Fig. 6

NDUFS3-silenced RAB7-downregulated cells are characterized by a less aggressive phenotype. (A) Lysates from YAPC cells transfected with control RNA (Scr) and with NDUFS3 siRNA#1 were subjected to SDS-PAGE and analyzed by immunoblotting using a specific antibody for vimentin, N-cadherin and Slug. An antibody against NDUFS3 was used to check the efficiency of silencing and an antibody against RAB7 was used to confirm the downregulation of this protein in silenced cells. (B-D) Relative abundance was quantified by densitometric analysis normalizing against HSP90 and it was reported as the ratio of NDUFS3 RNAi samples (#1) compared to their Scr control. (E) Lysates from MIA PaCa-2 cells transfected with control RNA (Scr) and with NDUFS3 siRNA#2 were subjected to SDS-PAGE and analyzed by immunoblotting using a specific antibody for vimentin and Snail. An antibody against NDUFS3 was used to check the efficiency of silencing and an antibody against RAB7 was used to confirm the downregulation of this protein in silenced cells. (F-G) Relative abundance was quantified by densitometric analysis normalizing against HSP90 and it was reported as the ratio of NDUFS3 RNAi samples (#2) compared to their Scr control. (H) Conditioned medium from NDUFS3-silenced YAPC (siRNA#1) and MIA PaCa-2 (siRNA#2) and relative control (Scr) was used to assess the activity of secreted MMP-2 and MMP-9 by gelatin zymography. Lysates from YAPC and MIA PaCa-2 NDUFS3-silenced cells and their respective control cells were subjected to SDS-PAGE and analyzed by immunoblotting using an antibody against NDUFS3 to check the efficiency of RNAi, an antibody against RAB7 to confirm the downregulation of this protein in silenced cells, and an antibody against HSP90 to verify the correct loading. (I) Viability of YAPC and MIA PaCa-2 cells transfected with control RNA (Scr) and with NDUFS3 siRNA#1 and siRNA#2, respectively. Data are presented normalized to T0 and the ratio of NDUFS3 RNAi samples (#1 and #2) compared to their Scr control was reported. (J-K) Capacity of YAPC and (L-M) MIA PaCa-2 cells transfected with control RNA (Scr) and with NDUFS3 siRNA#1 and siRNA#2, respectively, to form colonies in an anchorage-dependent manner. The ratio of NDUFS3 RNAi samples compared to their Scr control was reported. (N-O) YAPC cells transfected with control RNA (Scr) and with NDUFS3 siRNA#1 were imaged during the wound healing assay at an initial time point (T0) and 24 h after the scratch. Cell migration was measured as wound closure percentage after 24 h in Scr and in NDUFS3 siRNA#1 YAPC cells. Scale bar: 200 μm. (P-Q) Nuclei of migrating YAPC and MIA PaCa-2 cells transfected with control RNA (Scr) and with NDUFS3 siRNA#1 and siRNA#2, respectively, stained with 20 nM of PureBluTM Hoechst 33,342 Nuclear Staining Dye. Scale bar: 200 μm. (R-S) The mean number of migrating cells was calculated as the ratio of NDUFS3 RNAi samples (#1 and #2) compared to their Scr control. Values are the mean ± SEM of at least three independent experiments. *p ≤ 0.05, ** p ≤ 0.01 and *** p ≤ 0.001