Figure 5.

PARK7 is required for the autophagic targeting of R-HSPA5 and SQSTM1 in HCT116 cells treated with TNFSF10. (a) Cells were treated with 200 nM bafilomycin A₁ for 6 h or 10 ng/ml TNFSF10 for 4 h. Alternatively, the cells cultured in the presence of 200 nM bafilomycin A₁ for 2 h were additionally treated with 10 ng/ml TNFSF10 for 4 h. Immunostaining analysis was performed using antibodies to R-HSPA5 (red) and SQSTM1 (green), followed by confocal microscopy. Scale bar: 10 μm. (b) HCT116 cells stably expressing scrambled shRNA or shPARK7 were treated with 10 ng/ml TNFSF10 for 4 h or 200 nM bafilomycin A₁ (Baf) for 2 h and additionally treated with 10 ng/ml TNFSF10 for 4 h as described in A. (c) Quantification of R-HSPA5 cytosolic puncta in B. Error bars represent the mean ± SEM from each cell (***p < 0.001, n = 50). (d) Quantification of SQSTM1 cytosolic puncta in B. Error bars represent the mean ± SEM from each cell (***p < 0.001, n = 50). (e) Quantification of the colocalization of SQSTM1 cytosolic puncta in B with R-HSPA5 puncta. Error bars represent the mean ± SEM from each cell (***p < 0.001, n = 50). (f) Cells were treated with 10 ng/ml TNFSF10 for 4 h or 200 nM bafilomycin A₁ for 6 h. Alternatively, the cells cultured in the presence of 200 nM bafilomycin A₁ for 2 h were additionally treated with 10 ng/ml TNFSF10 for 4 h. The SQSTM1 and PARK7 oligomerization assay was followed by nonreducing SDS-PAGE and immunoblotting using antibodies to SQSTM1 and PARK7. (g) Cells were treated with tBHP at the indicated concentration. PARK7 oligomerization assay was followed by nonreducing SDS-PAGE and immunoblotting using an antibody to PARK7.