Fig. 4

SIX3 induces S phase arrest and reduces the recruitment of E2F1 on the promoters of AURKA and AURKB. a U251 cells were synchronized by double thymidine block and transfected with pEGFP-C1-SIX3 24 h before release. Western blotting analysis showed that SIX3 decreased the expression of AURKA, AURKB, phospho-AURKA (Thr288)/AURKB (Thr232)/AURKC (Thr198), phospho-histone 3 (S10), and p21 and delayed the aurora kinase peak approximately 5 h. b Flow cytometry showed that SIX3 induced S phase arrest in both U251 and U87 cells. Quantification of G1/S and G2/S ratios are shown on the right. c Western blotting analysis showed that SIX3 increased the expression of cyclin A and p27 and decreased the expression of E2F1 and p21 in U251 cells. d U87 cells were transfected with pEGFP-C1-SIX3 and siRNAs targeting SIX3. Western blotting analysis showed that SIX3 increased the expression of cyclin A, p27, and p21 and decreased expression of E2F1 (left). Knockdown of SIX3 increased the expression of E2F1 and decreased expression of cyclin A, p27, and p21. e ChIP assay indicated that SIX3 decreased the enrichment of E2F1 on the promoter regions of AURKA and AURKB in U251 cells. Chromatin samples were subjected to RT-qPCR analysis (*P < 0.05; **P < 0.01; ***P < 0.001)