample.
. Meta-analysis of original and replication attempt effect sizes: The replication data (mean and 95% confidence interval) will be plotted with the original reported data value plotted as a single point on the same plot for comparison.
The original NP40 cell lysis buffer was composed of: 150 mM NaCl, 50 mM Tris pH 7.5, 2 mM EDTA pH 8, 25 mM NaF, and 1% NP-40. The replication will use a commercial formula, which has the following composition: 250 mM NaCl, 50 mM Tris pH 7.4, 5 mM EDTA, 50 mM NaF, 1 mM Na 3 VO 4 , and 1% NP-40. 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. Infection efficiency will be determined for each replicate. The expression of the kinase of interest will be assessed using antibodies against the V5 tag as well as MAP3K8 and MEK1. All of the raw data, including the analysis files, will be uploaded to the project page on the OSF (https://osf.io/lmhjg/) 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/sptzv/

Protocol 1
The original data presented is qualitative (images of Western blots). We used Image Studio Lite v. 4.0.21 (LI-COR) to perform densitometric analysis of the presented bands to quantify the original effect size where possible. To identify a suitable sample size, power calculations were performed using different levels of relative variance.
Summary of estimated original data reported in Figure 3E: Power Calculations performed with G*Power software, version 3.1.7 (Faul et al., 2007). Partial h 2 calculated from (Lakens, 2013).
Comparisons are between DMSO and all PLX4720 doses (10, 1, and 0.1 mM) In order to produce quantitative replication data, we will run the experiment three times. Each time we will quantify band intensity. We will determine the standard deviation of band intensity across the biological replicates and combine this with the reported value from the original study to simulate the original effect size. We will use this simulated effect size to determine the number of replicates necessary to reach a power of at least 80%. We will then perform additional replicates, if required, to ensure that the experiment has more than 80% power to detect the original effect.

Protocol 3
The original data is from a single biological replicate. To identify a suitable sample size, power calculations were performed using different levels of relative variance. Figure 3D: In order to produce quantitative replication data, we will run the experiment three times. Each time we will calculate the GI 50 value. We will determine the standard deviation of GI 50 values across the biological replicates and combine this with the reported value from the original study to simulate the original effect size. We will use this simulated effect size to determine the number of replicates necessary to reach a power of at least 80%. We will then perform additional replicates, if required, to ensure that the experiment has more than 80% power to detect the original effect.  (Faul et al., 2007). ANOVA F test statistic and partial h 2 performed with R software, version 3.2.1 (Team, 2015). Partial h 2 calculated from (Lakens, 2013). In order to produce quantitative replication data, we will run the experiment eight times. Each time we will quantify band intensity. We will determine the standard deviation of band intensity across the biological replicates and combine this with the reported value from the original study to simulate the original effect size. We will use this simulated effect size to determine the number of replicates necessary to reach a power of at least 80%. We will then perform additional replicates, if required, to ensure that the experiment has more than 80% power to detect the original effect.

Protocol 5
The original data is from a single biological replicate. To identify a suitable sample size, power calculations were performed using different levels of relative variance. Figure 4E (provided by authors) Power Calculations performed with G*Power software, version 3.1.7 (Faul et al., 2007). ANOVA F test statistic and partial h 2 performed with R software, version 3.2.1 (Team, 2015). Power Calculations performed with G*Power software, version 3.1.7 (Faul et al., 2007). ANOVA F test statistic and partial h 2 performed with R software, version 3.2.1 (Team, 2015). In order to produce quantitative replication data, we will run the experiment four times. Each time we will quantify relative viability. We will determine the standard deviation of viability values across the biological replicates and combine this with the reported value from the original study to simulate the original effect size. We will use this simulated effect size to determine the number of replicates necessary to reach a power of at least 80%. We will then perform additional replicates, if required, to ensure that the experiment has more than 80% power to detect the original effect.

MEK1 and MEK1 DD values
The original data presented is qualitative (images of Western blots). We used Image Studio Lite v. 4.0.21 (LI-COR) to perform densitometric analysis of the presented bands to quantify the original effect size where possible.
Summary of estimated original data reported in Figure 4F: Power Calculations performed with G*Power software, version 3.1.7 (Faul et al., 2007). ANOVA F test statistic and partial h 2 performed with R software, version 3.2.1 (Team, 2015). Power Calculations performed with G*Power software, version 3.1.7 (Faul et al., 2007). ANOVA F test statistic and partial h 2 performed with R software, version 3.2.1 (Team, 2015). In order to produce quantitative replication data, we will run the experiment three times. Each time we will quantify band intensity. We will determine the standard deviation of band intensity across the biological replicates and combine this with the reported value from the original study to simulate the original effect size. We will use this simulated effect size to determine the number of replicates necessary to reach a power of at least 80%. We will then perform additional replicates, if required, to ensure that the experiment has more than 80% power to detect the original effect.