Fig. 7
Effect of rescued RAB7 levels on vimentin expression and mitochondrial morphology. (A) YAPC and MIA PaCa-2 NDUFS3-silenced cells and their respective controls were transfected with HA-RAB7 after 3 days of silencing. Levels of vimentin were analyzed by Western blot analysis respectively after 7 and 9 days from silencing. The same lysates were used to perform immunoblotting using antibodies against the HA tag to verify transfection, NDUFS3 to check the efficiency of RNAi, RAB7 to confirm the downregulation of this protein in silenced cells, HSP90 to show the correct loading. (B-C) Vimentin signal intensity was quantified by densitometric analysis by Image Lab Software version 6.0.1 in both cell lines and conditions and reported as the ratio of samples compared to their respective Scr control. (D) Representative image of GFP-RAB7 (green) and mitochondria (red, stained with MitoTracker Red CMXROS dye) in NDUFS3-silenced RAB7-downregulated YAPC cells transfected with GFP-RAB7 at day 3 of silencing. Nuclei were labeled with DAPI (blue). White boxes indicate zoomed areas on the right. In panel (a) mitochondria (red) in GFP-RAB7 transfected cells (green). In panel (b) mitochondria (red) in mock not transfected cells. Scale bar: 10 μm. The images were analyzed and quantified using the Mitochondria Analyzer in Fiji/ImageJ software (ImageJ). The measurements for (E) Mean Area (µm2), (F) Mean Perimeter (µm), (G) Aspect Ratio (au), (H) Form Factor (au), (I) Branch Junctions/Mito, (J) Branches/Mito, (K) Total Branch Length (µm) and (L) Mean Branch Length (µm) are presented. The numbers of mitochondria analyzed are n = 10,562 from 30 images for the Mock control group, n = 4555 from 15 images for GFP-RAB7 transfected cells. (M) Representative image of GFP-RAB7 (green) and mitochondria (red) in MIA PaCa-2 NDUFS3-silenced RAB7-downregulated cells transfected with GFP-RAB7 at day 3 of silencing. Mitochondria were stained with MitoTracker Red CMXROS dye. Nuclei were labeled with DAPI (blue). White boxes indicate zoomed areas on the right. In panel (a) mitochondria (red) in GFP-RAB7 transfected cells (green). In panel (b) mitochondria (red) in mock not transfected cells. Scale bar: 10 μm. The images were analyzed and quantified using the Mitochondria Analyzer in Fiji/ImageJ software (ImageJ). The measurements for (N) Mean Area (µm2), (O) Mean Perimeter (µm), (P) Aspect Ratio (au), (Q) Form Factor (au), (R) Branch Junctions/Mito, (S) Branches/Mito, (T) Total Branch Length (µm) and (U) Mean Branch Length (µm) are presented. The numbers of mitochondria analyzed are n = 4754 from 19 images for the Mock control group, and n = 2119 from 20 images for GFP-RAB7 transfected cells transfected cells. Values are the mean ± SEM of at least three independent experiments. Abbreviation: ns: not significant. *p ≤ 0.05, ** p ≤ 0.01 and *** p ≤ 0.001