Fig. 3

Regulatory effect of WASF2 on cell cycle, apoptosis, EMT, and actin polymerization. A Cell cycle analysis in Huh-7 and SNU475 cells following siWASF2 transfection, using flow cytometry. The percentage of cells in G2/M phase was analyzed using a FACSAria III flow cytometer. Western blot analyses of cell-cycle modulators in two HCC cell lines (mean ± SEM; n = 3, unpaired t-test). B Apoptosis analysis using flow cytometry. Dot plots indicate apoptosis ratios using PI and FITC-annexin V (left). Bar graph shows average percentage of apoptotic cells (right). Western blot analysis of PARP, cleaved PARP, caspase-3, cleaved caspase-3, caspase-9, and cleaved caspase-9. GAPDH was used as the loading control. C Representative cell images and bar charts showing the number of migrating and invading cells transfected with siWASF2 captured in three random fields following modified Boyden chamber motility assay (top) and Transwell invasion assay (bottom; mean ± SEM; n = 3, unpaired t-test). Scale bar = 50 μm. D Western blot analysis of EMT molecules in the presence or absence of TGF-β1 (20 ng/mL) in Huh-7 and SNU475 cells. E IF staining of F-actin and F Filopodia formation (white arrows) in SNU475 cells transfected with negative-control siRNA (NC) or siWASF2. Representative fluorescence images are shown. Scale bar = 100 μm. G Western blot analysis of molecules related to the actin cytoskeleton in Huh-7 and SNU475 cells. ***P < 0.001