Figure 4: TRB3 mediates insulin/IGF-induced p62 accumulation.

(a) HepG2 cells were treated with indicated stimulators for 12 h. The mRNAs were determined with RT–PCR using specific primer sets. Data are means±s.e.m of three independent assays. (b) HepG2 cells were incubated with cycloheximide (CHX) (10 μg ml⁻¹) for indicated time points after insulin/IGF-1 stimulation. Indicated proteins were detected with immunoblotting. Data are means±s.e.m of three independent assays. (c) Control or TRB3-silenced cells were incubated with CHX (10 μg ml⁻¹) for indicated time points after IGF-1 stimulation. Cell lysates were isolated for immunoblotting. Data are means±s.e.m of three independent assays. (d) HepG2 cells expressing p62- or control-siRNA were co-transfected with Ub^G76V-GFP. The cells were treated with IGF-1 for 12 h. The cell lysates were isolated for immunoblotting. Data are representative of immunoblots of three independent assays. (e) Cells expressing control-shRNA or TRB3-shRNAs were transfected with Ub^G76V-GFP plasmid plus or minus with p62-DDK-expressing plasmid. The lysates were isolated for immunoblotting. Data are representative immunoblots of three independent assays. (f) HepG2 cells expressing p62- or control-siRNA were infected with adenovirus containing mRFP-GFP-LC3-PE. After 24 h, the cells were treated with IGF-1 for 12 h, and the flux rate of autophagy was detected with Live Cell Imaging Microscopy. Data are representative images of three independent assays. Red colour shows autophagolysosomes and double-colour red/green shows autophagosomes. Scale bar, 10 μm. (g) Control or p62-silencing cells were treated with or without IGF-1 for 12 h. Indicated proteins were evaluated with immunoblotting. Data are representative immunoblots of three independent assays. (h) BALB/c nude mice were i.v. injected with HepG2 cells expressing control-shRNA or p62-shRNA (3 × 10⁶). Data are representative of number of metastases in lungs and lungs. Metastatic nodules were indicated by arrows (n=8 per group). (i) BALB/c nude mice were s.c. inoculated with HepG2 cells expressing control- or p62-shRNA (1.5 × 10⁶). Data are the mean volumes±s.e.m. at indicated times and representative tumours along with tumour weight (n=8 per group). Scale bar, 1.5 cm. Statistical significance was determined with Student’s t-test; *P<0.05.