Fig. 3: Depletion of NudCL2 impairs actin dynamics.

A549 cells transfected with siRNAs and vectors were subjected to the following analyses: a Western blotting analysis of the expression of NudCL2. β-actin was used as a loading control. b Cells were fixed and stained with phalloidin (red). DNA was visualized with DAPI (blue). Scale bar, 20 μm. c The percentage of cells with lamellipodia in (b) was calculated. More than 100 cells were counted in each experiment. d A sequence of phase-contrast time-lapse images of the cells were obtained with a LSM880 confocal microscope using a ×63 objective. Kymographs were produced and analyzed using MetaMorph software. The minimum intensity projection of a 250-frame movie (3 s per frame) is presented on the left. Pixel intensities along a one-pixel-wide line (white) were used to generate the kymograph presented on the right. Cells are outlined with dashed lines. Scale bar, 20 μm. e, f The velocity and persistence of lamellipodia protrusions in (d) are shown. g Cells were fixed and subjected to immunofluorescence staining with anti-paxillin (green) antibody. Scale bar, 20 μm. h Focal adhesions between cells in (g) were counted. About 20 cells were counted in each experiment. i Cells transfected with the indicated siRNA and vectors were subjected to western blotting analysis using the antibodies as shown. β-actin was used as a loading control. j–l Cells were fixed and stained with phalloidin (red) and anti-paxillin (green) antibody. DNA was visualized with DAPI (blue). Scale bar, 20 μm. The percentage of cells with lamellipodia and number of cellular focal adhesions were plotted respectively. More than 100 cells were counted in each experiment. m–o A sequence of phase-contrast time-lapse images of cells were obtained with a LSM880 confocal microscope using a ×63 objective. Kymographs were produced and analyzed using MetaMorph software. The minimum intensity projection of a 250-frame movie (3 s per frame) is presented on the left. Pixel intensities along a one-pixel-wide line (white) were used to generate the kymograph presented on the right. Cells are outlined with dashed lines. Scale bar, 20 μm. The velocity and persistence of lamellipodia protrusions are calculated. Quantitative data derived from at least three independent experiments are shown as the mean ± SD. n, sample size. *P < 0.05; **P < 0.01; ***P < 0.001. Student’s t-test.