Figure 6.

The autophagic targeting of R-HSPA5 and SQSTM1 is impaired in PARK7-deficient cells. (a) HCT116 cells stably expressing scrambled shRNA or shPARK7 were treated with 200 nM bafilomycin A₁ (Baf) for 6 h and immunostained for LC3-II (red) or PARK7 (green). LC3-II-positive autophagic vacuoles were examined using confocal microscopy. Scale bar: 10 μm. (b) Quantification of the number of LC3-II puncta in A. Error bars represent the mean ± SEM from each cell (***p < 0.001, n = 50). (c) HCT116 cells stably expressing scrambled shRNA or shPARK7 were treated with 200 nM bafilomycin A₁ for 2 h, followed by the treatment with 10 ng/ml TNFSF10 for 4 h. The cells were immunostained for LC3-II (red) or SQSTM1 (green). Puncta formation and colocalization of LC3-II and SQSTM1 were examined using confocal microscopy. Scale bar: 10 μm. (d) HCT116 cells stably expressing scrambled shRNA or shPARK7 were treated with 200 nM bafilomycin A₁ for 2 h, followed by treatment with 10 ng/ml TNFSF10 for 4 h. The cells were immunostained for PLEKHM1 (green). Puncta formation of PLEKHM1 was examined using confocal microscopy. Scale bar: 10 μm. (e) HCT116 cells stably expressing scrambled shRNA or shPARK7 were treated with 200 nM bafilomycin A₁ (Baf) for 6 h and immunoblotted with the indicated antibodies. (f) HCT116 cells stably expressing scrambled shRNA or shPARK7 were treated with 200 nM bafilomycin A₁ for 6 h and immunostained for WIPI2 (red). WIPI2-positive autophagic vacuoles were examined using confocal microscopy. Scale bar: 10 μm.