BACH2 modulates HIF-1α degradation by suppressing PHD3 under normoxia. (A) mRNA (top) and protein (bottom) levels of PHD3 were measured using qRT-PCR and immunoblots in BACH2^KO or BACH2^Con MCL cells. Each qRT-PCR value was normalized to GAPDH and represents the mean ± SD. GAPDH was used as a loading control for immunoblots. (B) Schematic of the experimental design (top). BACH2^Con or BACH2^KO SP53 MCL cells were treated with CoCl₂ (200 μmol/L) for 16 hours followed by removal of CoCl₂-containing medium and addition of cycloheximide (CHX; 50 μg/mL) or MG132 (5 μmol/L) at the indicated time. HIF-1α levels were determined by immunoblotting with GAPDH as a loading control (bottom). (C) PHD3 levels were measured in 293T cells transient transfected with BACH2 expression plasmids (pEGFP-BACH2) for 48 hours. The pEGFP-N1 empty vector was used as a control. GAPDH was used as a loading control. (D) 293T cells were transiently transfected with HIF-1α or BACH2 expression plasmids for 48 hours followed by addition of CHX (50 μg/mL) or MG132 (5 μmol/L) at the indicated time. The pEGFP-N1 empty vector was used as a control. HIF-1α stability was measured by immunoblotting with GAPDH as a loading control. (E) The knockout efficiency of CRISPR-Cas9–mediated deletion of PHD3 in SP53 MCL cells (left).The control cells were mock-transfected cells (PHD3^Con). PHD3^KO or PHD3^Con SP53 cells were treated with CoCl₂ (200 μmol/L) for 16 hours followed by removal of CoCl₂ and addition of CHX (50 μg/mL) at the indicated time. HIF-1α levels were determined by immunoblotting with GAPDH as a loading control. (F) The knockout efficiency of CRISPR-Cas9–mediated deletion of PHD3 in 293T cells (left). Manipulated 293T cells were transiently transfected with HIF-1α expression plasmids for 48 hours. Cells treated with CHX were harvested at the indicated times. HIF-1α levels were measured by immunoblotting with GAPDH as a loading control.