Fig. 6.

CALR is a direct HIF-1 target gene. (A) A candidate palindromic HIF-1 binding site was identified 227 bp 5′ to the transcription start site of the human CALR gene. (B) HCC1954 cells were exposed to 20 or 1% O₂ for 24 h, and ChIP assays were performed using IgG or antibody against HIF-1α or HIF-1β (mean ± SEM, n = 3; *P < 0.05 vs. 20% O₂; **P < 0.01 vs. 20% O₂). (C) HCC1954 cells were exposed to 20 or 1% O₂ for 24 h, and ChIP assays were performed using IgG or antibody against HIF-2α or HIF-1β (mean ± SEM, n = 3; ***P < 0.001 vs. 20% O₂). n.s., not significant. (D) HCC1954 and SUM159 cells were transfected with reporter plasmid pEZX-HRE, which contains a 55-bp oligonucleotide encompassing the putative hypoxia response element (HRE), and exposed to 20 or 1% O₂ for 24 h, and luciferase activity in cell lysates was determined (mean ± SEM, n = 3; *P < 0.05 vs. 20% O₂). (E) NTC and DKD subclones of HCC1954 were transfected with pEZX-HRE, exposed to 20 or 1% O₂ for 24 h, and luciferase activity was determined (mean ± SEM, n = 3, **P < 0.01 vs. NTC at 20% O₂; ^##P < 0.01 vs. NTC at 1% O₂).