Fig. 3: Knockdown of RCC1 reduces the protein stability of Skp2 in STS cells, leading to the accumulation of p27Kip1.
A qRT-PCR for mRNA expression analysis of proliferation marker genes (Skp2, cyclin D, cyclin R, MCM3, MCM6, MCM7, E2F1, p57, p27Kip1, p16Inkand PCNA) in RCC1 knockdown (shRCC1#1, shRCC1#2) SW872 cells. B Immunoblotting analysis of RCC1, Skp2, p27Kip1, E2F1, cyclin A2 and MCM3 in shScr and RCC1 knockdown (shRCC1#1, shRCC1#2) SW872 cells by lentivirus mediated transduction. Tubulin was used as the loading control. C Cycloheximide (CHX) assays showed that RCC1 knockdown accelerating the degradation rate of Skp2 in SW872 cells. Indicated cells were treated with cycloheximide (CHX, 35 μM) to inhibit protein synthesis, and harvested at indicated time-points for immunoblotting analysis. Tubulin was used as the loading control. Quantification of Skp2 and p27Kip1 abundance normalized to tubulin is shown alongside. D Proteasome degradation assay of protein Skp2 in RCC1 knockdown SW872 cells. Cells were treated with or without the proteasome inhibitor MG132 (20 μM) for 6 hours before harvest and western blot analysis. Tubulin was used as loading control. E FACS analyses of shScr or shRCC1 SW872 cells transduced with Skp2 overexpression plasmid for 48 hours. The cells belong to different cell cycle sub-phase was determined, with the percentage shown alongside using barchart. F The cell proliferation rate of Skp2 overexpression (OE-Skp2) in shScr or shRCC1 SW872 cells was analyzed by CCK8. Data is expressed as mean ± SD (n = 3). Significance (*p < 0.05, **p < 0.01, ***p < 0.001).
