Fig. 4

Compromised late endocytic trafficking of NDUFS3-silenced pancreatic cancer cell lines. (A-D) Relative protein abundance of RAB7, dynein, LAMP-1, TFEB, ATP6V1G1, and RAB9 was assessed by Western blotting in YAPC and MIA PaCa-2 cells transfected with control RNA (Scr) or with two different NDUFS3 siRNAs (indicated as #1 and #2). The antibody against NDUFS3 was used to check the efficiency of RNA interference (RNAi). Quantification of differences was performed by densitometric analysis, normalizing against HSP90, and it was reported as the ratio of NDUFS3-silenced samples (#1 and #2) compared to their Scr control. (E) Late endocytic acid compartments were live stained in NDUFS3-silenced YAPC and MIA PaCa-2 cells (indicated as #1 and #2) and relative Scr controls using LysoTracker DND-99 dye (red). Nuclei were labeled with PureBluTM Hoechst 33,342 Nuclear Staining Dye (blue). White boxes indicate zoomed areas below. Scale bar: 10 μm. (F-K) Corrected total cell fluorescence (CTCF), number and size of acid compartments were determined by ImageJ software in NDUFS3-silenced YAPC and MIA PaCa-2 cells and reported as the ratio compared to their Scr control. Each measure was obtained by analyzing at least 50 cells/samples from three or more independent experiments. Values are the mean ± SEM of at least three independent experiments. *p ≤ 0.05, ** p ≤ 0.01 and *** p ≤ 0.001