Fig 2: Knockdown of RPS6 in SKOV3 and HO-8910 cells using LV-shRNA. Lentivirus transduction in SKOV3 and HO-8910 cells was detected by fluorescence microscopy at a magnification of 100× (lower); scale bar: 200 μm. These results suggest that both SKOV3 and HO-8910 cells had high levels of GFP expression (a, d). Depletion of RPS6 mRNA in SKOV3 and HO-8910 cells lines treated with RPS6-shRPS6 was determined by qRT-PCR (b, e). Decreased expression of RPS6 protein was detected by western blotting after transfection (c, f)

Fig 3: TORC1 is strongly inhibited by phosphate starvation in C. glabrata, likely contributing to the ASR via its proximal kinase, Sch9.(A) A representative Western Blot for phosphorylated Rps6 (P-Rps6) and total Rps6 in both species under rich media, nitrogen starvation (-N) or phosphate starvation (-Pi) conditions. Red arrows point to the loss vs presence of the P-Rps6 band under -Pi in C. glabrata and S. cerevisiae, respectively. (B) Quantification of %P-Rps6 over total Rps6 (n = 3). *Bonferroni corrected P-values from Student’s t-tests were shown. (C) Inhibiting TORC1 by rapamycin induces Cta1-GFP in a dose-dependent manner. The dots, error bars and lines have the same meaning as before. (D) ASR for H2O2 with rapamycin as the primary treatment. Plotted are survival rates with or without rapamycin treatment (n = 12 for C. glabrata, n = 8 for S. cerevisiae). 60 mM and 6 mM of H2O2 were used as the secondary stress for the two species, resulting in a similar basal survival rate (P = 0.6). A Wilcoxon signed-rank test was used to compare the paired experiments with or without the primary treatment for each species. The raw, one-sided P-values were shown on the top. (E) Same plot as C, comparing Cta1-GFP induction in a phosphomimetic mutant of Sch9, a key proximal effector of TORC1, and a matching wild type Sch9 strain. (F) Same as D but with phosphate starvation (-Pi) as the primary stress, comparing the Sch9-3E mutant (n = 4) and the matching Sch9-wt control (n = 4). 100 mM and 40 mM of H2O2 were used as the secondary stress for the two genotypes, resulting in a similar basal survival rate (P = 0.7).

Fig 4: RSK-3 promotes cell proliferation by regulating rpS6 and autophagy. Effects of downregulated RSK-3 in MRL/MpJ CSPCs (A) and overexpressed (OE) RSK-3 in STR/Ort CSPCs (B) on the protein expression of p-rpS6, rpS6, p-mTOR and mTOR. *p < 0.05, **p < 0.01, ***p < 0.001 versus STR/Ort (n = 6 per group). (C) Immunolabeling of p-mTOR and LC3 in articular cartilage after DMM injury in normal and RSK-3-/- mice. **p < 0.01, ***p < 0.001 versus sham (RSK-3+/+), #p < 0.05, ###p < 0.001 versus DMM (RSK-3+/+) (n = 6 per group). Scale bars = 100 µm.

Fig 5: RSK-3 activates rpS6, but not autophagy, to regulate CSPCs proliferation. The proliferative ability of CSPCs from C57/BL6J mice after RSK-3 inhibitor (BI-D1870) and mTOR inhibitor (rapamycin) treatment detected by CCK-8 assay (A) and Ki67 labeling (B). (C,D) Protein expression of RSK-3, p-RSK-3, mTOR, p-mTOR, p-rpS6 and rpS6 in CSPCs from C57/BL6J mice and the corresponding quantifications. (E) p-RSK-3, p-mTOR and LC3 immunolabeling in CSPCs from C57/BL6J mice. *p < 0.05, **p < 0.01, ***p < 0.001 versus control, ##p < 0.01, ###p < 0.001 versus BI-D1870 (n = 6 per group). Scale bars = 100 µm.