Fig 2: Altered insulin signaling pathway in the livers of male young adult IUGR mice. (A) Representative ChIPSeq data for the H3K4me3 peak at the Akt1 gene in the control and IUGR-N mice. The mean number of reads was lower in the male IUGR-N mice than in the controls (n = 5–6 per group) and (B) this result was confirmed by the RNA analysis (n = 6–8 per group). (C) The levels of Pten mRNA were lower in all of the IUGR groups irrespective of the nutritional conditions during the lactation period (n = 5–8 per group). (D) Representative immunoblots for PTEN and ß-actin in the livers of the mice after the injection of saline (Sal) or insulin (Ins) (left panel). The hepatic PTEN content was higher in the IUGR-O mice than in the IUGR-R mice (n = 7–8 per group) after saline injection. *p < 0.05, **p < 0.01, and ***p < 0.001 between groups.

Fig 3: PTEN protein expression in wild-type and DICER mutant HCT116 cells depleted of PTEN ceRNAs.Wild-type (WT) and DICER mutant (DicerEx5) HCT116 cells were transfected with non-targeting control siRNA (siNC) or siRNA plasmids targeting SERINC1 (siSER), VAPA (siVAPA), CNOT6L (siCNO), or PTEN (siPTEN). Cells were harvested 72 hr later for Western blot analysis. (A) Relative protein expression (PTEN/HSP90) are presented for each condition. Western blot bands were quantified, PTEN levels were normalized to HSP90, with protein expression presented relative to siNC. Means reported and error bars represent SD from three independent biological repeats for wild-type HCT116 cells and four repeats for DicerEx5 HCT116 cells. Analysis of wild-type HCT116 cells: one-way ANOVA (equal variance) on PTEN/HSP90 expression: F(4,10) = 25.4, I=3.18×10-5. Planned contrasts between siNC and siSER: t(10) = 1.94, uncorrected I=0.082 with a priori Bonferroni adjusted significance threshold of 0.0125, Bonferroni corrected p=0.326; siNC and siVAPA: t(10) = 5.44, uncorrected p=2.85×10-4, Bonferroni corrected p=0.0011; siNC and siCNOT: t(10) = 3.69, uncorrected p=0.0042, Bonferroni corrected p=0.017; siNC and siPTEN: t(10) = 9.34, uncorrected p=2.97×10-6, Bonferroni corrected p=1.19×10-5. Analysis of DicerEx5 HCT116 cells: one-way ANOVA (unequal variance) on PTEN/HSP90 expression: F(4,6.0) = 19.3, p=0.0014. Planned comparisons: siNC and siSER: two-sample t-test, t(6) = 3.96, uncorrected p=0.0074 with a priori Bonferroni adjusted significance threshold of 0.0125, Bonferroni corrected p=0.030; siNC and siVAPA: two-sample t-test, t(6) = 0.896, uncorrected p=0.405, Bonferroni corrected p>0.99; siNC and siCNOT: Welch’s t-test, t(4.36) = 2.92, uncorrected p=0.039, Bonferroni corrected p=0.156; siNC and siPTEN: two-sample t-test, t(6) = 4.15, uncorrected p=0.0060, Bonferroni corrected p=0.024. (B) Representative Western blots probed with an anti-PTEN antibody and anti-HSP90 antibody. Additional details for this experiment can be found at https://osf.io/drcbw/.

Fig 4: Meta-analyses of each effect.Effect size and 95% confidence interval are presented for Tay et al., 2011, this replication study (RP:CB), and a random effects meta-analysis of those two effects. For each effect, Cohen’s d or Glass’ delta, which are standardized differences between the two indicated measurements, is reported. Sample sizes used in Tay et al., 2011 and RP:CB are reported under the study name. (A) These effects are related to the change in luciferase activity between the conditions reported in Figure 1 of this study and Figure 3C of Tay et al., 2011. Meta-analysis p values: siNC and siSER (p=0.374); siNC and siZNF (p=0.233); siNC and siVAPA (p=0.316); siNC and siCNO (p=0.253); siNC and siPTEN (p=0.079). (B) These effects are related to the change in luciferase activity between the conditions reported in Figure 2 of this study and Figure 3D of Tay et al., 2011. Meta-analysis p values: SER 3’U and EV (p=0.881); VAPA 3’U1 and EV (p=0.624); VAPA 3’U2 and EV (p=0.754); CNO 3’U1 and EV (p=0.790); CNO 3’U2 and EV (p=0.716); PTEN 3’U and EV (p=0.766). (C) These effects are related to the differences in PTEN protein expression between the conditions reported in Figure 3 of this study and Figure 3H of Tay et al., 2011. Meta-analysis p values: WT HCT116: siNC and siSER (p=0.091); siNC and siVAPA (p=2.99×10-7); siNC and siCNO (p=0.049); siNC and siPTEN (p=0.0041): DicerEx5 HCT116: siNC and siSER (p=2.78×10-4); siNC and siVAPA (p=0.215); siNC and siCNO (p=3.70×10-4); siNC and siPTEN (p=0.229). (D) These effects are related to the differences in cell growth between the conditions reported in Figure 4 of this study and Figure 5B of Tay et al., 2011. Meta-analysis p values: DU145: siVAPA and siNC (p=0.255); siCNO and siNC (p=0.554); siPTEN and siNC (p=0.082): WT HCT116: siVAPA and siNC (p=0.045); siCNO and siNC (p=0.287); siPTEN and siNC (p=0.082): DicerEx5 HCT116: siVAPA and siPTEN (p=0.075); siCNO and siPTEN (p=0.001); siPTEN and siNC (p=0.048). Additional details for these meta-analyses can be found at https://osf.io/xgrqp/.

Fig 5: Characterization of Pten398A/398A;MMTVneu knock-in mouse.A Survival rate in Pten+/+;MMTVneu and Pten398A/398A;MMTVneu mice (n = at least 10 mice/group). (***p = 0.0005; Log-rank test for comparisons of Kaplan–Meier survival curves). B Representative sections of Pten+/+;MMTVneu and Pten398A/398A;MMTVneu mammary tumors stained with a ?H2AX antibody (magnification, ×10; main image zoom, ×20, insets zoom ×80). C Semi-quantitatively expression scoring of Gamma-H2AX IHC stained tumors from Pten+/+;MMTVneu and Pten398A/398A;MMTVneu mice. D Immunoblot analysis of ?H2AX in Pten+/+;MMTVneu and Pten398A/398A;MMTVneu mammary tumor samples. Vinculin was used as a loading control. E Quantification of ?H2AX levels, normalized to vinculin, from the blots shown in panel (E) (t test, *p = 0.0332).