Figure 2. Hyperactivation of mTORC1 in MPS chondrocytes suppresses autophagy via a Beclin 1–Vps34–UVRAG kinase complex.

(A) Western blot analysis of mTORC1 signaling upon amino acid stimulation in Gusb^–/– and Gusb^–/– Rpt^+/– primary chondrocytes. (B) Quantification of S6K1 phosphorylation in primary chondrocytes isolated from mice of the indicated genotypes. n = 3. (C) Representative images of an RFP-GFP-LC3 assay in primary chondrocytes from mice of the indicated genotypes. Scale bars: 10 μm. (D and E) Quantitative analysis of RFP⁺GFP⁺ puncta (D) and RFP⁺GFP^– puncta (E). Horizontal bars indicates the mean value. n = 3 chondrocytes donors/phenotype; n = 30 cells analyzed. (F) Western blot analysis of the indicated proteins in Gusb^–/– and Gusb^–/– Rpt^+/– primary chondrocytes. β-Actin was used as a loading control. (G) Quantification of protein levels in cells from mice of the indicated genotypes. n = 3. (H) Representative blot of a UVRAG IP assay testing UVRAG interaction with Rubicon in control and Gusb-KO RCS chondrocytes. (I) Quantification of Rubicon co-IP with UVRAG. n = 3. (J) GFP-2xFYVE transfection in control and Gusb-KO RCS chondrocytes. Gusb-KO cells were treated with Torin1 (1 μM; 6 h) or cotransfected with Myc-UVRAG. Cells were costained for endosomes (EEA1). Boxed area in the bottom right-hand panel shows positive myc staining. Scale bars: 10 μm. (K) Quantitative analysis of PI3P puncta. n = 3; n = 25 cells analyzed. (L and M) Western blot analysis of SQSTM1/p62 in Gusb-KO cells transfected with Myc-UVRAG (L) or treated with Torin1 (1 μM, 6 h) and SAR405 (10 μM, 6 h) (M). β-Actin was used as a loading control. Bar graphs show quantification of protein levels. n = 3 (L) and n = 2 (M). Data represent the mean values derived from the indicated number of independent experiments. Error bars indicate the SEM. *P ≤ 0.05, **P ≤ 0.005, and ***P ≤ 0.0005, by ANOVA followed by Tukey’s post-hoc test (B, D, E, G, and K) and unpaired Student’s t test (I and L).