Fig. 6

SETDB1 methylation of PLK3 promotes PLK3 phosphorylation and decreases PLK3 activity. A IB of the phosphorylation and lysine methylation of PLK3, and also determined the expression of HIF1α, SOX2, and CD44 in control and SETDB1-knockdown MDA-MB-231 and BT549 BCSCs. Lysates were assessed by immunoprecipitation (IP) with anti-PLK3 and immunoblotting with anti-Phospho-(Ser/Thr) and anti-Lys-Mono-Methyl. B, C Effects of ectopic expression of PLK3 WT or 2KM (K106M/K200M) mutant on the phosphor-PLK3 and the expression of HIF1α, SOX2, and CD44 in MDA-MB-231 /sh115 (B) and MDA-MB-231/shSETDB1 BCSCs (C). EV, an empty vector control. Lysates were assessed by immunoprecipitation (IP) with anti-PLK3 and immunoblotting with anti-Phospho-(Ser/Thr). D In vitro GST pull-down analysis. Purified GST-HIF1α protein was incubated with cell extracts from MDA-MB-231 BCSCs. E and F Effects of ectopic expression of PLK3 WT or 2KM mutant on HIF-1α protein degradation in MDA-MB-231/shC or MDA-MB-231/sh115 BCSCs with cycloheximide (CHX, 100 µM) treatment (0, 10, 30, 60, and 120 min). G Effects of PLK3 WT or 2KM mutant on HIF-1α ubiquitination in MDA-MB-231/sh115 BCSCs. H Effects of ectopic expression of PLK3 WT or 2KM mutant on HIF-1α ubiquitination in MDA-MB-231/shSETDB1 BCSCs.Data are representative of two or three independent experiments with similar results. Error bars, ± SD. **P < 0.01, by paired two-way Student’s t-test