Supplementary Figure 1: mTORC1 is nonessential for sestrin function in senescent T cells.

Measurement of (a) sestrin1, (b) sestrin2 or (c) sestrin3 expression by both immunoblots (top) and quantitative PCR (bottom) in senescent CD27^- CD28^- CD4⁺ T cells transduced as indicated. Immunoblots are representative of 2 independent experiments. Samples for quantitative PCR were analyzed from 3 different donors and normalized against housekeeping GAPDH. Knockdown efficiency was evaluated 96 hours post-transduction. Results presented relative to those of cells transduced with shCtrl, set as 1. (d) Immunoblots of mTOR and S6K1 expression in cells gated as in Fig. 1b (n = 3). Total ERK, loading control. Sesn3, internal Sestrin control. See also Fig. 1b. (e) Ki-67 (top panel), mTOR expression (middle panel) and p-S6K1 (bottom panel) in senescent CD4⁺ T cells transduced as indicated, then examined by phospho-flow 96h later. Representative overlay (left) and pooled data (right; n=3) are shown. Results presented relative to those of cells transduced with shCtrl, set as 1. (f) Effectiveness of the mTOR inhibitor rapamycin (20 ng/mL) in cells as in (e). (g) Calcium abundance, (h) IL2, (i) telomerase activity and (j) γ-H2Ax phosphorylation in CD27^- CD28^- CD4⁺ T cells transduced as indicated then treated with rapamycin for 18h, presented as above. *p<0.05 **p<0.01 and ***p< 0.001 values were assessed by a paired Student’s t test (a-c) or ANOVA for repeated measures with a Bonferroni post-test correction (d-j). Errors bars depict s.e.m.