Fig. 6: LATS1-induced Beclin-1 ubiquitination is required for Beclin-1 stabilization and autophagy regulation.

a, b Mutation of lysine residues K32/263 in Beclin-1 blocks LATS1-induced Beclin-1 stabilization. HEK293T/17 cells were transfected with the same amount of vector encoding for Flag-tagged wild-type or K32/263R lysine mutant Beclin-1 together with a vector encoding for HA-tagged LATS1 or empty vector as indicated. Seventy-two hours later cells were analyzed by immunoblotting with indicated antibodies. Representative immunoblots (a) and quantification of the relative Flag-tagged Beclin-1 (b) (normalized to GAPDH) from three independent experiments are shown. Statistical significance was calculated using one-way ANOVA. c Huh7 cells were transfected with indicated siRNAs followed by transfection with HA-tagged siRNA-refractory wild-type or K32/263R lysine mutant Beclin-1 cDNAs. Cells were analyzed by immunoblotting with indicated antibodies. Results represent three independent experiments. d, e Huh7 cells were transfected with indicated siRNAs followed with siRNA-refractory wild-type or K32/263R mutant Beclin-1 cDNAs. Cells were then treated with DMSO or sorafenib (Srf; 6 μM for Huh7) for 24 h and/or in combination with chloroquine (CQ; 20 μM) for 2 h before fixation. Representative images (d) and quantification of LC3B-puncta numbers (e) pooled from three independent experiments are shown. Statistical analysis was calculated by one-way ANOVA. Scale bars, 25 μm. f U2OS-GFP-LC3 Cells were transfected with indicated siRNAs followed with siRNA-refractory wild-type or K32/263R lysine mutant Beclin-1 cDNAs. Cells were then treated with rapamycin (Rapa; 100 nM) for 16 h and/or in combination with chloroquine (CQ; 10 μM) for 2 h before analysis. Results represent three independent experiments. g, h Huh7 cells were transfected with indicated siRNAs followed with siRNA-refractory wild-type or K32/263R lysine mutant Beclin-1 cDNAs. Cells were then treated with sorafenib for 24 h. Cells were analyzed by immunoblotting. Representative images (g) and quantification of relative p62 levels (h, treated with DMSO or 6 μM sorafenib) pooled from three independent experiments are shown. Statistical analysis was calculated by one-way ANOVA. Scale bars, 25 μm. i, j Cells were transfected with indicated siRNAs followed with siRNA-refractory wild-type or K32/263R mutant Beclin-1 cDNAs. i Cells then treated with sorafenib (Srf; 6 μM) for 48 h for immunoblotting (i) or for 7–10 days for colony formation assays (j). Results represent three independent experiments.