Figure 4. Blimp1 is regulated by hypoxia/Hif1 in murine and human pancreatic cancer cells.

(A) The GFP^pos PDAC cell state is enriched for hypoxia-induced genes and Hif-targets.

(B) Enrichment for the Hif1-binding site in the promoters of genes up-regulated in the GFP^pos cell state.

(C) Significant enrichment for hypoxia-regulated genes in GFP^pos cells. ES, enrichment score; FC, fold change.

(D) Volcano plot of the gene expression differences between GFP^pos and GFP^neg cancer cell populations. Fold change and adjusted p-values were generated by taking paired samples into consideration. Red dots denote annotated Hif1 target genes.

(E,F) Hypoxia (0.5% O₂ for 24 hours) induces Blimp1 expression in two mouse PDAC cell lines (E) and four human PDAC cell lines (F). *, p-value < 0.05; **, p-value < 0.005; ***, p-value < 0.0005 by Student's t test. Mean +/- SD of triplicate wells in shown.

(G,H) Knockdown of Hif1a in the murine 688M cells (G) and HIF1a in two human PDAC cell lines (H) reduces hypoxia-induced Blimp1 and BLIMP1 upregulation, respectively. Mean +/- SD of triplicate wells in shown. *, p-value < 0.05; **, p-value < 0.005 by Student's t test.

(I) Expression of stabilized HIF1α increases BLIMP1 expression in two human PDAC cell lines. *, p-value < 0.05; **, p-value < 0.0005 by Student's t test. Mean +/- SD of triplicate wells in shown.

(J) Chromatin immunoprecipitation (ChIP) qPCR-quantified Hif1α binding at an HRE-containing region 240 kb upstream of the Blimp1 TSS (site6). 688M cells were cultured under normoxia or hypoxia before ChIP. Six HRE were quantified for enrichment of Hif1a binding by qPCR. An HRE region in the Vegfa locus is a positive control for hypoxia-induced Hif1α binding. *, p-value < 0.05; **, p-value < 0.005 by Student's t test.

(K) The wildtype site6 (WT-HRE), but not site6 with all three HREs mutated (mut-HRE), conferred hypoxia responsiveness on a SV40 promoter or a 1.6kb Blimp1 promoter. Representative results of 688M cells transfected with indicated reporters cultured under normoxia or hypoxia. Means +/- SD of triplicated ratios of firefly luciferase normalized to co-transfected Renilla luciferase reporter are shown.