Fig 2: Differential p70S6K stretch response in isolated C57 and ?-SG-/- muscles. EDL muscles from C57 and ?-SG-/- mice were maintained in oxygenated high glucose DMEM and subjected to passive stretching for 30 or 90 min. (A) Immunoblot of ?-SG following immunoprecipitation with anti-P-Tyr or lysate only, showing ?-SG phosphorylation in response to 30 min of stretch. (B) Representative immunoblots of P-p70S6K (T389 and T421/S424 sites) and tubulin. (C, D) Quantification of P-p70S6K T389 (C) and T421/S424 (D), normalized to tubulin. Samples for each time point were run on separate immunoblots and normalized to C57 NS. n = 3-5 muscles per genotype, condition, and time point. Bars represent mean ± standard error. All datasets were tested by two-way ANOVA. Stretch was statistically significant for T421/S424 after 30 min of stretch; genotype, stretch, and the interaction between them were statistically significant T389 after 90 min of stretch, by two-way ANOVA. *Significantly different from C57 S at 90 min by two-way ANOVA with Tukey’s multiple comparisons test. NS, non-stretched; S, stretched.

Fig 3: Changes in mTOR signalling pathway-related proteins after psoralen treatment in L02 cells and mice. L02 cells were treated with DMSO or psoralen and incubated for various time periods. When the cells were just beginning to completely adhere (defined as 0 h), the total protein was collected successively every 3 and 6 h during a 48 h period. All experiments were performed three times independently. (A,B) Representative immunoblots for the protein levels of total AMPK, AKT, mTOR, p70S6K, and 4EBP1 and their phosphorylated forms. (C) The correspondingly relative protein levels were quantified by scanning band intensity and normalised to ß-actin. The density of immunoreactive bands was quantified using ImageJ software. Density values were normalised to ß-actin and then expressed as the relative expression levels between DMSO-treated and psoralen-treated. C57BL/6 mice were treated with 0.5% CMC-Na solution (vehicle group), or 400 mg/kg or 800 mg/kg psoralen for 24 h. (D) Representative immunoblots for the protein levels of total AMPK, AKT, and p70S6K, and their phosphorylated forms. (E) The correspondingly relative protein levels were quantified by scanning band intensity and normalised to ß-actin (n = 6). Data are means ± SDs (n = 6). *p < 0.05, **p < 0.01, ***p < 0.001 versus DMSO group or vehicle group.

Fig 4: C1orf74 positively regulates the AKT/mTORC1 signaling pathway.(A) The Gene Set Enrichment Analysis results indicated that the gene sets of ‘HALLMARK_PI3K_AKT_MTOR_SIGNALING’ and ‘HALLMARK_MTORC1_SIGNALING’ were enriched with high expression of C1orf74. (B) Knockdown of C1orf74 inhibited the levels of phosphorylation of AKT and p70S6K compared with those in the control siRNAtransfected group. Overexpression of C1orf74 enhanced the phosphorylation of AKT and p70S6K. (C) Knockdown of C1orf74 inhibited the expression of cyclin D1; (D) Knockdown of C1orf74 enhanced the conversion of LC3-I to LC3-II and the degradation of p62 compared with those in the control siRNA group, meanwhile overexpression of C1orf74 had opposite effects on autophagy. NES, normalized enrichment score; FDR, false discovery rate; siRNA, small interfering RNA; mTORC1, mTOR complex 1; p-, phosphorylated.

Fig 5: C1orf74 enhances EGF-induced phosphorylation of AKT and p70S6K in lung adenocarcinoma cells.The results of the western blot analysis demonstrated that C1orf74 knockdown suppressed the basal level and EGF-induced phosphorylation of AKT and p70S6K in A549, H1993 and HCC827 cells compared with those in the control siRNA-transfected cells, and that overexpression of C1orf74 enhanced the phosphorylation of AKT and p70S6K. siRNA, small interfering RNA.