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Figure 4 | Journal of Hematology & Oncology

Figure 4

From: Azacytidine sensitizes acute myeloid leukemia cells to arsenic trioxide by up-regulating the arsenic transporter aquaglyceroporin 9

Figure 4

Transcription factor HNF1A was involved in azacytidine-induced up-regulation of AQP9. (A). HNF1A mRNA and protein expressions were markedly up-regulated after treatment of 5′Aza as examined by semi-quantitative and quantitative RT-PCR (upper panel) and western immunoblotting (lower panel). (B) Diagram depicting the regions of the HNF1A promoter analyzed by Methylation-specific PCR (MSP) and Combined Bisulfite Restriction Analysis (COBRA). (C) The methylation status of HNF1A gene promoter was extensively studied and determined using combined bisulfite restriction analysis. 5′Aza treatment resulted in demethylation of the HNF1A gene promoter. (C: control; IVD: universal methylated DNA). (D) Methylation-specific PCR (MSP) was performed using PCR primers specific for methylated or unmethylated HNF1A gene promoter. The results showed that the HNF1A gene promoter was highly methylated, and 5′Aza treatment led to demethylation of the HNF1A gene promoter. (E) Specific HNF1A siRNA abrogated 5′Aza-induced AQP9 up-regulation as determined by RT-PCR (upper panel) and quantitative RT-PCR (lower panel). (F) In K562 cells treated with 5′Aza, specific HNF1A siRNA almost completely blocked the intracellular entry of As2O3 as compared with the control siRNA-treated cells. The results showed that HNF1A was the mediator for up-regulation of AQP9 after 5′Aza treatment.

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