Fig 2: p62 interacts with G3BP1 and G3BP2. (a) NP40-soluble lysates were prepared from G3BP1-KD (G3BP1) HeLa or its control cells (NT) and immunoprecipitated with anti-p62 antibody or normal rabbit IgG. Cell lysate (Input) and immunoprecipitate (IP) were subjected to a Western blot analysis with anti-USP10, anti-G3BP1, anti-G3BP2 and anti-p62 antibodies. Asterisks indicate non-specific bands. (b) HeLa cells were lysed with ice-cold NP40-lysis buffer containing RNase A, and soluble cell lysates were immunoprecipitated with anti-p62 antibody or normal rabbit IgG (IgG). The cell lysate (Input) and immunoprecipitate (IP) with or without RNase treatment were subjected to a Western blot analysis with anti-USP10, anti-G3BP1 or anti-p62 antibody. (c,d) HeLa cells were transfected with FLAG-G3BP1 or its mutant plasmid (G3BP1/1-138, G3BP1/139-466, G3BP1/222-466 or G3BP1/338-466). Cell lysates (Input) and IP with the anti-p62 antibody (IP) were subjected to a Western blot analysis with anti-FLAG (G3BP1) and anti-p62 antibodies.

Fig 3: USP10-knockdown reduces Tau/TIA1-SG in HT22 cells. (a) Knockdown of USP10 (USP10-KD) in HT22 cells was performed using the lentivirus vector encoding USP10/shRNA (USP10-3, USP10-4) or the control (NT). The expression of USP10, Tau, TIA1 and β-actin in these cells was measured by Western blotting using respective antibodies. The asterisk indicates a nonspecific band. (b,c) USP10-KD HT22 cells and the control (NT) were treated with 5 µM MG-132 for 4 h, and SG formation was evaluated by anti-Tau (TAU-5) and anti-TIA1 antibodies as well as Hoechst for nuclear staining using a fluorescent microscope. Arrowheads indicate the colocalization of Tau with TIA1. The population (%) of cells with TIA1-SGs or TIA1/Tau-SGs is presented as the mean and SD from three independent experiments in (c). *P < 0.05; **P < 0.01. Scale bar: 10 µm.

Fig 4: p62 and USP10 mediate G3BP1-KD-induced CFTR-ΔF508 aggregation. (a–f) HeLa cells were transfected with G3BP1-siRNA (G3BP1), USP10-siRNA (USP10), p62-siRNA (p62), their combination (G1/U10 or G1/p62) or control (NT), and cells were then transfected with the GFP-CFTR-ΔF508 plasmid. Whole-cell lysates were subjected to a Western blot analysis using anti-GFP (CFTR), anti-USP10, anti-G3BP1, anti-G3BP2, anti-p62, anti-ubiquitin and anti-β-actin antibodies. The ratios of the GFP-CFTR band or the ubiquitin one to the β-actin one were presented as the means and SD from three experiments in (b,c,e,f). (g,h) HeLa cells were transfected with G3BP1-siRNA (G3BP1), USP10-siRNA (USP10), p62-siRNA (p62), a control (NT) or their combination (G1/U10 or G1/p62) by lipofection, and cells were then transfected with the GFP-CFTR-ΔF508 plasmid. The cells were stained with anti-HDAC6 (red) and Hoechst33258 (blue), and GFP fluorescence, anti-HDAC6 and Hoechst33258 staining was evaluated by a microscope. The percentages of cells with GFP/HDAC6-positive aggresomes in the cells (Aggresome (%)) were presented as the mean and SD. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. NS: not significant.

Fig 5: G3BP1 inhibits proteasome activity. (a–c) HeLa cells were transfected with G3BP1-siRNA (G3BP1), p62-siRNA (p62), their combination (G1/p62) or the control (NT) and then transfected with the YFP-CL1 plasmid. Whole-cell lysates were subjected to a Western blot analysis using anti-GFP (YFP-CL1), anti-G3BP1, anti-G3BP2, anti-p62, anti-ubiquitin or anti-β-actin antibodies. The ratios of the YFP-CL1 band or the ubiquitin one to the β-actin one were presented as the means and SD from three experiments in (b,c). (d) G3BP1, USP10 and p62 control cytotoxicity of GFP-CFTR-ΔF508. HeLa cells were transfected with G3BP1-siRNA (G3BP1), G3BP1-siRNA plus USP10-siRNA (G1/U10), G3BP1-siRNA plus p62-siRNA (G1/p62) or control (NT) and then further transfected with the GFP-CFTR-ΔF508 plasmid. To measure the metabolic activity of cells (Cell viability) by Cell Counting Kit-8 (CCK-8), cells were treated with CCK-8 solution for 1 h, and the absorbance (450 nm) of culture medium was measured by TriStar LB 941 Microplate Reader. Absorbance was normalized to the value of cells transfected with control siRNA (NT) (without GFP-CFTR∆F508 plasmid), and the ratio was presented as the mean and SD from three experiments. *P < 0.05; **P < 0.01; ****P < 0.0001. NS: not significant.