Fig. 3

ANXA2 activates HIF1A gene transcription by upregulating MYC in ESCC cells. a Western blot analysis of MYC expression in ESCC cells transfected with non-silencing or ANXA2-specific siRNA. GAPDH was used as the loading control. b ESCC cells stably expressing ANXA2-shRNA were transfected with pcDNA3.1-ANXA2-Y23A, pcDNA3.1-ANXA2-Y23D, or empty vector. Cell lysates were immunoblotted for the indicated proteins. c Real-time RT-PCR measurement of HIF1A mRNA levels in ESCC cells transiently transfected with MYC-specific siRNA or non-silencing siRNA. d Schematic diagram of MYC binding sites in the human HIF1A promoter and the primers used in the ChIP assay (top). The numbers listed for the ANXA2 promoter region indicate the nucleotide position relative to the transcriptional initiation site. ChIP assay in KYSE30 and KYSE150 cells (bottom). e-f HIF1A reporter activities were determined with luciferase reporter assay. e ESCC cells were co-transfected with pGL3-HIF1A and ANXA2 siRNA or MYC siRNA. f ESCC cells were transfected with pGL3-Basic, pGL3-HIF1A or pGL3-HIF1A-Del construct. g ESCC cells stably expressing ANXA2-shRNA were transfected with pcDNA3.1-MYC or empty vector. Cell migration and invasion abilities were examined with transwell assays. Representative results (left) and statistical plots (right) are shown. **, P < 0.01; ***, P < 0.001