Figure S4.
Vinculin is not required for cell cycle progression in non-polarized cells. (A) Scheme of the DNA vector used and the procedures followed in this figure. (B) Bar graph showing the mRNA level of vinculin measured by RT-qPCR 24 hpe of two different shRNAs targeting human vinculin into HEK-293 cultures. Mean ± SEM. One-way ANOVA plus Dunnett’s multiple comparisons test, n = 3 (3 RT-qPCR reactions using three independent HEK-293 transfections). (C and D) HEK-293 (C) and HeLa (D) cells transfected with shControl or sh2VCL, stained with anti-vinculin (magenta or gray scale), GFP (green) indicates transfection, and DAPI (blue) shows the nucleus. Scale bar = 10 μm. (E) Images of HeLa cultures transfected with shControl or sh2VCL, stained with anti PH3 (magenta); GFP (green) shows transfection. Arrowheads indicate transfected mitotic cells (PH3+/GFP+). Scale bar = 100 μm. The right panels show a magnification of an example of a transfected mitotic cell (PH3+/GFP+), the top panel shows PH3 and GFP channels together and the two bottom ones show them separately. Scale bar = 10 μm. (F) Bar graphs showing the percentage of mitotic (PH3+) among the transfected population (GFP+) in HEK-293 (left graph) or HeLa (right graph) cultures transfected with shControl or sh2VCL. Mean ± SEM. t test, n = 1,438 cells for HEK and n = 12,650 cells for HeLa coming from three independent transfections. (G) Flow cytometry analysis profiles showing DNA content (Hoechst fluorescence intensity) and cell number of GFP+ population of HEK293 cultures transfected with shControl or shVCL. A minimum of 5,000 cells were analyzed for each condition. (H) Bar graph showing the percentage of cells in each cell cycle phase from the profiles shown in Fig. 8 G. Mean ± SEM. t test, n = 3 independent transfections (I) Same experiment as in Fig. 8 G but performed in HeLa cells. (J) Bar graph showing the percentage of cells in each cell cycle phase from the profiles shown in Fig. 8 I. Mean ± SEM. t test, n = 3 independent transfections. ns = non-significant. **** = P < 0.0001.

Vinculin is not required for cell cycle progression in non-polarized cells. (A) Scheme of the DNA vector used and the procedures followed in this figure. (B) Bar graph showing the mRNA level of vinculin measured by RT-qPCR 24 hpe of two different shRNAs targeting human vinculin into HEK-293 cultures. Mean ± SEM. One-way ANOVA plus Dunnett’s multiple comparisons test, n = 3 (3 RT-qPCR reactions using three independent HEK-293 transfections). (C and D) HEK-293 (C) and HeLa (D) cells transfected with shControl or sh2VCL, stained with anti-vinculin (magenta or gray scale), GFP (green) indicates transfection, and DAPI (blue) shows the nucleus. Scale bar = 10 μm. (E) Images of HeLa cultures transfected with shControl or sh2VCL, stained with anti PH3 (magenta); GFP (green) shows transfection. Arrowheads indicate transfected mitotic cells (PH3+/GFP+). Scale bar = 100 μm. The right panels show a magnification of an example of a transfected mitotic cell (PH3+/GFP+), the top panel shows PH3 and GFP channels together and the two bottom ones show them separately. Scale bar = 10 μm. (F) Bar graphs showing the percentage of mitotic (PH3+) among the transfected population (GFP+) in HEK-293 (left graph) or HeLa (right graph) cultures transfected with shControl or sh2VCL. Mean ± SEM. t test, n = 1,438 cells for HEK and n = 12,650 cells for HeLa coming from three independent transfections. (G) Flow cytometry analysis profiles showing DNA content (Hoechst fluorescence intensity) and cell number of GFP+ population of HEK293 cultures transfected with shControl or shVCL. A minimum of 5,000 cells were analyzed for each condition. (H) Bar graph showing the percentage of cells in each cell cycle phase from the profiles shown in Fig. 8 G. Mean ± SEM. t test, n = 3 independent transfections (I) Same experiment as in Fig. 8 G but performed in HeLa cells. (J) Bar graph showing the percentage of cells in each cell cycle phase from the profiles shown in Fig. 8 I. Mean ± SEM. t test, n = 3 independent transfections. ns = non-significant. **** = P < 0.0001.

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