Extended Data Figure 4: HIF1α is a co-regulator of XBP1.
From: XBP1 promotes triple-negative breast cancer by controlling the HIF1α pathway
a, Identification of XBP1 motif in MDA-MB-231 ChIP-seq data set. Matrices predicted by the de novo motif-discovery algorithm Seqpos. P < 1.08 × 10−30. b, Induction of XBP1 splicing by hypoxia. RT–PCR analysis of the ratio of XBP1s to total XBP1 in MDA-MB-231 cells cultured under normoxic condition or hypoxic condition (0.1% O2) for 24 h. Data are shown as mean ± s.d. of technical triplicates. **P < 0.01. c, Induction of XBP1 splicing by glucose deprivation. RT–PCR analysis of the ratio of XBP1s to total XBP1 in MDA-MB-231 cells cultured in normal medium or glucose-free medium for 24 h or 48 h. Data are shown as mean ± s.d. of technical triplicates. *P < 0.05. d, Induction of XBP1 splicing by oxidative stress. RT–PCR analysis of the ratio of XBP1s to total XBP1 in MDA-MB-231 cells untreated or treated with different doses of H2O2 for 1 h, 4 h or 24 h. Data are shown as mean ± s.d. of technical triplicates. e, Western blotting analysis of XBP1s expression in nuclear extract of MDA-MB-231 cells cultured under normoxia or hypoxia (0.1% O2) and glucose-free condition for 16 h. Lamin B was used as loading control. f, Venn diagram showing the overlap between XBP1 targets in MDA-MB-231 breast cancer cells cultured under normoxic conditions (untreated) or hypoxia and glucose deprivation conditions (treated). g, Motif enrichment analysis in the XBP1 binding sites in untreated or stressed (0.1% O2 and glucose deprivation: HG) MDA-MB-231, Hs578T or T47D cells. The 1-kb region surrounding the summit of the XBP1 peak is equally divided into 50 bins. The average HIF1α motif occurrence over top 1,000 XBP1 peaks in each bin is plotted. The corresponding P values of each condition are listed as follows: M231_Un, 7.78 × 10−20; M231_HG, <1.08 × 10−30; Hs578T_HG, <1.08 × 10−30; T47D_HG, 6.14 × 10−6. h, Flag-tagged HIF1α and XBP1s were co-expressed in 293T cells, and the cells were treated in 0.1% O2 for 16 h. Co-immunoprecipitation was performed with M2 anti-Flag antibody. Western blot was carried out with anti-XBP1s antibody, anti-Flag antibody or anti-HIF1α antibody. Empty vector was used as control. i, Nuclear extracts from Hs578T cells treated with tunicamycin (1 μg ml−1, 6 h) in 0.1% O2 (16 h) were subjected to co-immunoprecipitation with anti-HIF1α antibody or rabbit IgG. Western blot was carried out with anti-XBP1s antibody or anti-HIF1α antibody. j, HA-tagged HIF2α and XBP1s were co-expressed in 293T cells, and the cells were treated in 0.1% O2 for 16 h. Co-immunoprecipitation was performed with anti-HA antibody. Western blot was carried out with anti-XBP1s antibody or anti-HA antibody. Empty vector was used as control. k, Localization of XBP1s and HIF1α in MDA-MB-231 cells. Western blotting analysis of XBP1s and HIF1α expression in cytosolic extracts and nuclear extracts of MDA-MB-231 cells cultured under 0.1% O2 condition for 24 h. HSP90 and TBP were used as control. l, XBP1u is not expressed in MDA-MB-231 cells. Western blotting analysis of XBP1u in MDA-MB-231 cells untreated or treated with 1 μM or 10 μM MG132 for 4 h. HSP90 was used as loading control. m, Schematic diagram of full-length and truncated forms of XBP1s protein. n, A GST pull-down assay was performed using GST-tagged XBP1s proteins and 293T cell lysates overexpressing HA-tagged HIF1α. Western blotting was performed with an anti-HA antibody. Lower panel: Coomassie blue staining of GST-tagged different truncated forms of XBP1s proteins.
