Fig 2: The ZRF1 phosphorylation mutant blunts the senescence programme Immunoblot analysis of nuclear and cytoplasmic fractions extracted from liver of WT mice starved overnight and refed for 4 h. When indicated, mice were injected intraperitoneally with 5 mg/kg of the rapamycin derivative temsirolimus 1 h before refeeding.Representative bright‐field images of SA β‐galactosidase staining. Tsc1 fl/fl primary MEFs (passage P1) were transduced with lentivirus shScramble or shZRF1 followed by transduction with lentivirus expressing ZRF1 or ZRF1 A mutant (ZRF1S47AS49A) or the empty vector. Cells were finally transduced with GFP or GFP‐Cre adenovirus (at passage P3). Seven to nine days post‐adenoinfection (P6 and P7), cells were fixed and β‐galactosidase staining was performed. Quantification of β‐galactosidase‐positive cells was performed on three independent experiments. Data are presented as mean ± SEM; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, n.s. not significant, two‐way ANOVA, Tukey's multiple comparisons test. Tsc1 fl/fl primary MEFs were transduced with lentivirus shScramble or shZRF1 followed by transduction with lentivirus expressing ZRF1 or ZRF1 A mutant (ZRF1S47AS49A) or the empty vector. Cells were finally transduced with GFP or GFP‐Cre adenovirus and proteins were analysed by Western blot using the indicated antibodies. Data are expressed as relative values normalized to β‐actin, with respect to the control. Data are reported as mean ± SEM of n = 3; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, two‐way ANOVA, Tukey's multiple comparisons test.

Fig 3: ZRF1 phosphorylation in tissue and cultured cells ARepresentative immunoblot analysis of proteins extracted from skeletal muscles of WT and S6K1 −/− S6K2 −/− mice that were starved overnight or starved overnight and refed for 4 h (n = 3 mice for each genotype).B, CDensitometric analyses of the blots in Fig 6D and E, respectively. The phosphorylation level of ZRF1 is normalized to tubulin or actin protein level and densitometric results are presented as fold changes relative to the control. Data are reported as mean ± SEM of n = 3; *P < 0.05, **P < 0.01, ANOVA multiple comparisons.

Fig 4: Sequence homology of ZRF1 phosphorylation motif across speciesAlignment of amino acid sequences of ZRF1 from different species encompassing the S6K consensus phosphorylation site motif (R/K‐X‐R/K‐X‐X‐S/T) underlined; the arrow indicates the phosphorylation site.

Fig 5: ZRF1 is a novel S6 kinase substrate in mouse tissues in vivo and in primary MEFs Immunoblot analysis of proteins extracted from liver of WT mice that were starved overnight and refed for 4 h. When indicated, mice were injected intraperitoneally with 5 mg/kg of the rapamycin derivate temsirolimus 1 h before refeeding (n = 3 mice for each condition).Immunoblot analysis of proteins extracted from liver and WAT of WT and S6K1 −/− S6K2 −/− mice that were starved overnight or starved overnight and refed for 4 h. Proteins were analysed by Western blot using the indicated antibodies (n = 3 mice for each genotype).Immunoblot analysis of proteins extracted from liver of WT, S6K1 −/−, S6K2 −/− and S6K1 −/− S6K2 −/− mice starved overnight and refed for 4 h (n = 3 mice for each genotype).Representative immunoblot of Tsc2 −/− p53 −/− and Tsc2 +/+ p53 −/− immortalized MEFs kept in serum, starved for serum overnight or starved for serum overnight and amino acids for 2 h. Proteins extracts were analysed by Western blot using the indicated antibodies (n = 2 cultures). Densitometric analyses of phosphorylation level of ZRF1 normalized to total tubulin are shown in Fig EV2B.Representative immunoblot analysis of ZRF1 phosphorylation levels in Tsc1 fl/fl and Tsc1 fl/fl S6K1 −/− S6K2 −/− primary MEFs transduced with GFP or GFP‐Cre adenovirus and harvested at passage P7. Densitometric analyses of phosphorylation level of ZRF1 normalized to total β‐actin protein levels are shown in Fig EV2C.