Fig. 5

Suppressing S403 phosphorylation rescues the defective aggresome formation caused by unphosphorylated SQSTM1 (T269/S272). A SQSTM1 knockout AD293 cells stably expressing FLAG-SQSTM1(WT), FLAG-SQSTM1(T269A/S272A), or FLAG-SQSTM1(T269A/S272A/S403A) were treated with 1 μM MG132 for 14 h. The aggresome formation was analyzed by immunostaining with anti-UB-K48 (Red) and anti-FLAG (Green) antibodies. Nuclei were stained with DAPI (blue). Scale bar: 20 μm. B Quantitative analysis of results in A. C SQSTM1 knockout AD293 cells stably expressing FLAG-SQSTM1(T269A/S272A) were treated with 1 μM MG132 alone or combined with CX-4945 (5 μM) for 14 h. The aggresome formation was analyzed by immunostaining with anti-UB-K48 (Red) and anti-FLAG (Green) antibodies. Nuclei were stained with DAPI (blue). Scale bar: 20 μm. D Quantitative analysis of results in C. E AD293 cells were treated with MG132 (1 μM), Doramapimod (50 μM), and CX-4945 (5 μM), alone or in combination for 14 h. The aggresome formation was analyzed by immunostaining with anti-UB-K48 (Green) antibodies. Scale bar: 20 μm. F Quantitative analysis of results in E. G A375 cells were treated with MG132 (1 μM), Doramapimod (50 μM), and CX-4945 (5 μM), alone or in combination for 14 h. The aggresome formation was analyzed by immunostaining with anti-UB-K48 (Red) and anti-SQSTM1 (Green) antibodies. Nuclei were stained with DAPI (blue). Scale bar: 20 μm. H Quantitative analysis of results in G. For quantitative analysis of aggresome formation, at least 50 cells were randomly selected from each group to score for aggresomes. Data are mean ± SEM of three independent experiments. **P < 0.01