Fig. 5: SKA3 enhances PLK1 protein stability by blocking its ubiquitylation.

A FD-LSC-1 and TU-177 cells were transfected with siRNAs targeting SKA3 or negative-control siRNAs for 48 h, and then treated with the protein synthesis inhibitor cycloheximide (CHX, 40 µg/ml) for 0, 1, 3, 5, and 8 h. One group of cells was simultaneously treated with CHX (40 µg/ml) and the proteasome inhibitor MG132 (20 µM) for 8 h. Expression of SKA3 and PLK1 was measured by western blot analysis. The protein bands were quantified by ImageJ program. The data are expressed as means ± SD of three independent experiments. *P < 0.05; **P < 0.001. B, C Effect of SKA3 overexpression or knockdown on the ubiquitylation of PLK1. Cells were cotransfected with the SKA3-overexpression plasmid or SKA3-specific siRNA and HA-tagged ubiquitin expression plasmid for 48 h. CoIP was performed using an anti-PLK1 antibody followed by western blot analysis to assess the effects of SKA3 overexpression (B) or knockdown (C) on the ubiquitylation of PLK1 protein. D, E Representative images of IHC of SKA3 and PLK1 expression in 165 ANM (D) and LSCC (E) tissues. F Pearson correlation analysis of PLK1 with the SKA3 protein level in 165 LSCC samples using the IHC score. G Kaplan–Meier survival analysis of the association of PLK1 levels with the overall survival of LSCC patients. The PLK1-expression levels were divided into low or high groups according to the median IHC score (n = 165). H Representative whole-mount IHC staining images of PLK1 in the embryos of WT (+/+) and KO (+/−) mice on day 16.5 of gestation.