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Replication Study: Transcriptional amplification in tumor cells with elevated c-Myc

  1. L Michelle Lewis
  2. Meredith C Edwards
  3. Zachary R Meyers
  4. C Conover Talbot Jr
  5. Haiping Hao
  6. David Blum
  7. Reproducibility Project: Cancer Biology  Is a corresponding author
  1. University of Georgia, Bioexpression and Fermentation Facility, United States
  2. Johns Hopkins University, Deep Sequencing and Microarray Core Facility, United States
Replication Study
Cite this article as: eLife 2018;7:e30274 doi: 10.7554/eLife.30274
3 figures, 3 tables and 2 additional files

Figures

Induction of c-Myc in P493-6 cells and impact on total RNA levels.

P493-6 cells were grown in the presence of tetracycline (Tet) for 72 hr and switched into Tet-free growth medium to induce c-Myc expression. Cells were cultured in two separate lots of serum. (A) Representative Western blot using an anti-c-Myc antibody (top panels) or an anti-ß-Actin antibody (bottom panel). Two exposures of the anti-c-Myc antibody are presented to facilitate detection of c-Myc. (B) Quantification of total RNA levels (ng of total RNA per 1,000 cells) for cells at 0, 1, and 24 hr after release from Tet. Means reported and error bars represent s.e.m. from three independent biological repeats. For serum lot one, one-way ANOVA on total RNA levels of all groups; F(2, 6)=1.25, p=0.353. Planned contrast between 0 hr and 24 hr; t(6) = 1.02, p=0.347 with a priori alpha level = 0.05. For serum lot two, one-way ANOVA on total RNA levels of all groups; F(2, 6)=21.87, p=0.00176. Planned contrast between 0 hr and 24 hr; t(6) = 5.03, p=0.0024 with a priori alpha level = 0.05. Additional details for this experiment can be found at https://osf.io/tfd57/.

https://doi.org/10.7554/eLife.30274.002
Figure 2 with 2 supplements
Digital gene expression analysis.

P493-6 cells grown in the presence of tetracycline (Tet) for 72 hr for repression of the conditional pmyc-tet construct, were switched into Tet-free growth medium to induce c-Myc expression. Cells were cultured in two separate lots of serum. Transcripts/cell estimates from NanoString nCounter gene expression assays (1369 genes assay) for active (left) and silent (right) genes at 0, 1, and 24 hr after release from Tet. Active genes expressed greater than one transcript/cell. Silent genes expressed less than 0.5 transcript/cell. Box and whisker plots with median represented as the line through the box and whiskers representing values within 1.5 IQR of the first and third quartile. Cells grown in serum lot one: active genes = 708, silent genes = 580. Cells grown in serum lot two: active genes = 719, silent genes = 573. Confirmatory analysis is reported in Table 1 and exploratory statistical analysis is reported in Table 2 and Table 3. Additional details for this experiment can be found at https://osf.io/fn2y4/.

https://doi.org/10.7554/eLife.30274.003
Figure 2—source data 1

List of Reporter CodeSets and gene expression values.

https://doi.org/10.7554/eLife.30274.006
Figure 2—figure supplement 1
Logarithmic expression of genes.

This is the same experiment as in Figure 2. (A–B, E–F) Gene expression data plotted on a log2 transformed scale for active (A, E) and silent (B, F) genes at 0, 1, and 24 hr after release from Tet for both lots of serum. (C–D, G–H) Box and whisker plots showing gene expression changes (log2 ratio) between the indicated times for active (C, G) and silent (D, H) genes. Median represented as the line through the box and whiskers representing values within 1.5 IQR of the first and third quartile. Additional details for this experiment can be found at https://osf.io/fn2y4/.

https://doi.org/10.7554/eLife.30274.004
Figure 2—figure supplement 2
Comparison of gene expression data as continuous.

This is the same experiment as in Figure 2. (A–C, E–G) Scatter plots of log2 transformed gene expression data for all genes analyzed at the indicated times on the y and x axes for both lots of serum. Active genes are blue, silent genes are red, and genes that are neither active or silent (expression was more than 0.5 transcript/cell and less than one transcript/cell at time 0 hr) are white. (D, H) Box and whisker plots showing gene expression changes (log2 ratio) between the indicated times for all genes analyzed for both lots of serum. Median represented as the line through the box and whiskers representing values within 1.5 IQR of the first and third quartile. Additional details for this experiment can be found at https://osf.io/fn2y4/.

https://doi.org/10.7554/eLife.30274.005
Meta-analyses of each effect.

Effect size and 95% confidence interval are presented for Lin et al., 2012, this replication study (RP:CB), and a random effects meta-analysis of those two effects. Cohen’s d is the standardized difference between the two measurements, with a larger positive value indicating total RNA levels are increased at 24 hr compared to 0 hr. The effect size r is a standardized measure of the correlation (strength and direction) of the association between gene expression and c-Myc induction, with a larger positive value indicating gene expression increased during the course of c-Myc induction. Sample sizes used in Lin et al., 2012 and this replication attempt are reported under the study name. (A) Total RNA levels in P493-6 cells 0 hr compared to 24 hr after release from tetracycline (meta-analysis p = 0.0488). (B) Gene expression of active or silent genes are shown for all comparisons. Active genes: 0 hr compared to 1 hr (meta-analysis p = 1.12x10-7), 0 hr compared to 24 hr (meta-analysis p = 7.01x10-4), 1 hr compared to 24 hr (meta-analysis p = 0.0129). Silent genes: 0 hr compared to 1 hr (meta-analysis p = 0.203), 0 hr compared to 24 hr (meta-analysis p = 7.10x10-17), 1 hr compared to 24 hr (meta-analysis p = 0.0571). Additional details for these meta-analyses can be found at https://osf.io/5yscz/.

https://doi.org/10.7554/eLife.30274.010

Tables

Table 1
Confirmatory statistical tests.
https://doi.org/10.7554/eLife.30274.007
GenesComparisonStudyZ valueP valueSample size (n)
Active0 hr vs 1 hrRP:CB Lot 114.861.36e-55708
RP:CB Lot 211.832.83e-34719
Lin et al. (2012)21.171.05e-130755
0 hr vs 24 hrRP:CB Lot 19.9223.67e-24708
RP:CB Lot 212.773.77e-40719
Lin et al. (2012)23.263.33e-184755
1 hr vs 24 hrRP:CB Lot 14.7421.92e-06708
RP:CB Lot 210.049.91e-25719
Lin et al. (2012)23.263.33e-184755
Silent0 hr vs 1 hrRP:CB Lot 112.617.11e-40580
RP:CB Lot 27.059.29e-13572
Lin et al. (2012)−1.9980.0457274
0 hr vs 24 hrRP:CB Lot 18.3282.22e-17579
RP:CB Lot 28.1561.03e-16572
Lin et al. (2012)3.1790.00144276
1 hr vs 24 hrRP:CB Lot 10.68530.493580
RP:CB Lot 24.4368.35e-06573
Lin et al. (2012)5.8064.11e-09236
  1. These confirmatory statistical tests relate to the data presented in Figure 2. Wilcoxon signed-rank test, which treat the data as paired, were conducted for the original study (Lin et al., 2012) and this replication attempt (RP:CB). Uncorrected p values are reported with an a priori significance threshold of. 0167. Sample sizes reported are based on the sample size used in the tests. Additional details for this experiment can be found at https://osf.io/fn2y4/.

Table 2
Exploratory statistical tests.
https://doi.org/10.7554/eLife.30274.008
GenesComparisonStudyW valueP valueSample size (n)
Active0 hr vs 1 hrRP:CB Lot 12703780.01031416
RP:CB Lot 22746960.03951438
Lin et al. (2012)3187996.67e-051510
0 hr vs 24 hrRP:CB Lot 13007747.16e-111416
RP:CB Lot 23245644.74e-171438
Lin et al. (2012)4009991.16e-421510
1 hr vs 24 hrRP:CB Lot 12816795.45e-051416
RP:CB Lot 23089541.45e-101438
Lin et al. (2012)3727144.11e-251510
Silent0 hr vs 1 hrRP:CB Lot 11876820.0006381160
RP:CB Lot 21746950.06021146
Lin et al. (2012)1271040.2361028
0 hr vs 24 hrRP:CB Lot 11858040.002031160
RP:CB Lot 21844700.0002891146
Lin et al. (2012)1320820.9971028
1 hr vs 24 hrRP:CB Lot 11661220.7161160
RP:CB Lot 21736080.09181146
Lin et al. (2012)1364430.2951028
  1. These exploratory statistical tests relate to the data presented in Figure 2. Wilcoxon rank sum tests, which treat the data as unpaired, were conducted for the original study (Lin et al., 2012) and this replication attempt (RP:CB). Uncorrected p values are reported. Sample sizes reported are based on treating genes as unpaired between conditions. Additional details for this experiment can be found at https://osf.io/fn2y4/.

Table 3
Exploratory statistical tests.
https://doi.org/10.7554/eLife.30274.009
ComparisonStudyW valueP valueSample size (n)
0 hr vs 1 hrRP:CB Lot 13038977.78e-501288
RP:CB Lot 22786461.1e-271292
Lin et al. (2012)3493511.27e-1301269
0 hr vs 24 hrRP:CB Lot 12724415.18e-241288
RP:CB Lot 22928657.4e-391292
Lin et al. (2012)3681821.14e-1631269
1 hr vs 24 hrRP:CB Lot 12350777.45e-061288
RP:CB Lot 22720283.73e-231292
Lin et al. (2012)3320695.72e-1041269
  1. These exploratory statistical tests relate to the data presented in Figure 2. Wilcoxon rank sum tests were conducted for the original study (Lin et al., 2012) and this replication attempt (RP:CB) on the difference in expression of active genes during c-Myc induction (e.g. from 0 hr to 24 hr) compared to the difference in expression of silent genes over that same period (e.g. from 0 hr to 24 hr). Uncorrected p values are reported. Sample sizes reported are based on number of active and silent genes used in the tests. Additional details for this experiment can be found at https://osf.io/fn2y4/.

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