Fig. 1

SIX3 directly suppresses the transcription activity of AURKA and AURKB. a Schematic diagrams of promoters of AURKA and AURKB. TSS = 0; the numbers 1–8 refer to regions for ChIP assay; P1, P2, and P3 represent E2F1-binding regions, and the scores are the ChIP-seq data from the ENCODE project. b Promoter regions of AURKA (−2939 to −528 bp) and AURKB (−1715 to −105 bp) were inserted into pGL3-enhancer vector and co-transfected with pEGFP-C1-SIX3 into U251 cells. Luciferase reporter assay indicated that SIX3 suppressed the transcriptional activity of AURKA and AURKB promoters. c U251 cells were transfected with p3xFLAG-CMV-10-SIX3 plasmid. Anti-Flag antibody was used for immunoprecipitation. ChIP assay showed differential recruitment of SIX3 to the promoter regions of AURKA and AURKB. Immunoprecipitated chromatin was subjected to PCR and amplified for 29 cycles according to the data from RT-qPCR and then analyzed by agarose gel electrophoresis. The numbers 1–8 correspond to the numbers in the schematic diagram in a. d Promoter regions of AURKA (−2939 to −2600 bp) and AURKB (−1650 to −1350 bp) were inserted into pGL3-enhancer vector and co-transfected with pEGFP-C1-SIX3 into U251 cells. Luciferase reporter assays indicated that SIX3 suppressed the transcriptional activity of AURKA and AURKB promoters. e Analysis of REMBRANDT showed inverse correlation between SIX3 and AURKA and SIX3 and AURKB in glioma. f U251, U87, and U118 cells were transfected with pEGFP-C1-SIX3. RT-qPCR showed that SIX3 reduced the mRNA level of AURKA and AURKB (normalized with GAPDH). g U251 and U87 cells were transfected with different doses of pEGFP-C1-SIX3. Western blotting analysis showed that SIX3 decreased the expression of AURKA, AURKB, and phospho-histone 3 (H3-pS10) in a dose-dependent manner. h U87 and U118 cells were transfected with two different siRNAs targeting SIX3. Western blotting analysis showed that the knockdown of SIX3 increased the expression of AURKA, AURKB, and phospho-histone 3 (H3-pS10) (*P < 0.05; **P < 0.01; ***P < 0.001)