Fig. 3.

WDR76 suppresses proliferation, transformation, motility and invasive properties of liver cancer cells through RAS destabilization. a IBs of RAS in Flag-WDR76 or shWDR76-transfected Huh7, SK-Hep1, PLC/PRF/5, Hep3B, and HepG2 cells. b Schematic representation of WDR76 and its NLS deleted forms. c SK-Hep1 cells stably expressing GFP-WDR76FL or GFP-WDR76ΔNLS were analyzed. Immunocytochemical analyses data showing localizations of RAS and WDR76 were obtained by using the anti-RAS (red) or anti-GFP (green) antibody. The nuclei were stained with DAPI (blue). Scale bars, 20 µm. d–f SK-WT cells stably expressing GFP, or SK-KO cells stably expressing GFP, GFP-WDR76FL, or GFP-WDR76ΔNLS were analyzed (d), or were transfected with 3XFlag-Ub (f) and then treated with ALLN (e, f). WCLs were immunoprecipitated with antibody recognizing RAS (e, f). g, h Cells were cultured and MTT assay (g), and foci formation assays (h) were performed. Data are presented as the mean ± SD (n = 3 biological replicates). Two-sided Student’s t test, ***p < 0.001. i Single-cell migratory behavior was monitored using real-time imaging microscopy at least five independent times. Data are presented as the mean ± SD. j Invaded cells through the Matrigel were stained with crystal violet. Representative images were captured, and the total numbers of invaded cells were counted (bottom). Data are presented as the mean ± SD (n = 3 biological replicates). Two-sided Student’s t test, ***p < 0.001. Scale bar, 200 µm