Fig. 4: Knockdown of RCC1 elevates the poly-ubiquitination of Skp2 by promoting its nucleus retention.

A In vivo exogenous ubiquitination assay to detect ubiquitination of Skp2 influenced by RCC1 knockdown on SW872 cell. Each cell group was co-transduced with Myc-Skp2, His-Ubiquitin (His-Ub) and shScr/shRCC1 plasmids as indicated. B Immunofluorescence analysis was performed to determine the subcellular localization of Skp2 (red) in SW872 cells with shRCC1 and RCC1 overexpression (OE-RCC1). DAPI was used to counterstain the cells nuclei (blue). The X-Y scatter plot of 200 cells from control (blue dots), shRCC1 (red dots) and OE-RCC1 (green dots) groups based on the quantification of nucleus and cytoplasm fluorescence intensity of Skp2 is shown alongside. Scale bars: 10 μm. Data is expressed as mean ± SD (n = 3). Significance (**p < 0.01, ***p < 0.001). C Immunoblotting analysis of subcellular localization of Skp2 in shRCC1 or OE-RCC1 SW872 cells. Cells were starved for 48 hours and subsequently released into full culture medium for 12 hours. Lamin B1 was used as nuclear control and tubulin was used as cytoplasmic control. D Immunoblotting analysis was conducted to investigate the subcellular localization regulation of Skp2 in RCC1 knockdown SW872 cells throughout the cell cycle process. SW872 cells were transduced with the control plasmid (shScr) or RCC1 knockdown plasmid (shRCC1) were starving for 48 hours and subsequently released into full culture medium for several time period (0, 4, 8, 12, 18, and 24 hours). Cytoplasmic and nuclear extracts were analyzed by immuno-blotting. Lamin B1 was used as nuclear control and tubulin was used as cytoplasmic control. In vivo protein co-immunoprecipitation of endogenous Skp2 and RCC1 in SW872 cells (E) and exogenous binding in HEK 293 T cells (F).