Extended Data Fig. 3: Analysis of mTOR inhibition and VPF treatment on ER stress in senescent cells.
From: The YAP–TEAD complex promotes senescent cell survival by lowering endoplasmic reticulum stress
a, Cell viability as assessed by direct cell counting of proliferating (P) or ETIS WI-38 fibroblasts that were either left untreated or treated with 100 nM Torin1 for 72 h. b, Representative micrographs showing the differences in viability in the conditions from (a). Scale bar, 100 µm. c, Western blot analysis of the levels of ATF6, XBP1s, and ACTB in the conditions described in (a), at 48 h instead. d, RT-qPCR analysis of the levels of PUMA, PMAIP1, and TNFRSF10B mRNAs in the conditions described in (c). Untreated P cells were included as baseline controls. e, Direct cell counting after treating as indicated for 72 h (1.5 µM VPF, 100 nM Torin1, or both) in ETIS WI-38 cells. f, RT-qPCR analysis of the indicated transcripts for the treatments described in (e), in this case for 48 h. g, Dot plot representation of the values calculated for the ER-positive relative area per cell (60 cells per condition) in ETIS WI-38 cells either untreated or treated with VPF (1.5 µM) or Torin1 (100 nM) for 48 h. h, Micrographs showing the areas corresponding to the endoplasmic reticulum (ER) in red for the indicated treatments as in (g). Phosphatidylcholine (PtdCho) was simultaneously supplemented at 50 µM where indicated. Scale bar, 100 µm. i, Heat map representation of the differences in SASP mRNA levels represented by row Z-Score for the indicated transcripts when comparing the conditions described in (g). Untreated P WI-38 cells were included as baseline controls. j, Heat map of the row Z-Score calculated for the differences in the secretion of the indicated SASP members among the groups described in (g), including proliferating (P) WI-38 cells as a control for baseline secretion. k, UMAP plot representation of the scRNA-seq data from ETIS WI-38 cells (no VPF treatment) showing the expression score specified in the legends, associated with the indicated gene sets (SASP, a custom gene set of 132 markers; ER stress, GOBP: Response to ER stress; and Oxidative Phosphorylation, Hallmark: Oxidative Phosphorylation). l, Western blot analysis of phosphorylated EIF2A (S51) and ACTB levels in WI-38 cells transfected with the indicated siRNAs, rendered senescent by treatment with etoposide for 6 days, and then either left untreated or treated with 100 nM Torin1 for 48 h. m, Cell viability measurement by direct cell counting of the conditions described in (l), here treated for 72 h. n, o, RT-qPCR analysis (n) and Bioplex analysis of the conditioned media (o) to assess SASP production and secretion in WI-38 cells transfected with siCtrl or siRELA, and rendered senescent with etoposide for 6 days. Proliferating controls transfected with siCtrl siRNA were included. Graphs in (a, d–f, m, n) represent the means and individual values (dots) of n = 3 independent replicates; plot in (g) shows the individual values of 20 different cells from each of the 3 independent replicates analyzed, making a total 60 individual values; Significance (*P < 0.05, **P < 0.01, ***P < 0.001) was calculated using two-tailed Student’s t-test.
