FIGURE 7:

Treatment with MitoQ and JNK inhibitor decreases accumulation and colocalization of autophagic markers with LDs in COPI-depleted cells. U2OS and PC3 cells were transfected with siCONT, siCOPA, siCOPZ1, or mock transfection. MitoQ, SP600125, or vehicle control was added 48 h posttransfection; 72 h posttransfection, cells were either fixed or lysed for IF or Western blotting analysis. (A) Western blotting determination of the level of LC3-II and SQSTM1/p62 in COPA-depleted or COPZ1-depleted PC3 and U2OS cells treated with MitoQ. (B) Western blotting determination of the level of LC3-II and SQSTM1/p62 in COPZ1-depleted PC3 and U2OS cells treated with JNK inhibitor. (C) Quantification of LC3-positive LDs. The total number of LDs and the number of droplets positive for LC3 were determined. Plot represents the average of quantification of at least 12 different fields from three independent transfections, ± SEM. (D) Representative immunofluorescent images of LDs (BODIPY, green) and LC3 (red) in the control or siCOPZ1-transfected PC3 cells, treated with MitoQ or JNK inhibitor. Images of U2OS and DU145 cells are presented in Supplemental Figure S6. Scale bar = 25 μm.