Extended Data Figure 7: Overexpression of constitutively activated HIF1α rescues XBP1 knockdown phenotype.
From: XBP1 promotes triple-negative breast cancer by controlling the HIF1α pathway
a, Immunoblotting analysis of cell lysates of MDA-MB-231 cells infected with retrovirus encoding control vector or HA–HIF1α dPA was performed using anti-HA or anti-actin antibody. b, XBP1 splicing is not affected by HIF1α or HIF2α activation. RT–PCR analysis of the ratio of XBP1s to total XBP1 in MDA-MB-231 cells expressing control vector, HA–HIF1α dPA or HA–HIF2α dPA. Expression of HA–HIF1α dPA is shown in a and expression of HIF2α is shown in the right panel. c, XBP1 splicing is not affected by HIF1α or HIF2α depletion. RT–PCR analysis of the ratio of XBP1s to total XBP1 in MDA-MB-231 cells infected with control, shHIF1α or shHIF2α lentivirus. Knockdown efficiency of HIF1α or HIF2α is shown in the middle and right panels. d, e, Expression of constitutively activated HIF1α doesn’t affect XBP1 expression (d), but restores HIF1α targets expression (e). RT–PCR analysis of XBP1 total (d), XBP1s (d) and VEGFA, PDK1, DDIT4 (e) in control shRNA (shCtrl), XBP1 shRNA (shXBP1), or XBP1 shRNA plus constitutively activated HIF1α (shXBP1+HIF1α dPA) infected MDA-MB-231 cells. Data (b–e) are shown as mean ± s.d. of technical triplicates. *P < 0.05, **P < 0.01. f, Quantification of soft agar colony formation in control shRNA (shCtrl), XBP1 shRNA (shXBP1), or XBP1 shRNA plus constitutively activated HIF1α (shXBP1 + HIF1α dPA) infected MDA-MB-231, MDA-MB-468, or MDA-MB-157 cells. Experiments were performed in triplicate and data are shown as mean ± s.d. **P < 0.01. g, Quantification of mammosphere formation in control shRNA (shCtrl), XBP1 shRNA (shXBP1), or XBP1 shRNA plus constitutively activated HIF1α (shXBP1+HIF1α dPA) infected MDA-MB-231 cells. Experiments were performed in triplicate and data are shown as mean ± s.d. **P < 0.01. h, CD31 immunostaining of tumours formed by MDA-MB-231 cells infected with control shRNA (shCtrl), XBP1 shRNA (shXBP1) or XBP1 shRNA plus constitutively activated HIF1α (shXBP1+HIF1α dPA). i, Silencing of HIF1α inhibits the XBP1s-sustained mammosphere-forming ability. Quantification of mammosphere formation in MCF10A cells expressing control vector, XBP1s, XBP1s plus control shRNA or XBP1s plus HIF1α shRNA. Experiments were performed in triplicate and data are shown as mean ± s.d. **P < 0.01.
