Fig 4. eNEMAL is upregulated under hypoxia in breast cancer cells.

(A) eRNA expression determined by RT-qPCR following in cells grown under normoxic (21% O₂) and hypoxic (1% O₂) conditions for 24 hours, normalized to MCF10A normoxic expression. MCF10A, non-tumorigenic immortalized; MDA-MB-157 and MDA-MB-231, triple-negative; MCF7 and T47D, luminal A; BT474 and MDA-MB-361, triple positive; SKBR3 and MDA-MB-453, HER2-enriched. (B) Hypoxic response elements (HREs) in the eNEMAL promoter. (C) MCF7 cells were transfected with siRNA targeting HIF1A or HIF2A for 48 hours then maintained under hypoxia for additional 24 hours. The expression levels of HIF1A, HIF2A and eNEMAL were determined by qPCR. Note that we observed HIF2A upregulation upon HIF1A siRNA transfection, which is consistent with the previous finding of a repressive role of HIF1A on HIF2A expression [20]. Data shown as mean ± SD, n = 3, * P < 0.05, ** P < 0.01, *** P < 0.001.