Extended Data Figure 6: XBP1 regulates HIF1α targets.
From: XBP1 promotes triple-negative breast cancer by controlling the HIF1α pathway
a, Plot from GSEA showing enrichment of the HIF1α-mediated hypoxia response pathway in XBP1-upregulated genes. b, Quantitative RT–PCR analysis of VEGFA, PDK1, GLUT1 and DDIT4 expression after knockdown of XBP1 in MDA-MB-231 under both normoxic or hypoxic conditions. c, Quantitative RT–PCR analysis of VEGFA, PDK1, GLUT1, MCT4, JMJD1A and XBP1 expression after knockdown of XBP1 in Hs578T cells treated with 0.1% O2 for 24 h. Results are presented relative to β-actin expression. Experiments were performed in triplicate and data are shown as mean ± s.d. *P < 0.05, **P < 0.01. d, Plot from GSEA showing no enrichment of the HIF1α-mediated hypoxia response pathway in XBP1-regulated genes in T47D cells (P = 0.1684). e–k, Cooperative binding of XBP1 and HIF1α on common targets. e, Immunoblotting analysis of control MDA-MB-231 cell lysates and XBP1 knockdown lysates (treated with 0.1% O2 for 24 h) were performed using anti-HIF1α or anti-HSP90 antibody. f, g, Chromatin extracts from control MDA-MB-231 cells or XBP1 knockdown MDA-MB-231 cells (treated with 0.1% O2 for 24 h) were subjected to ChIP using anti-XBP1s antibody (f), or anti-RNA polymerase II antibody (g). The primers used to detect ChIP-enriched DNA were the peak pair of primers in JMJD1A, DDIT4, NDRG1, PDK1 and VEGFA promoters (Supplementary Table 3). Primers in the β-actin region were used as control. h, i, Chromatin extracts from control MDA-MB-231 cells or HIF1α knockdown MDA-MB-231 cells (treated with 0.1% O2 for 24 h) were subjected to ChIP using anti-HIF1α antibody (h), or anti-RNA polymerase II antibody (i). j, k, Chromatin extracts from control MDA-MB-231 cells or XBP1 knockdown MDA-MB-231 cells (treated with 1% O2 for 24h) were subjected to ChIP using anti-HIF1α antibody (j), and anti-XBP1s antibody (k). All ChIP data (f–k) are shown as mean ± s.d. of technical triplicates. Results show a representative of two independent experiments. *P < 0.05; **P < 0.01. l, Quantitative RT–PCR analysis of VEGFA, PDK1, GLUT1 and JMJD1A after knockdown of XBP1 in MDA-MB-231 cells treated with 1% O2 for 24 h. Results are presented relative to β-actin expression. Data are shown as mean ± s.d. of technical triplicates. *P < 0.05, **P < 0.01.
