Fig. 3

RIT1 interacts with SMC3 in HCC cells. A Immunofluorescence staining of Myc-tag (red) and DAPI (blue) shows the distribution of RIT1 at different cell cycle phases in HCC-LY10 and Huh7 cells transfected with Myc-RIT1 plasmid. Scale bars, 20 μm. B Heat map of mitotic chromosome segregation-related proteins across Myc-RIT1-precipitated compared to IgG-precipitated group in HCC-LY10, Huh7, and Hep3B cells transfected with Myc-RIT1 plasmid. C Co-IP analysis of RIT1 and SMC3 in HCC-LY10, Huh7 and Hep3B cells transfected with Myc-RIT1 plasmid. Cell lysates from indicated cells were immunoprecipitated using an anti-Myc antibody (upper section) and an anti-SMC3 antibody (lower section). D Interaction of RIT1 and SMC3 was analyzed by Co-IP analysis in 293T cells co-transfected with Myc-RIT1 and Flag-SMC3 plasmids. Cell lysates were immunoprecipitated using an anti-Myc antibody (upper section) and an anti-Flag antibody (lower section). E Schematic presentation of structural domain of RIT1. F Co-IP analysis of the indicated RIT1 truncation and wild-type with SMC3 for their interaction position in 293T cells co-transfected with Myc-RIT1 (WT, ΔN or ΔC) and Flag-SMC3 plasmids. G Co-IP analysis of RIT1 and SMC3 in Huh7 and HCC-LY10 cells with RIT1 overexpression during asynchronous and synchronized to mitosis (Full indicates asynchronization treatment; Mitosis indicates cells were enriched in mitosis). H Colocalization of RIT1 and SMC3 during interphase and different phases of mitosis was analyzed by co-immunofluorescence staining (Myc-RIT1: red, SMC3: green, DAPI: blue) in HCC-LY10 (left) and Huh7 (right) cells transfected with Myc-RIT1 plasmid. Scale bars, 20 μm