Fig 2: SRC-mediated p53 repression and p38 activation promotes prosurvival responses in early senescence.(A) Heatmap representation of p53 levels (top) quantified by densitometry from signals on Western blots (bottom, representative panel) in IMR-90 cells transfected with siCtrl or siSRC siRNAs, either untreated or treated with 50 μM etoposide for 48 hours. (B) Heatmap representation of densitometry quantification of signals on Western blots detecting phosphorylated residues, normalized to protein levels assessed in cells transfected with siCtrl or siSRC siRNAs that were either left untreated or treated with 50 μM etoposide for 48 hours. (C) Representative Western blot analysis of the data shown in (B). (D) Quantification of PUMA, PMAIP1, BCL2L2, and BCL2L1 mRNAs by RT-qPCR analysis. Data were normalized to the levels of ACTB mRNA. (E) Representative Western blot analysis of the levels of PUMA and BCL2L2 proteins. (F) Levels of phosphorylated SRC (Y419), total SRC, myc tag, phospho-p38 (T180/Y182), p38, and p53 in the different conditions tested. The levels of ACTB were assessed to evaluate loading differences. Values indicate means ± SD from signals obtained in three independent experiments. (G) Quantification of PUMA and BCL2L2 mRNAs by RT-qPCR analysis; data were normalized to the levels of ACTB mRNA. (H) Representative Western blot analysis of the levels of proteins expressed from the transcripts in (F). (I) Cell viability was assessed in IMR-90 fibroblasts treated with 50 μM etoposide and either left without further treatment or treated with PP1 or PP2 for 48 hours or 10 days. (J) The levels of PUMA and BCL2L2 mRNAs were quantified by RT-qPCR analysis in cells treated with 50 μM etoposide for the times shown; data were normalized to the levels of ACTB mRNA. Values shown in (D), (G), (I), and (J) are means ± SD; significance (*P < 0.05, **P < 0.01, ***P < 0.001) was determined using two-tailed Student’s t test.

Fig 3: Developmental changes in the Akt–FoxO1–PUMA pathway in regulating physiological and sevoflurane-induced apoptosis. Immunoblots and quantitation of phosphorylated Akt (A,B), phosphorylated FoxO1 (C,D), and PUMA (E,F) from the S1 region of control and sevoflurane-anesthetized mice at P5 and P9, respectively. *, sevoflurane group compared to control group of the same age; #, P5 compared to P9 in each treatment condition; ***, P < 0.001; ###, P < 0.001, using two-way analysis of variance followed by Tukey’s multiple comparisons test. Four mice were used per condition. Data are shown as the mean ± standard error of the mean.

Fig 4: Vascular cell types are vulnerable to inhibition of prosurvival BCL-2 family proteins.(A) BH3 profiling of hiPSCs, hiPSC-ECs, hiPSC-VSMCs, primary HCAECs, primary human FFs, and HeLa cells for sensitizer BH3 peptides BAD, HRK, MS1, and PUMA; n = 3 to 4 biological replicates. (B) AxV/PI staining of hiPSCs, hiPSC-ECs, hiPSC-VSMCs, HCAECs, FFs, and HeLa cells in response to BH3 mimetics alone or in combination, 24 hours after treatment start. All combinations were performed with the same concentration of each drug, and treatment was given simultaneously; n = 3 to 8 biological replicates. Cmax arrows indicate what concentration is comparable to maximum patient plasma concentration. Significance: ns, not significant, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Fig 5: Essential factors for cell fate determination are regulated by signaling through SRC.(A and B) GSEA plots (A) and heatmap (B) depicting microarray analysis of mRNAs encoding proapoptotic (A) or prosurvival (B) proteins in untreated or etoposide-treated IMR-90 fibroblasts; data in (B) are row z score of log2-normalized transcript values (n = 3). (C) Validation of the top microarray targets PUMA, PMAIP1, BCL2L2, and BCL2L1 mRNAs by RT-qPCR analysis (see also fig. S2A). (D) Western blot analysis of proteins expressed in cells treated as in (A) to (C). (E) Viability (direct counts) of cells transfected with the siRNAs shown, then treated with 200 μM etoposide (left) or left untreated (right). Significant differences are indicated above (siPUMA versus siCtrl) and below (siPMAIP1 versus siCtrl) the curves. (F) Viability (direct counts) of cells that were transfected with the siRNAs shown, either treated with 50 μM etoposide (left) or left untreated (right). Significant differences are indicated above (siBCL2L1 versus siCtrl) and below (siBCL2L2 versus siCtrl) the curves. (G) Viability (direct counts) of cells transfected with the siRNAs shown, then treated with 50 μM etoposide (left) or left untreated (right). (H) RT-qPCR analysis of PUMA and BCL2L2 mRNAs (normalized to ACTB mRNA) after etoposide treatment for different times. (I) Western blot analysis of the proteins shown; means ± SD from densitometric quantification of phosphorylated SRC(Y419) (n = 3) is shown. (J) Enrichr analysis of terms (columns, hub proteins) and input proteins (rows); filled cells designate row proteins associated with hub proteins (Materials and Methods). (K) Heatmap representation (top) of quantified densitometry signals from p53 by Western blot analysis (bottom) in IMR-90 cells treated as indicated. (L and M) Heatmap representation (L) of the quantified densitometry signals on Western blots (M) detecting phosphorylated protein relative to corresponding total protein. Data represent log2 of the normalized values. Graphs (C) and (E) to (I) represent means ± SD (n = 3 experiments); significance (*P < 0.05, **P < 0.01, ***P < 0.001) was determined using two-tailed Student’s t test.