Fig. 6
From: Ubiquitination of Rheb governs growth factor-induced mTORC1 activation
Rheb ubiquitination regulates cell autophagy, cell proliferation, and cell size. a Cell autophagy induced by glucose starvation was analyzed in WT and RNF152−/− MEF cells. Cells were harvested after treated with Bafilomycin A1 (100 nM) for 4 h. RNF152-KO MEF cells were analyzed by genomic DNA PCR in Supplementary information, Fig. S3s. b The protein levels of LC3II were analyzed in HEK293T cells after glucose starvation and Bafilomycin A1 treatment as in (a). c Under glucose starvation, Rheb-K8R showed stronger blocking effects on autophagy than Rheb-WT. Bafilomycin A1 treatment was performed as in (a). d USP4−/− MEF cells showed higher autophagy levels than USP4+/+ MEF cells. Cells were treated with Bafilomycin A1 treatment as (a). e The effects of USP4 on cell autophagy were analyzed by examining the LC3II protein levels. Cells were treated with Vialinin A (2 μmol) for indicated time periods together with Bafilomycin A1 as in (a). f, g The effects of WT or USP4 mutants on autophagy were detected in HEK293T cells. h USP4 knockdown and Vialinin A treatment blocked cell proliferation in Rheb-WT-expressed, but not Rheb-K8R expressed SW620 cells. Data were analyzed by two-way ANOVA. P < 0.05 was considered statistically significant. *P < 0.05 and ***P < 0.001, respectively, ns not significant. i, j Cell size was measured in USP4 knockdown cells (i) and Vialinin A-treated cells (j)
