HIF-1α and heme are responsible for BACH2 repression under hypoxia. (A) 293T cells were transfected with truncated promoter plasmids with or without HIF-1α expression plasmids (left) or in the presence or absence of CoCl₂ (24 hours prior to harvest) (right). An empty pGL2-basic plasmid was used as a negative control. The Renilla luciferase reporter pRL-SV40 was used as an internal control for normalization. Luciferase activity was measured 48 hours after transfection. The results are presented as the relative luciferase activity compared with the cells without HIF-1α expression or without CoCl₂ treatment. (B) 293T cells were transfected with truncated promoter plasmids. During a 48-hour transfection, cells were cultured at 1% O₂ for 4 hours with or without HIF-1α inhibitor KC7F2 (40 μmol/L), and luciferase activity was measured. Relative luciferase activity compared with that of cells cultured in normal conditions or with dimethyl sulfoxide (DMSO) (control) are indicated. (C) DNA fragments (a-g) amplified by PCR following ChIP assays are indicated (top). SP53 cells were cultured at 1% O₂ for 4 hours. Chromatin was immunoprecipitated with antibodies against HIF-1α or control immunoglobulin G (IgG). Total chromatin before immunoprecipitation (input) was used as positive control for PCR. Samples processed under normoxia were used as negative controls. Further quantitative analyses of the ChIP assays in SP53 cells following hypoxia were analyzed using qRT-PCR and are presented as the mean ± SD values from 2 independent experiments (bottom). (D) Intracellular heme content was measured in MCL cells cultured under hypoxia at the indicated time. Each condition was run in duplicate with the values normalized to the controls (at 0 hours). (E) HO-1 protein levels were detected by immunoblotting using MCL cells cultured under hypoxia at the indicated time, with GAPDH as a loading control. HO-1/GAPDH values in each lane are indicated. (F) Intracellular heme levels of CD45⁺ human cells in subcutaneous tumors, SP, or BM from xenograft mice bearing MCL cells (n = 2). Values normalized to subcutaneous tumors are indicated. (G) CD19⁺ B cells isolated from PB, SP, or BM samples (n = 6) were used to measure the cellular heme levels. The values normalized to PB are indicated. NS, not significant; *P < .05; **P < .01 (vs control cells; Student t test).