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Fig. 2 | Journal of Hematology & Oncology

Fig. 2

From: SIX3, a tumor suppressor, inhibits astrocytoma tumorigenesis by transcriptional repression of AURKA/B

Fig. 2

SIX3 reactivates the p53 pathway dependent on suppressing of AURKA and AURKB. a U251 and U87 cells were transfected with pEGFP-C1-SIX3 and siRNAs targeting SIX3. Western blotting analysis showed that SIX3 did not affect p53 expression in p53 mutant U251 cells. Overexpression of SIX3 increased p53 expression, and knockdown of SIX3 reduced p53 expression in p53 wild-type U87 cells. b U251 and U87 cells were transfected with two different siRNAs targeting AURKA and AURKB. Western blotting analysis showed that knockdown of AURKA or AURKB did not affect p53 expression in p53 mutant U251 cells but increased p53 expression in p53 wild-type U87 cells. c p53 activity reporter and siRNAs targeting SIX3 were co-transfected into p53 wild-type U87 cells. Luciferase activity analysis showed that knockdown of SIX3 expression decreased the activity of p53 in p53 wild-type U87 cells. d U87 cells were transfected with pEGFP-C1-SIX3. ChIP assay indicated overexpression of SIX3 enhanced the recruitment of p53 protein to the promoter regions of well-known p53 targets, including p21, PUMA, and GADD45. Chromatin samples were subjected to RT-qPCR. e U87 cells were transfected with pEGFP-C1-SIX3, pEGFP-C1-SIX3 and p3xFlag-CMV-10-AURKA, and pEGFP-C1-SIX3 and p3xFlag-CMV-10-AURKB, respectively, and pEGFP-C1-p53 was the positive control. Luciferase activity analysis and Western blot showed that co-transfection of AURKA or AURKB with SIX3 counteracted the effect of SIX3 on increasing the expression and activity of p53 in U87 cells (*P < 0.05; **P < 0.01; ***P < 0.001)

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