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. 2022 Apr 8;8(14):eabm0756. doi: 10.1126/sciadv.abm0756

Fig. 5. SRC-FAK activation influences cell viability by signaling through p38 and p53.

Fig. 5.

(A) Viable IMR-90 cells (direct counts) following 48 hours of treatment with etoposide (50 μM) plus p38i (AL 8697, 10 μM), p53a (Nutlin-3a, 10 μM), p53a + p38i, PP1 (5 μM), or PP2 (5 μM); see also fig. S3B. (B) RT-qPCR analysis of PUMA and BCL2L2 mRNAs (normalized to ACTB mRNA) in cells treated as in (A). (C) Western blot analysis of the proteins shown (and loading control ACTB). (D) Cell viability (direct counting) after 48-hour treatment as indicated; doxycycline-induced SRC(CA) expression was performed as in Fig. 1R simultaneously with the other treatments. (E) RT-qPCR analysis of PUMA and BCL2L2 mRNAs (normalized to ACTB mRNA) in cells treated as in (D). (F) Analysis of proteins preferentially phosphorylated at 50 μM etoposide ranked CAS-SRC-FAK at the top using CORUM database and Enrichr analysis using data from Fig. 1H. (G) Western blot analysis of p-FAK (activating phosphorylation Y576/Y577), FAK, and loading control ACTB. (H and I) Viability (direct counting) of cells transfected with the siRNAs shown and treated with etoposide (H), and Western blot analysis (I) of the proteins shown (p-p38 is phosphorylated at T180/Y182). (J and K) GSEA (J) and heatmap (K) analyses of the levels of mRNAs encoding EMT and integrin proteins (z scores of the log2-normalized expression values, n = 3). (L and M) GSEA of relevant gene sets in the conditions indicated after expressing SRC(DN) (see also fig. S3E) (L) or SRC(CA) (M). (N) Proposed model (created using BioRender). Senescence-causing, low-level DNA damage (lower half) activates SRC, decreasing p53 function and activating p38 (lowering PUMA, elevating BCL2L2). Apoptosis-causing, high-level DNA damage (top half) does not activate SRC and instead activates p53 and represses p38, in turn increasing PUMA and lowering BCL2L2 levels and leading to apoptosis. Values shown in (A), (B), (D), (E), and (H) are means ± SD (n ≥ 3); significance (*P < 0.05, **P < 0.01, ***P < 0.001) was determined by using two-tailed Student’s t test.