• No power calculations required.

Notes:

• This protocol contains information described in Li et al. (2009).

• ^*Based on the number of tumor cells provided by the original authors, inject at least two mice in order to maintain the tumor tissue and provide sufficient cells for downstream experiments.

1. Anesthetize 6–8 week old male NOD/SCID mice with Ketamine/xylazine.

   • a. ^#Ketamine (50 mg/ml) and xylazine (20 mg/ml) are mixed with saline (2:0.42:5.91), sterilized with 0.22 µm filter and 0.08–0.1 ml is injected per mouse.

   • b. The anesthesia may be supplemented with inhalant isoflurane mixed 50/50 with mineral oil when the mouse responds to squeezing of the hind foot or shows response during the procedure.

2. Shave the dorsal hair of each mouse and prep the skin with Betadine and then 70% ethanol. Make a small 0.5 cm incision in the dorsal skin of the mouse, right under the head. Open a pocket under the skin with surgical scissors and insert a prepared testosterone pellet. Staple the incision to close.

   • a. Prepare the testosterone pellet by directly compressing testosterone propionate powder with a 2 mm × 4 mm die set.

3. Immediately after testosterone pellet implantation, inject tumor cells into mice.

   • a. Resuspend 2 × 10⁶ cells in 30 µl IMDM supplemented with 10% FBS and chill on ice.

   • b. Mix with 30 µl concentrated Matrigel.

   • c. Inject the mixture (60 µl total) into the flank of the mouse using an insulin syringe.

   • d. Implant at least 2 and if possible 3 mice with tumor cells.

4. Inject the mouse subcutaneously with 0.02 ml of banamine followed by 1 ml of sterile saline to aid in recovery from the anesthesia. Put the mouse cage on a heating pad. Monitor the mouse until it starts to move.

5. After 7 days of surgery, remove the surgical staples.

6. Monitor tumor development starting from the second week after the surgery.

   • a. Measure tumor incidence (number of tumors/number of injections).

   • b. Measure latency (time from injection to detection of palpable tumors).

   • c. Measure tumors weekly with caliper measurements (height, width, and depth to determine volume by the formula V = 1/2*[length × width^2]).

7. Terminate animals at 4–6 months after tumor cell injection when tumor size reaches 1.5 cm in diameter and aseptically dissect out tumors.

   • a. Lab will record euthanasia method.

   • b. Weigh tumors.

8. Mince each dissected tumor into 1–2 mm³ pieces using a razor blade.

   • a. Use the tumor tissue for purifying LAPC4 cells (Protocol 2).

• Data to be collected:

  • ○ Record LAPC4 xenograft tumor maintenance (passages, tumor incidence, latency, tumor caliper measurements (and calculated volume), tumor weight at harvest, time of harvest, and euthanasia method, calculated density of each tumor based on volume and weight).

• Sample delivered for further analysis:

  • ○ Minced LAPC4 tumor tissue for purification of LAPC4 cells. (Protocol 2).

• Statistical Analysis of the Replication Data:

  • ○ None required.

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

  • ○ None required.

The replicating lab will use a thoracentesis puncture needle to implant tumor tissue into recipient mice instead of making incisions. The replicating lab routinely uses this technique to implant tumor tissue into mice.

Tumor development characteristics will be recorded for each mouse. All data obtained from the experiment—raw data, data analysis, control data and quality control data—will be made publicly available, either in the published manuscript or as an open access dataset available on the Open Science Framework (https://osf.io/gb7sr/).

• No power calculations required.

Note: This protocol contains information described in papers from Patrawala and colleagues (2005), (2006), (2007); Li and colleagues (2009).

1. Use tumor aseptically dissected out of animals in Protocol 1. Weigh tumor tissue in a pre-tared sterile Petri dish and record weight. Mince tumor into ∼1 mm³ pieces using a razor blade in 1–2 ml cold IMDM + 15% FBS.

2. Transfer cell slurry to a 50 ml tube using a cut 1 ml pipette tip and add ∼20× the volume of cold IMDM with GLUTAMAX + 10% FBS. Invert tube to mix and centrifuge at 130×g for 5 min at 4°C. Discard supernatant.

3. Rinse twice in cold IMDM with GLUTAMAX + 10% FBS and once in PBS to wash out serum. Invert tube to mix and centrifuge at 130×g for 5 min at 4°C. Discard supernatant.

4. Incubate slurry in 1× Accumax Cell Dissociation solution at 10 ml per 0.5 g tumor tissue in PBS for 30 min at room temperature under rotating conditions.

   • a. Before experiment, bring Accumax to room temperature.

5. Briefly vortex tissue solution and allow residual undissociated tumor pieces to precipitate to the bottom of tubes for ∼2 min. Collect cells from the supernatant into new tube.

   • a. Residual tumor pieces can be subjected to one or more rounds of Accumax digestion (step 3). Store already-digested cells on ice. *Repeat until no large undissociated pieces remain.

   • b. Pool dissociated cells.

6. Spin pooled cells at 130×g for 5 min.

7. Filter supernatant through a 40 µm cell strainer to obtain a cell suspension.

   • a. Pre-wet strainer with medium.

   • b. Collect into a 50 ml centrifuge tube and resuspend in 1–3 ml IMDM with GLUTAMAX + 10% FBS.

8. Count viability of cells on a hemocytometer using erythrosine B.

   • a. If a large number of cells are obtained (>50 million live cells), then only a portion of that number should be used for further processing (flow cytometry time and sorting speed will put a limit on analyzing a maximum of ∼20 million cells).

   • b. Adjust cell suspension to 1–1.5 million cells (dead + live) per ml with IMDM with GLUTAMAX + 10% FBS.

9. Load 3 ml of cell suspension gently onto a 3 ml layer of Histopaque-1077 gradient in a 15 ml tube and centrifuge at 400×g for 30 min at room temperature.

   • a. Load with a 1 ml pipette tip.

   • b. For larger volumes, 15 ml of cell suspension can be added to 15 ml of Histopaque in a 50 ml tube at a concentration of no more than 1 × 10⁶ cells/ml (dead + live).

10. Collect live nucleated epithelial cells at the interface of the two layers (opaque layer) using a 1 ml pipette tip to transfer to a new tube. Spin at 130×g for 5 min at RT.

    • a. If multiple tubes were used in Step 9, combine the live cells from all tubes before spinning.

11. Resuspend cells in 0.1–5 ml of IMDM with GLUTAMAX + 10% FBS, count the cell number, and spin again at 130×g for 5 min at RT.

    • a. Wash cells again as per Miltenyi's instructions.

12. Deplete cell mixture of lineage-positive host (mouse) cells using the MACS Lineage Cell Depletion Kit. Refer to additional details in protocol from Miltenyi. (Volumes given are for 5 million cells.)

    • a. Suspend cell pellet in 40 µl of staining buffer (PBS, pH 7.2, 0.5% FBS, 5 µg/ml insulin, 0.5% BSA).

    • b. Add 10 µl of supplied biotin-antibody cocktail (CD5, CD45R, CD11b, anti-Ly-6G, 7-4, and Ter-119).

    • c. Mix well and incubate for 10 min at 4°C in the dark.

    • d. Add 30 µl of staining buffer.

    • e. Add 20 µl supplied anti-biotin microbeads.

    • f. Mix well and incubate for additional 15 min at 4°C in the dark.

    • g. Wash cells by adding 10–20× labeling volume in cold PBS and centrifuge at 130×g for 5 min at 4°C. Pipette off supernatant completely.

    • h. Resuspend up to 10⁷ cells in 500 µl staining buffer.

    • i. Assemble MACS apparatus by attaching the MACS Separator on the stand and placing the MACS MS column in the magnetic field with a 15 ml collection tube below the column.

    • j. Prepare the column by rinsing with 500 µl of staining buffer.

    • k. Apply cell suspension to the column. Allow cells to pass by gravity and collect the effluent as fraction with unlabeled cells, representing the enriched human tumor cell fraction depleted of mouse cells.

    • l. Wash the column with 500 µl of staining buffer and collect the effluent in the same tube as effluent of step k. Perform 3×, each time once the column reservoir is empty.

    • m. Centrifuge the effluent at 130×g for 5 min at 4°C and discard supernatant.

13. ^#Confirm the purity of the cells using FACS.

    • a. Prepare the PE-labeled human EpCAM following the manufacture's instruction for the primary and isotype staining, calculating enough total volume to add 100 µl per sample Ab stain in cold Stain buffer.

    • b. Use cold Stain buffer (PBS, pH 7.2, 0.5% FBS, 0.5% BSA) when preparing primary antibodies and isotypes.

    • c. Add 100 µl per sample of diluted antibodies to the 5 ml FACS tube and resuspend cells by gently pipetting.

    • d. Incubate for 45 min at 4°C, protected from light.

    • e. Add 200 µl cold Stain Buffer to each tube and centrifuge at 1300 rpm (240×g) × 5 min at 4°C.

    • f. Remove supernantant and resuspend in cold BD Stain Buffer at 200 µl/well and then pellet at 1300 rpm (240×g) × 5 min at 4°C. Repeat wash two times.

    • g. Add 100 μl cold Stain Buffer/tube to resuspend cells and analyze samples immediately using BD FACSCalibur. Cell debris and cell aggregates will be gated out based on the FSC/SSC parameters.

14. Resuspend cells in 0.1–5 ml of IMDM with GLUTAMAX + 10% FBS and count the cell number.

15. Use resuspended cells for further analysis (Protocol 3, Protocol 4, and Protocol 7).

• Data to be collected:

  • ○ Weight of tumor tissue.

  • ○ Cell viability counts before and after Histopaque gradient and after MACS depletion.

  • ○ All FACS plots from assessing the purity of the cells.

  • ○ Percentage of LAPC4 cells in enriched cell population.

• Sample delivered for further analysis:

  • ○ Resuspended purified LAPC4 cells (for Protocols 3, 4 and 8).

• Statistical Analysis of the Replication Data:

  • ○ None required.

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

  • ○ None required.

• None noted.

The purity of the cells after depletion of lineage-positive cells will be determined by FACS. All data obtained from the experiment—raw data, data analysis, control data and quality control data—will be made publicly available, either in the published manuscript or as an open access dataset available on the Open Science Framework (https://osf.io/gb7sr/).

• The original data presented is qualitative. Based on power calculations with a range of possible variance, we will perform the experiment three times for a final estimated power of at least 82.2%.

  • ○ See Power calculations for details.

• Each experiment consists of two cohorts:

  • ○ Cohort 1: CD44⁺ LAPC4 cells.

  • ○ Cohort 2: CD44⁻ LAPC4 cells.

  • ○ Within each cohort, assess levels of three miRNAs:

    • ■ Let-7b.

    • ■ miR-34a.

    • ■ miR-103.

      • These qRT-PCR reactions are run in technical triplicate.

1. CD44-based purification by FACS (volumes given below are for 5 million cells). Note: This step contains information described in Patrawala and colleagues (2006), (2007) and Li and colleagues (2009).

   • a. Centrifuge the single cell suspension from Protocol 2 at 380×g for 5 min at 4°C and resuspend cells in 90 µl of cold staining buffer (PBS, pH 7.2, 0.5% BSA, 5 µg/ml insulin).

     • i. Remove 10 µl from cell suspension and add to 90 µl staining buffer in another tube for isotype control staining.

   • b. Add 20 µl of FcR blocking reagent to the remaining 80 µl of cell suspension and incubate at 4°C in the dark for 10 min.

   • c. Add 10 µl of FITC-conjugated monoclonal anti-CD44 to the cell suspension, mix by tapping, and incubate at 4°C in the dark for 15 min.

     • i. Note: antibody comes prediluted from the manufacturer and no additional dilution is necessary.

     • ii. Gently tap every 5 min to prevent cells from settling down.

     • iii. For isotype control use 10 µl FITC-mouse IgG2b isotype control immunoglobulin.

   • d. Add 5 ml of cold PBS and spin at 380×g for 5 min at 4°C. Resuspend cells in cold serum-free IMDM with GLUTAMAX + 1% pen/strep at a concentration of 2 × 10⁶ cells/ml in a 5 ml polystyrene tube at no more than 2.5 ml/tube.

     • i. Keep on ice and shielded from light.

   • e. Prepare collection tubes using 5 ml polypropylene tubes containing 1 ml of FBS + 1% pen/strep.

   • f. Add 7-AAD to 1 µg/ml (stock = 100 µg/ml) to the sort tube 10 min before analysis.

   • g. Analyze and sort by FACS (Coulter Epics Elite flow cytometer), electronically gating out cell debris and cell aggregates. Collect only the top 10% of the most brightly stained cells for the positive population (Cohort 1: CD44⁺) and only the bottom 10% most dimly stained cells for the negative population (Cohort 2: CD44⁻).

     • i. Re-run the isolated cells to confirm their purity.

2. qRT-PCR analysis. Note: This step contains information described in Wiggins and colleagues (2010).

   • a. Isolate total RNA from sorted cells.

     • i. Measure RNA concentrations and record the A_260/280 and A_260/230 ratios using a NanoDrop 2000^#.

   • b. Heat-denature 10 ng of total RNA and miRNA-specific RT-primers at 70°C for 2 min and reverse transcribe using M-MLV reverse transcriptase following manufacturer's instructions.

   • c. Determine gene expression levels by real-time PCR using Platinum Pfx DNA Polymerase and the Stratagene MX3005P Real-time PCR system^#. All reactions should be optimized^# and run in triplicate.

     • i. miR-34a specific primers/probe.

     • ii. hsa-let-7b specific primers/probe.

     • iii. Internal references.

       • miR-103 specific primers/probe.

   • d. Analyze:

     • i. Define the threshold cycle (Ct) as the fractional cycle number at which fluorescence exceeds the fixed threshold of 0.2.

     • ii. Analyze using the dCt (the Ct value normalized to miR-103 miRNA levels) and ddCt (difference between the dCt of marker positive population and that of the negative population) values for each of the miRNAs and markers.

     • iii. Convert ddCt to percentage of expression using the formula (2^-ddCt).

3. Repeat experiment two more times.

• Data to be collected:

  • ○ FACS plots of CD44 purification.

  • ○ Post-sort FACS analysis of CD44 purity.

  • ○ Measured RNA concentrations after small RNA purification as well as A_260/280 and A_260/230 ratios.

  • ○ Raw Ct qRT-PCR values, dCt, ddCT, and percentage of expression.

  • ○ Graph of percentage of expression for each condition (Compare to Figure 1B.)

• Statistical Analysis of the Replication Data:

  • ○ At the time of analysis, we will perform the Shapiro–Wilk test and generate a quantile–quantile (q–q) plot to attempt to assess the normality of the data and also perform Levene's test to assess homoscedasiticity. If the data appears skewed, we will attempt a transformation in order to proceed with the proposed statistical analysis listed below and possibly perform the appropriate non-parametric test.

    • ■ Compare percentage expression levels of miR-34a to levels of let-7b.

      • unpaired two-tailed Student's t-test.

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

  • ○ Because the original data is qualitative, a meta-analysis cannot be performed. The replication data will be plotted as means and 95% confidence intervals and the original data will be presented as a single point on the same plot for comparison.

• The replication will be restricted to LAPC4 cells only.

• The replicating lab will use a Stratagene MX3005P Real-time PCR system in place of the ABI Prism 7900 SDS used by the original authors.

• Based on the recommendation of the original authors, total RNA will be isolated in Step 2a instead of isolating only the small RNA fraction.

RNA sample purity (A_260/280 and A_260/230 ratios) will be reported for each sample. An isotype control will be used during the FACS experiment. All data obtained from the experiment—raw data, data analysis, control data and quality control data—will be made publicly available, either in the published manuscript or as an open access dataset available on the Open Science Framework (https://osf.io/gb7sr/).

• This experiment will be performed with 7 mice per group for a final power of 81.2%.

  • ○ See Power Calculations section for details.

• The experiment consists of three cohorts of NOD/SCID mice:

  • ○ Cohort 1: injected with uninfected LAPC4 cells (additional control).

    • ■ N = 7.

      • Based on power calculations performed for the original two groups.

  • ○ Cohort 2: injected with control-infected LAPC4 cells.

    • ■ N = 7.

  • ○ Cohort 3: injected with miR-34-infected LAPC4 cells.

    • ■ N = 7.

Notes:

• HEK293 cells are maintained in IMDM with GLUTAMAX + 7% FBS without antibiotics.

• HT1080 cells are maintained in EMEM + 10% FBS with antibiotics.

  • ○ All cells are maintained at 37°C with 5% CO₂.

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

1. Generate lentivirus. Note: This step contains information described in Jeter and colleagues (2009).

   • a. Grow and prep all plasmids using Sigma Maxiprep kit according to manufacturer's instructions.

     • i. Total DNA needed for one round of transfection:

       • ■ RRE: 12 µg.

       • ■ REV: 8 µg.

       • ■ VSVG: 8 µg.

       • ■ Lenti-ctl: 6 µg.

       • ■ Lenti-miR-34a: 6 µg.

   • b. Seed HEK293 packaging cells at 6 million cells per 15 cm dish for each transfection.

   • c. The next day transfect with 6 µg RRE, 4 µg REV, and 4 µg VSVG packaging plasmids, along with the 6 µg of the lentiviral vector (lenti-ctl or lenti-miR-34a) using Lipofectamine 2000 at a 1:2 ratio of DNA (µg) to transfection reagent (µl) following manufacturer's instructions.

   • d. After 36–48 hr, collect media containing virus and add fresh media.

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

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

   • f. Concentrate viral stock.

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

     • ii. Filter the supernatant through a 0.45 µm syringe filter.

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

   • g. Determine lentivirus titer for GFP using HT1080 cells.

     • i. 1 day before harvesting viral supernatant, plate 1.2 × 10⁵ HT1080 cells per well of a six well dish.

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

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

     • iv. Using a fluorescent microscope, count the number of GFP-positive cells per well; roughly 1–10% of cells will be GFP positive.

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

       • ■ Divide the number of GFP-positive cells by the number of total cells counted.

       • ■ Multiply that by the number of cells at the time of infection.

       • ■ Multiply that by 1 divided by the final viral solution in µl multiplied by 1000.

       • ■ This will yield the number of transfection units per ml.

2. Infect LAPC4 single cell suspension from Protocol 2 at a multiplicity of infection (MOI) of 10.

   • a. *Keep LAPC4 cells at 37°C/5% CO₂ in IMDM with GLUTAMAX + 10% FBS in ultra-low attachment T75 flasks.

3. 24 hr post infection, harvest 10,000 cells per xenograft injection and harvest the remaining cells for qRT-PCR measurement.

   • a. At time of harvest, visually examine the percentage of transduced cells by assessing GFP expression using a fluorescent microscope; ensure that greater than 90% of cells are GFP positive.

     • i. Perform cell counts and record percentage of transduced cells. If percentage is less than 90%, discard cells and repeat transduction to obtain a 90% transduced population.

   • b. 210,000 cells total for 21 injections.

     • i. 7 injections per group.

       • ■ 70,000 uninfected LAPC4 cells.

       • ■ 70,000 lenti-ctl infected LAPC4 cells.

       • ■ 70,000 miR-34a infected LAPC4 cells.

   • c. Snap freeze remaining cells for RNA extraction and store at −80°C until use in Protocol 6.

     • i. Snap-freeze an equal number of uninfected, lenti-ctl infected and miR-34a infected LAPC4 cells.

     • ii. Split each batch of cells into three aliquots as technical replicates for Protocol 6.

4. Subcutaneous (s.c.) injection of LAPC4 cells. Note: This step contains information described in Patrawala and colleagues (2005), (2006), (2007) and Li and colleagues (2009).

   • a. For each injection, centrifuge 10,000 cells at 380×g for 5 min at RT and resuspend in 20 µl culture medium (IMDM + 15% FBS).

   • b. Mix with 20 µl Matrigel on ice.

   • c. Hold a 6–8 week old male NOD/SCID mice and inject the cell/Matrigel mixture into the flank by using an ultra-fine II insulin syringe, ½ cc, 31G needle.

   • d. Repeat for remaining mice.

5. Monitor tumor development starting from the second week after injection.

   • a. Measure tumor incidence (numbers of tumors/number of injections).

   • b. Measure latency (time from injection to detection of palpable tumors) (additional parameter).

   • c. Measure tumors weekly with caliper measurements (height, width, and depth to determine volume) (additional parameter).

6. Terminate all animals when the average weight of the control group tumors reaches 0.54 g; in the original study, this was at the 69 day mark. Dissect out tumors, and image.

   • a. To convert caliper measurements to approximate weight, use data on tumor volume and tumor weight from Protocol 1 to calculate the mean density of the control group tumors. Use this density to calculate weight of the current tumors from measured volume.

     • i. Only control group tumors will be analyzed at this stage to assess when to terminate all animals in the experiment.

   • b. After dissection, weigh tumors.

   • c. Image whole tumors by brightfield imaging.

     • i. Please include a ruler to provide a size scale.

   • d. Divide tumor into two pieces: one for protein extraction (Protocol 5) and one for RNA extraction (Protocol 6).

     • i. Tumor tissue can be snap frozen for RNA and protein extraction.

• Data to be collected:

  • ○ Record of LAPC4 xenograft tumor maintenance (tumor incidence, latency [additional parameter], tumor caliper measurements [and volume (additional parameter)], tumor weight at harvest, time of harvest) (Compare to S5C).

  • ○ Images of tumors by brightfield microscopy (Compare to Figure 5C).

• Sample delivered for further analysis:

  • ○ Tumor tissue processed for protein extraction (Protocol 5) and RNA extraction (Protocol 6).

  • ○ Snap frozen cells for RNA extraction (Protocol 6).

• Statistical Analysis of the Replication Data:

  • ○ At the time of analysis, we will perform the Shapiro–Wilk test and generate a quantile–quantile (q–q) plot to attempt to assess the normality of the data and also perform Levene's test to assess homoscedasiticity. If the data appears skewed, we will attempt a transformation in order to proceed with the proposed statistical analysis listed below and possibly perform the appropriate non-parametric test.

    • ■ Compare mean tumor weight in miR-34a expressed tumors relative to lenti-ctl control tumors. Also compare to uninfected control tumors (additional control).

      • One-way ANOVA followed by planned comparisons using Fisher's LSD.

        • ○ Lenti-control vs miR-34a.

        • ○ Uninfected vs miR-34a.

    • ■ Compare tumor incidence in miR-34a expressed tumors relative to lenti-ctl tumors. Also compare to uninfected control tumors (additional control).

      • One-way ANOVA followed by planned comparisons using Fisher's LSD.

        • ○ Lenti-control vs miR-34a.

        • ○ Uninfected vs miR-34a.

    • ■ Compare tumor latency in miR-34a expressed tumors relative to lenti-ctl tumors. Also compare to uninfected control tumors (additional control).

      • Fisher's Exact Test.

    • ■ Compare tumor volume over time in miR-34a expressed tumors relative to lenti-ctl tumors. Also compare to uninfected control tumors (additional control).

      • Calculate area under the curve (AUC) for each mouse and perform a one-way ANOVA followed by planned comparisons using Fisher's LSD.

      • Lenti-control vs miR-34a.

      • Uninfected vs miR-34a.

• 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.

• This protocol will use the samples derived from Protocol 4.

  • ○ Original data presented is qualitative; representative images. The replication attempt is exploratory in nature and not looking for a specific effect.

• The experiment consists of three cohorts:

  • ○ Cohort 1: tumors from uninfected LAPC4 cells.

  • ○ Cohort 2: tumors from control-infected LAPC4 cells.

  • ○ Cohort 3: tumors from miR-34-infected LAPC4 cells.

  • ○ Each cohort is probed by Western blot for:

    • ■ Anti-CD44.

    • ■ Anti-beta-actin.

Note: at authors' recommendation, the replicating lab will use their standard western blot protocol.

1. Make a total protein lysate from tumor tissue from Protocol 4.

   • a. Transfer a piece of tumor sample into a 5 ml polypropylene tube containing 800–1000 µl lysis buffer.

     • i. Lysis buffer: Add 1 tablet of complete-EDTA free tablet, 100 µl Phosphatase Inhibitor Cocktail II and II to 10 ml RIPA buffer.

   • b. Homogenize the tumor sample on ice with setting on 6 (equal to 30,000 rpm) for 5 s on and 5 s off until it is homogenized completely.

   • c. Transfer the lysate into a 1.5 ml tube and sonicate on ice for 15 s.

   • d. Spin at 13,000 rpm for 20 min at 4°C to remove tissue debris.

   • e. Spin the resultant supernatant at 13,000 rpm for 20 min at 4°C.

   • f. Aliquot the supernatant and store at −80°C.

2. Quantify total protein concentration.

   • a. Prepare BCA standard (0, 25 μg/ml, 125 μg/ml, 250 μg/ml, 500 μg/ml, 750 μg/ml, 1000 μg/ml, 1500 μg/ml, 2000 μg/ml) according to the user manual.

   • b. Dilute tumor lysate sample with PBS at 1:10 ratio.

   • c. Pipette 25 µl of each standard or sample duplicate into 96-well plate.

   • d. Add 200 µl of the working reagent (50:1, Reagent A:B) to each well and mix plate thoroughly on a plate shaker for 30 s.

   • e. Cover plate and incubate at 37°C for 30 min.

   • f. Cool plate to RT. Measure the absorbance at or near 562 nm on a plate reader. Calculate total protein amount of each sample.

3. Preparation of sample:

   • a. Mix tumor lysate with 4× LDS sample buffer plus reducing agent. Boil at 70°C for 5 min. Cool down on ice before centrifuge briefly.

4. Preparation of gel:

   • a. Unpack NuPAGE 4–12% Bis-Tris gel and wash wells with running buffer. Assemble the gel into the cassette. Fill the inner and outer chamber with running buffer.

5. Running the gel:

   • a. Load samples. Run with constant voltage (150–200 V). Stop running when the dye is close to the lower edge, about 1.5 hr.

6. Membrane transfer:

   • a. Pre-activate PVDF membrane in ethanol or methanol for 1 or 2 min, followed by pre-wetting it and filter papers in cold transfer buffer.

   • b. Assemble ‘sandwich’ for Bio-Rad's semi-dry transblot. Layer from bottom to top: filter paper—membrane—gel—filter paper.

   • c. Semi-dry transfer for 90 min at 25 V.

7. When the transfer has finished, immerse PVDF membrane in 5% non-fat milk TBS/0.1%T solution and gently shake for 1 hr at RT.

8. Incubate with primary antibody diluted in antibody dilute solution at 4°C overnight.

   • a. Anti-CD44: 1:1000.

   • b. Anti-beta-actin: 1:5000.

     • i. Loading control.

9. Wash membrane in TBS with 0.1% Tween-20, 5 min × 3.

10. Add IRDye 800CW/680LT Goat anti-rabbit/mouse IgG (diluted 1:5000 with 0.5% non-fat milk TBS/0.1%T solution) and incubate 1 hr at RT, protected from light.

11. Wash membrane in TBS/0.1%T, 5 min × 3, protected from light. Soak in TBS to remove Tween-20.

12. Detect with Odyssey at 800 and 700 channel.

• Data to be collected:

  • ○ Full images, including ladder, of western blots for CD44 and beta-actin for each tumor sample.

  • ○ Calculate band intensity for each band as ratio to loading control band.

• Statistical Analysis of the Replication Data:

  • ○ At the time of analysis, we will perform the Shapiro–Wilk test and generate a quantile–quantile (q–q) plot to attempt to assess the normality of the data and also perform Levene's test to assess homoscedasiticity. If the data appears skewed, we will attempt a transformation in order to proceed with the proposed statistical analysis listed below and possibly perform the appropriate non-parametric test.

    • ■ Compare CD44 protein across all three conditions.

      • One way ANOVA followed by planned comparisons using Fisher's LSD:

        • ○ CD44 protein levels in miR-34a expressed tumors vs to lenti-ctl tumors.

        • ○ CD44 protein levels in miR-34a expressed tumors vs to uninfected control tumors (additional control).

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

  • ○ Meta-analysis will not be performed as this experiment is exploratory in nature and there is no matching original data with which to statistically compare the replication data.

• While the original study performed Western blot analysis on DU145 and PC3 xenograft tumor tissue, this experiment will perform the same analysis on LAPC4 xenograft tumor tissue.

• Uninjected LAPC4 cells and tumors derived from uninjected LAPC4 cells have been added as an additional control.

• Because of these changes, the experiment is not directly confirmatory in nature, but rather exploratory. Thus, we will not directly power the experiment, but we have determined our sensitivity based on the number of samples we will use.

All data obtained from the experiment—raw data, data analysis, control data and quality control data—will be made publicly available, either in the published manuscript or as an open access dataset available on the Open Science Framework (https://osf.io/gb7sr/).

• This protocol will use samples derived from Protocol 4.

  • ○ Original data presented is qualitative; representative images. The replication attempt is exploratory in nature and not looking for a specific effect.

• The experiment consists of six cohorts:

  • ○ RNA derived from uninfected LAPC4 cells.

  • ○ RNA derived from lenti-ctl LAPC4 cells.

  • ○ RNA derived from lenti-miR-34a infected LAPC4 cells.

    • ■ Each cohort of cells has three technical replicates.

  • ○ RNA derived from uninfected LAPC4 xenograft tumor tissue.

  • ○ RNA derived from lenti-ctl LAPC4 xenograft tumor tissue.

  • ○ RNA derived from lenti-miR-34a infected LAPC4 xenograft tumor tissue.

    • ■ Each cohort of tumors has seven biological replicates.

  • ○ In each cohort, qRT-PCR will be performed for:

    • ■ miR-34a.

    • ■ miR-103 (housekeeping miRNA, additional control).

Note: This procedure contains information described in Wiggins and colleagues (2010).

1. Isolate total RNA from frozen cells and tumor tissue from Protocol 4.

   • a. Measure and record RNA concentrations as well as A_260/280 and A_260/230 ratios.

2. Heat denature 10 ng of total RNA and miRNA-specific RT-primers at 70°C for 2 min and reverse transcribe using M-MLV reverse transcriptase following manufacturer's instructions.

3. Determine gene expression levels by real-time PCR using Platinum Pfx DNA Polymerase and the Stratagene MX3005P Real-time PCR system^#. All reactions should be optimized^# and run in triplicate.

   • i. miR-34a specific RT primer.

   • ii. Internal references.

     • a. miR-103 specific RT primer.

4. Analyze qPCR raw data.

   • a. Define the threshold cycle (Ct) as the fractional cycle number at which fluorescence exceeds the fixed threshold of 0.2.

   • b. Analyze using the dCt (the Ct value normalized to miR-103 levels) values for each of the miRNAs and markers.

• Data to be collected:

  • ○ Measured RNA concentrations after small RNA purification, including the A_260/280 and A_260/230 ratios.

  • ○ Raw Ct qRT-PCR values and calculated dCt.

  • ○ Graph of miR-34a levels (dCt) for each condition.

• Statistical Analysis of the Replication Data:

  • ○ At the time of analysis, we will perform the Shapiro–Wilk test and generate a quantile–quantile (q–q) plot to attempt to assess the normality of the data and also perform Levene's test to assess homoscedasiticity. If the data appears skewed, we will attempt a transformation in order to proceed with the proposed statistical analysis listed below and possibly perform the appropriate non-parametric test.

    • ■ One way ANOVA of miR-34a levels across all three conditions in cells before injection followed by planned comparisons using Fisher's LSD:

      • Lenti-clt control cells compared to miR-34a expressed cells.

      • Uninfected cells compared to miR-34a expressed cells.

    • ■ One way ANOVA of miR-34a levels across all three conditions in tumors followed by planned comparisons using Fisher's LSD:

      • Lenti-clt control tumors compared to miR-34a expressed tumors.

      • Uninfected tumors compared to miR-34a expressed tumors.

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

  • ○ Meta-analysis will not be performed as this experiment is exploratory in nature and there is no matching original data with which to statistically compare the replication data.

• The replication will be restricted to LAPC4 cells.

• We will run the qRT-PCR analysis on tumors derived from the treated LAPC4 cells as well as on the infected LAPC4 cells directly.

• Uninjected LAPC4 cells and tumors derived from uninjected LAPC4 cells have been added as an additional control.

• Because of these changes, the experiment is not directly confirmatory in nature, but rather exploratory. Thus, we will not directly power the experiment, but we have determined our sensitivity based on the number of samples we will use.

RNA sample purity (A_260/280 and A_260/230 ratios) will be reported for each sample. All data obtained from the experiment—raw data, data analysis, control data and quality control data—will be made publicly available, either in the published manuscript or as an open access dataset available on the Open Science Framework (https://osf.io/gb7sr/).

• ■ The experiment will be repeated a total of 16 times for a final power of 80.4%.

• ■ Each experiment consists of two cohorts;

  • ○ Cohort 1: DU145 cells transfected with pMIR-CD443′ UTR.

  • ○ Cohort 2: DU145 cells transfected with pMIR-CD44M1M23′ UTR.

• ■ Each cohort is treated with miR-34a or a non-coding control and levels of luciferase activity are measured.

Notes:

• DU145 cells are maintained in RPMI1640 supplemented with 10% FBS.

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

1. Transform, grow and maxiprep the following plasmids using an endo-free Maxiprep kit according to the manufacturer's instructions.

   • a. pMIR-CD443′ UTR.

   • b. pMIR-CD44M1M23′ UTR.

   • c. phRL-CMV.

2. Sequence inserts to confirm vector identity and run on an agarose gel to confirm vector integrity.

3. Seed 3 × 10⁴ DU145 cells per well in 24-well plates (27 wells total).

   • a. Let grow overnight.

4. 24 hr after plating, transfect cells with pMIR-3′ UTR or pMIR-CD44mut3′ UTR, miR-34a oligo or NC oligo and phRL-CMV using Lipofectamine 2000 according to the manufacturer's instructions.

   • a. 1 µg pMIR-CD443′ UTR + 24 pmoles miR-34a oligo + 1 ng phRL-CMV.

   • b. 1 µg pMIR-CD443′ UTR + 24 pmoles NC oligo + 1 ng phRL-CMV.

   • c. 1 µg pMIR-CD44M1M23′ UTR + 24 pmoles miR-34a oligo + 1 ng phRL-CMV.

   • d. 1 µg pMIR-CD44M1M23′ UTR + 24 pmoles NC oligo + 1 ng phRL-CMV.

   • e. Untransfected cells.

   • f. Empty wells (media only).

   • g. Each condition should be run in quadruplicate (four wells).

5. 48 hr later, read the ratio of luciferase to renilla using the Promega dual luciferase assay according to the manufacturer's instructions.

6. Repeat experiment from Step 3 onwards an additional 15 times.

• Data to be collected:

  • ○ Sequence data confirming vector identity.

  • ○ Whole gel images of agarose gels confirming vector integrity.

  • ○ Raw luciferase and renilla readings for each well.

  • ○ Normalized renilla and luciferase readings based on empty well and cells-only wells.

  • ○ Ratio of luciferase to renilla averaged across the four wells of each condition.

• At the time of analysis, we will perform the Shapiro–Wilk test and generate a quantile–quantile (q–q) plot to attempt to assess the normality of the data and also perform Levene's test to assess homoscedasiticity. If the data appears skewed, we will attempt a transformation in order to proceed with the proposed statistical analysis listed below and possibly perform the appropriate non-parametric test.

  • ○ Statistical Analysis of the Replication Data:

    • ■ Two way ANOVA (2 × 2 factorial) followed by Bonferroni corrected pairwise comparisons:

      • Wt+NC vs wt+34a.

• 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.

• The replication attempt will use only the wild-type CD44 3′ UTR anad the 3′ UTR with both miR-34a seed regions mutated; it will exclude the two constructs containing one mutated seed region.

All data obtained from the experiment—raw data, data analysis, control data and quality control data—will be made publicly available, either in the published manuscript or as an open access dataset available on the Open Science Framework (https://osf.io/gb7sr/).

• STR profiling and mycoplasma testing results.

• Sequencing results and agarose gel images confirming vector integrity.

• Original data is qualitative; only one experiment is shown without any error bars.

• Values were estimated from graph.

• Since no variance or sample size data were provided, power calculations will be performed with a range of variances and an assumed N per group of 3.

• Unpaired two-tailed Student's t-test, alpha error = 0.05.

• Note: Calculations were performed using G*Power software (version 3.1.7) (Faul et al., 2007).

In order to produce quantitative replication data, we will run the replication experiment three times. We will then determine the replication attempt's mean and variance from those three replicates. We will combine the replication variance with the original data, whose single datapoint we are assuming represents a mean. Combining the original mean and the replication variance will generate a simulated effect size. Using this simulated effect size, we will then determine the number of replicates required to reach 80% power and will perform additional replicates if necessary to ensure the replication is powered to at least 80%.

• Note: Original values presented in Supplemental Figure 5C. Authors confirmed tumor incidence was 100%.

• One-way ANOVA followed by planned comparisons using Fisher's LSD.

• Calculations were performed using R software, version 3.1.2 (R Core Team, 2014) and G*Power software (version 3.1.7) (Faul et al., 2007).

• Note: the additional control group of uninfected cells was assumed to have the same mean and variance as the lenti-control group for calculation purposes.

• Calculations were performed using R software, version 3.1.2 (R Core Team, 2014) and G*Power software (version 3.1.7) (Faul et al., 2007).

• Based on the sample size from Protocol 4, with α of 0.05, we will be powered to 80% to detect an effect size f of 0.7379139 (ANOVA: Fixed effects, omnibus, one-way) and with α of 0.05, we will be powered to 80% to detect an effect size d of 1.6317141 (unpaired two-tailed t-test) for tumors.

• Based on the sample size from Protocol 4, with α of 0.05, we will be powered to 80% to detect an effect size f of 1.3573433 (ANOVA: Fixed effects, omnibus, one-way) and with α of 0.05, we will be powered to 80% to detect an effect size d of 3.0708923 (unpaired two-tailed t-test) for tumors.

• Note: data provided by original authors.

• Two-way ANOVA (2 × 2 factorial) followed by Bonferroni corrected pairwise comparisons:

  • ○ Wt+NC vs wt+34a.

• Calculations were performed using GraphPad PRISM v6 for Mac and G*Power software (version 3.1.7) (Faul et al., 2007).

• Reproducibility Project: Cancer Biology