Figure 3: Enhanced TRB3 compromises selective autophagy and clearance of UPS.

(a) BEAS-2B cells were infected with TRB3-Myc-adenovirus expressing Myc-Tagged TRB3 with an independently expressing GFP. GFP-adenovirus was used as control. Indicated molecules were detected by immunoblotting. Data are representatives of four independent assays. (b) TRB3-silenced cells were treated with IGF-1 (100 nM) for 12 h and indicated proteins were detected by immunoblotting. Data are representatives of four independent assays. (c) Control or TRB3-silenced HepG2 cells were infected with adenovirus with mRFP-GFP-LC3-PE. After 24 h, the cells were treated with IGF-1 (100 nM) for 12 h and the flux rate of autophagy was detected with Live Cell Imaging Microscopy. Data are schematic drawing of the autophagy process (upper) and microscopy images showing red-coloured autophagolysosomes or red/green double-coloured autophagosomes (lower). Scale bar, 10 μm. See also Supplementary Movies 1–6. (d) Silencing TRB3 increased the number of autophagosomes (Avi) and autolysosomes (Avd) in HepG2 cells treated with or without IGF-1 (100 nM). Data are representative images of TEMs of three independent assays. Single arrows denote Avi; double arrows indicate Avd. Scale bar, 500 nm. The ratio of autophagic vacuole area to the cytoplasmic area was determined by morphometric analysis. Data are means±s.e.m. (e) TRB3-silenced cells were treated with IGF-1 (100 nM) for 12 h. Expression of indicated proteins was detected by immunoblotting. Data are representatives of four independent assays. (f) TRB3-silenced HepG2 cells were treated with bafilomycin (left) or MG132 (right) for 12 h. Indicated proteins were detected by immunoblotting. Data are representatives of four independent assays. (g) TRB3-silenced or control cells were transfected with Ub^G76V-GFP plasmid. The cells were treated with or without IGF-1 for 12 h and the expression of Ub^G76V-GFP was immunoblotted with anti-GFP antibody (Ab; left) or detected with flow cytometry (right). Data are means±s.e.m. (n=4). (h,i) HepG2 cells stably expressing TRB3-shRNA and ATG5-shRNA simultaneously were generated. The proliferative and invasive capacities of cells were evaluated with Edu (h) and transwell assays (i). Statistical significance was determined with one-way analysis of variance (ANOVA); *P<0.05; **P<0.01; ***P<0.001.