Fig. 5

Late endocytic pathway impairment in NDUFS3-silenced RAB7 downregulated cells. (A) The presence of Cathepsin D immature forms was evaluated by Western blot analysis on cell lysates from NDUFS3-silenced YAPC and MIA PaCa-2 cells (indicated as #1 and #2) and their respective controls using an anti-Cathepsin-D antibody. An antibody against NDUFS3 was used to verify silencing, while an antibody against HSP90 was used to verify correct loading. (B) Protein levels were quantified by densitometry with Image Lab Software version 6.0.1, and we quantified the ratio between immature forms (52/44 kDa) and mature form (32 kDa). The difference is reported as the ratio of NDUFS3-silenced cells compared to their Scr control. (C) Representative images of DQ-Green BSA assay in YAPC and MIA PaCa-2 silenced cells using NDUFS3 siRNA #1 and #2 and their respective Scr controls. Nuclei were labeled with DAPI (blue). White boxes indicate zoomed areas below. Scale bar: 10 μm. (D-G) For each cell line, CTCF intensity and DQ-BSA puncta were quantified by ImageJ software. Data are reported as the ratio of NDUFS3-silenced samples (indicated as #1 and #2) compared to their Scr control. Measures were obtained by analyzing at least 50 cells/samples derived from three or more independent experiments. (H) Representative images of megamitochondria stained with MitoTracker Red CMXROS dye in MIA PaCa-2 cells NDUFS3-silenced (with siRNA #1 and #2) and relative control (Scr). Nuclei were labeled with DAPI (blue). White boxes indicate zoomed areas below. Scale bar: 10 μm. (I) Quantification of cells with megamitochondria in MIA PaCa-2 silenced cells and control compared with the total number of cells analyzed. (J) Relative protein abundance of RAB7 and LAMP-1 was assessed by Western blotting in YAPC and MIA PaCa-2 cells after treatment with Rotenone (1 µM) for 48 h and 72 h, respectively. An antibody against HSP90 was used to verify correct loading. (K-L) Protein levels were quantified by densitometry with Image Lab Software version 6.0.1. (M) Relative protein abundance of RAB7 and LAMP-1 was assessed by Western blotting in YAPC and MIA PaCa-2 cells after treatment with CCCP (10 µM) for 18 h. An antibody against HSP90 was used to verify correct loading. (N-O) Protein levels were quantified by densitometry with Image Lab Software version 6.0.1. Values are the mean ± SEM of at least three independent experiments. *p ≤ 0.05, ** p ≤ 0.01 and *** p ≤ 0.001