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[…] PC9 cells are grown in RPMI supplemented with 4.5 g/l glucose, 5% FBS and 1% penicillin/streptomycin at 37˚C in a humidified atmosphere at 5% CO2.Generate PC9-derived DTPs with 2 µM erlotinib in 10 cm tissue culture dishes, as described in protocol 2.Treat cells with DMSO (vehicle) or 1 µM AEW541 for 2 hr.Stock concentration used will be determined from assessing DMSO solvent toxicity on PC9 cells (protocol 1, step 3) and drug-withdrawn DTPs (protocol 1, step 6) to determine final DMSO concentration.Stock concentration used will be determined from assessing DMSO solvent toxicity on PC9 cells (protocol 1, step 3) and drug-withdrawn DTPs (protocol 1, step 6) to determine final DMSO concentration.Dissociate cells from plates and count. Harvest drug naïve PC9 and PC9-derived DTPs in complete lysis buffer following replicating labs standard procedure.The detachment method identified in protocol 1, step 4 will be used.Complete lysis buffer: RIPA lysis buffer supplemented with 1X phosphatase inhibitor, protease inhibitor cocktail, 1 mM PMSF.The detachment method identified in protocol 1, step 4 will be used.Complete lysis buffer: RIPA lysis buffer supplemented with 1X phosphatase inhibitor, protease inhibitor cocktail, 1 mM PMSF.Normalize gel loading to total cell number, add 4X LDS sample buffer supplemented with reducing agent, and denature at 70˚C for 10 min.Separate equivalent number of cells (~10–60 µg of protein) per lane with protein ladder and transfer to a membrane using the replicating labs standard procedures.After transfer, block non-specific binding and immunoblot membrane with the following combinations of primary antibodies at the dilution/concentration recommended by the supplier.Rabbit anti-phospho-IGF-1R (Y1165/1166) (95 kDa) at a 1:500-1:2000 dilution and mouse anti-ERK1/2 (42/44 kDa) at a 1:2000 dilution.Rabbit anti-phospho-IGF-1R (Y1165/1166) (95 kDa) at a 1:500-1:2000 dilution and mouse anti-IGF-1R at 0.1-2 µg/ml.Rabbit anti-phospho-IGF-1R (Y1165/1166) (95 kDa) at a 1:500-1:2000 dilution and mouse anti-ERK1/2 (42/44 kDa) at a 1:2000 dilution.Rabbit anti-phospho-IGF-1R (Y1165/1166) (95 kDa) at a 1:500-1:2000 dilution and mouse anti-IGF-1R at 0.1-2 µg/ml.Wash and apply appropriate secondary antibodies for 1 hr at RT with constant agitation and detect signal using Odyssey imaging system.Analyze bands with Image Studio software and normalize to loading controls.pIGF-1R (Y1165/1166) normalized to ERK1/2 (total)pIGF-1R (Y1165/1166) normalized to IGF-1R (total) [additional]pIGF-1R (Y1165/1166) normalized to ERK1/2 (total)pIGF-1R (Y1165/1166) normalized to IGF-1R (total) [additional]Repeat independently two additional times. [...] PC9 cells are grown in RPMI supplemented with 4.5 g/l glucose, 5% FBS and 1% penicillin/streptomycin at 37˚C in a humidified atmosphere at 5% CO2.Generate PC9-derived DTPs with 2 µM erlotinib in 10 cm tissue culture dishes, as described in protocol 2.Treat cells with DMSO (vehicle) or 1 µM AEW541 for 24 hr.Stock concentration used will be determined from assessing DMSO solvent toxicity on PC9 cells (protocol 1, step 3) and drug-withdrawn DTPs (protocol 1, step 6) to determine final DMSO concentration.Stock concentration used will be determined from assessing DMSO solvent toxicity on PC9 cells (protocol 1, step 3) and drug-withdrawn DTPs (protocol 1, step 6) to determine final DMSO concentration.Dissociate cells from plates and count. Harvest drug naïve PC9 and PC9-derived DTPs in complete lysis buffer following replicating labs standard procedure.The detachment method identified in protocol 1, step 4 will be used.Complete lysis buffer: RIPA lysis buffer supplemented with 1X phosphatase inhibitor, protease inhibitor cocktail, 1 mM PMSF.The detachment method identified in protocol 1, step 4 will be used.Complete lysis buffer: RIPA lysis buffer supplemented with 1X phosphatase inhibitor, protease inhibitor cocktail, 1 mM PMSF.Normalize gel loading to total cell number, add 4X LDS sample buffer supplemented with reducing agent, and denature at 70˚C for 10 min.Separate equivalent number of cells (~10–60 µg of protein) per lane with protein ladder and transfer to a membrane using the replicating labs standard procedures.After transfer, block non-specific binding and immunoblot membrane with the following combinations of primary antibodies at the dilution/concentration recommended by the supplier.Rabbit anti-KDM5A (200 kDa) at a 1:2000 to 1:10,000 dilution and mouse anti-ERK1/2 (42/44 kDa) at a 1:2000 dilution.Rabbit anti-KDM5A (200 kDa) at a 1:2000 to 1:10,000 dilution and mouse anti-ERK1/2 (42/44 kDa) at a 1:2000 dilution.Wash and apply appropriate secondary antibodies for 1 hr at RT with constant agitation and detect signal using Odyssey imaging system.Analyze bands with Image Studio software and normalize to loading controls.KDM5A normalized to ERK1/2 (total)KDM5A normalized to ERK1/2 (total)Repeat independently two additional times. [...] Performed with G*Power software, version 3.1.7 (). [...] 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. The data presented in Figures 1E, upper right panel, and Figure 2B were unable to be quantified for all bands and are thus considered exploratory in nature. Due to the lack of raw original data, and inability to quantify some of the Western blots, we are unable to perform power calculations for all comparisons. However, because the same samples will be used to detect each protein of interest (3 total), the alpha error will be adjusted to account for multiple comparisons.Summary of original data quantified from the image reported in Figure 4A, upper panel: 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 using the values quantified from the reported image as the mean. At each level of variance the effect size was estimated and used to calculate the needed sample size to achieve at least 80% power with the indicated alpha error. The achieved power is reported.H3K14Ac normalized signal: [...] Performed with G*Power software, version 3.1.7 (). 2% variance: 15% variance: 28% variance: 40% variance: CD133 and pEGFR (Y1068) normalized signals: [...] Performed with G*Power software, version 3.1.7 (). In order to produce quantitative replication data, we will run the experiment four 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. [...] Performed with G*Power software, version 3.1.7 ().ANOVA F test statistic and partial η2performed with R software, version 3.1.2 (). [...] Performed with G*Power software, version 3.1.7. () [...] 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 original data quantified from the image reported in Figure 7A: 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 using the values quantified from the reported image as the mean. At each level of variance the effect size was estimated and used to calculate the needed sample size to achieve at least 80% power with the indicated alpha error. The achieved power is reported. […]

Pipeline specifications

Software tools G*Power, Image Studio
Applications Miscellaneous, DNA fingerprinting
Diseases Neoplasms