Fig 2: Analogue‐specific S6K1 mutation to screen for direct in vivo substrates HEK293 cells stably expressing myc‐WT‐S6K1 (WT) or myc‐AS‐S6K1 (AS), or U2OS transduced with adeno‐myc‐WT‐S6K1 (WT) or adeno‐myc‐AS‐S6K1 (AS), were transfected with tagged forms of candidates (lamin A, ZRF1, CUX1, NPM1). In vivo kinase assay were performed in the presence of 6‐Bn‐ATP‐γ‐S. After immunoprecipitation, the thiophosphorylation was revealed by Western blot using an anti‐thiophosphate ester antibody. * indicates unspecific band.HEK293 cells stably expressing myc‐WT‐S6K1 (WT) or myc‐AS‐S6K1 (AS) were transfected with Flag‐tagged forms of ZRF1 or a mutant of ZRF1, ZRF1S47A. In vivo kinase assay was performed in the presence of 6‐Bn‐ATP‐γ‐S. After immunoprecipitation using an anti‐Flag antibody, the thiophosphorylation was revealed by Western blot using an anti‐thiophosphate ester antibody.HEK293 was transfected with Flag‐ZRF1WT or Flag‐ZRF1S47A mutant plasmids. Twenty‐four hours post‐transfection, cells were starved overnight and treated for 3 h with Torin 1 (100 nM). After immunoprecipitation with anti‐Flag antibody, an in vitro kinase assay was performed with a recombinant active S6K1. ZRF1 phosphorylation was analysed by immunoblotting.

Fig 3: 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.

Fig 4: Therapeutic blocking p113-ZRF1 interaction inhibits NB progression. a 3D structure and sequences of inhibitory peptides (ZIP-12) for blocking interaction between p113 and ZRF1, and those of mutant control (Ctrl) peptides. b Confocal images showing the distribution of synthesized FITC-labeled Ctrl or ZIP-12 peptides (20 μmol·L− 1, arrowheads) within cultured BE(2)-C cells, with nuclei and cellular membranes staining with DAPI or Dil. Scale bars: 10 μm. c Co-IP and western blot assays indicating the interaction of p113 with ZRF1 or BRD4 in BE(2)-C cells treated with Ctrl or ZIP-12 peptides (20 μmol·L− 1) for 24 h. d Relative fatty acid levels, complex I activity, NAD+/NADH ratio, and ATP levels in BE(2)-C cells treated with Ctrl or ZIP-12 peptides (20 μmol·L− 1) for 24 h. e In vivo images (left upper panel), growth curve (right panel), and weight at the end points (right panel) of xenografts formed by subcutaneous injection of BE(2)-C cells in nude mice (n = 5 per group) that were treated with intravenous injection of Ctrl or ZIP-12 peptides (5 mg·kg− 1) as indicated (left lower panel). f In vivo imaging (left panel), lung metastatic colonization (right lower panel), and Kaplan–Meier curves (right lower panel) of nude mice (n = 5 for each group) treated with tail vein injection of BE(2)-C cells, Ctrl or ZIP-12 peptides (5 mg·kg− 1) as indicated (right upper panel). Student’s t test or ANOVA compared the difference in d-f. Log-rank test for survival comparison in f. *P < 0.05, **P < 0.01 vs. Ctrl. Data are shown as mean ± s.e.m. (error bars) and representative of three independent experiments in b-d

Fig 5: 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.