Fig. 4. S6K1 and/or S6K2 deletion does not bypass senescence but dampens SASP induction in MEFs.
a, Experimental scheme. MEFs were generated from S6K1 WT/KO, S6K2 WT/KO and S6K1/2 WT/DKO embryos and were assessed for replicative senescence. MEFs were generated from 3-5 independent pairs of embryos from at least three different mothers. b–d, Cumulative population doublings of S6K1 WT (n = 5) and KO (n = 4) MEFs (b), S6K2 WT (n = 4) and KO (n = 5) MEFs (c) and S6K1/2 WT (n = 3) and DKO (n = 5) MEFs (d). e–h, Quantification (e) and representative images (f–h) of SA-β-gal staining in young (passage 2) and old (passage 8) MEFs from S6K1 WT and KO (young n = 3; old n = 3), S6K2 WT and KO (young n = 3; old n = 3) and S6K1/2 WT (young n = 3; old n = 3) and DKO (young n = 4; old n = 5) cells. Scale bar, 100 μm. i–k, Relative mRNA expression for Ink4a (i), Il1b (j) and Il1a (k) assessed by RT–qPCR from young (passage 3) and old (passage 8) MEFs from S6K1 WT (n = 4) and KO (n = 4), S6K2 WT (n = 4) and KO (n = 5) as well as S6K1/2 WT (n = 3) and DKO (n = 5) cells. mRNA expression was normalized to the Rps14 housekeeping gene. l, Experimental scheme. MEFs of the indicated genotypes were stably transduced with a retroviral vector containing the EV or expressing HRASG12V. MEFs were generated from three independent pairs of embryos from three different mothers. m,n, Relative mRNA expression for Il1b (m) and Il1a (n) assessed by RT–qPCR from MEFs transduced with EV or HRASG12V (RAS) from S6K1 WT (n = 3) and KO (n = 3), S6K2 WT (n = 3) and KO (n = 3) as well as S6K1/2 WT (n = 3) and DKO (n = 3) cells. mRNA expression was normalized to the Rps14 housekeeping gene. o, Experimental scheme. MEFs of the indicated genotypes were treated with DMSO or 5 μM etoposide for 7 d. MEFs were generated from three independent pairs of embryos from three different mothers. p,q, Relative mRNA expression for Il1b (p) and Il1a (q) assessed by RT–qPCR from MEFs treated with DMSO or 5 μM etoposide from S6K1 WT (n = 3) and KO (n = 3), S6K2 WT (n = 3) and KO (n = 3) as well as S6K1/2 WT (n = 3) and DKO (n = 3) cells. mRNA expression was normalized to the Rps14 housekeeping gene. Data are expressed as mean ± s.e.m. Statistical significance was calculated using repeated two-way ANOVA with Sidak’s multiple comparison test (b–d) or by two-way ANOVA with Tukey’s multiple comparison test (e,i–k,m,n,p,q). n denotes individual MEF replicates derived from different embryos. Etopo., etoposide; O, old; P, passage; Y, young.