Fig. 4: Hypoxia-inducible factor (HIF)-1α binds directly to the glutaminase 1 (GLS1) gene in hypoxic cells. | Cell Death & Disease

Fig. 4: Hypoxia-inducible factor (HIF)-1α binds directly to the glutaminase 1 (GLS1) gene in hypoxic cells.

From: Glutaminase 1 expression in colorectal cancer cells is induced by hypoxia and required for tumor growth, invasion, and metastatic colonization

Fig. 4

a levels of GLS1 mRNA were analyzed by reverse transcription and quantitative real-time PCR (RT-qPCR) in Caco2 subclones (non-targeting control (NTC), sh1α-1, and sh1α-2), which exposed to 20 or 1% O2 for 24 h (mean ± SEM; n = 3). **p < 0.01, ***p < 0.001 vs. NTC at 20% O2; ###p < 0.001 vs. NTC at 1% O2 (ANOVA with Bonferroni post-test). b Immunoblot assays were conducted using lysates prepared from Caco2 subclones exposed to 20 or 1% O2 for 48 h. c HIF-binding sites in the 5′-flanking region of the human GLS1 gene were identified by ChIP as described below. GLS exons and hypoxia response element (HRE) are indicated by black bars and arrow. HRE nucleotide sequence is shown. d, e HT29 cells were exposed to 20 or 1% O2 for 16 h and ChIP assays were performed using IgG or antibodies against HIF-1α (d) and HIF-1β (e). Primers flanking the HRE were used for qPCR and results were normalized to lane 1 (mean ± SEM; n = 3). ***p < 0.001 vs. 20% O2 (analysis of variance (ANOVA) with Bonferroni post-test). f–g ChIP assays (HIF-1α and HIF-1β) for a known HIF-binding site in the PDK1 gene are shown as positive controls. ***p < 0.001 vs. 20% O2 (ANOVA with Bonferroni post-test)

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