Extended Data Figure 2. Effect of XBP1 on tumor relapse and CD44^high/CD24^low cells.

a, Tumor growth of MDA-MB-231 cells untreated or treated with paclitaxel (20mg/kg), or paclitaxel + control shRNA, or paclitaxel + XBP1 shRNA in nude mice. TX: treatment with paclitaxel or paclitaxel+ shRNA. Data are shown as mean ± SD of biological replicates (n=5). b, Number of mammospheres per 1,000 cells generated from day 20 xenograft tumors under different treatments as indicated under normoxic or hypoxic condition (0.1% O₂). Data are shown as mean ± SD of biological replicates (n=3). * denotes significantly different from paclitaxel+shCtrl control in each treatment, *p<0.05, **p<0.01. c, Effect of XBP1 knockdown on cell death in hypoxic regions (assessed by CA9 and cleaved caspase3 staining) or accumulation of CD44^high/CD24^low cells (assessed by CD44 staining). Immunohistochemical staining of CA9 and Cleaved Caspase 3 (consecutive sections) showed that cell death was not induced in hypoxic region in XBP1 knockdown tumors. Immunohistochemical staining of CD44 showed significant reduction of CD44 expression in XBP1 knockdown tumors. All tumor sections are from MDA-MB-231 derived xenograft with different treatment as indicated. d, Knockdown efficiency of XBP1 in MCF10A-ER-Src cells. Data are shown as mean ± SD of technical triplicates. e, Left panel: Flow cytometry analysis of CD44 and CD24 expression of TAM treated (36h) MCF10A ER-Src cells infected with control shRNA or XBP1 shRNA encoding lentivirus. Right panel: Percentage of CD44^high/CD24^low cells in TAM (4-OH-tamoxifen) treated MCF10A-ER-Src cells infected with control shRNA or XBP1 shRNA encoding lentivirus. Experiments were performed in triplicate and data are shown as mean ± SD. *p<0.05. f, Number of mammospheres per 1,000 cells generated by TAM treated MCF10A ER-Src cells uninfected, or infected with control shRNA or XBP1 shRNA encoding lentivirus. Experiments were performed in triplicate and data are shown as mean ± SD. g, Cell viability assay (Cell-titer Glo) on CD44^high/CD24^low or CD44^low/CD24^high cells isolated from TAM treated MCF10A-ER-Src cells infected with control shRNA or XBP1 shRNA encoding lentivirus (72h after infection). Data were normalized to the control (cell infected with shCtrl). Experiments were performed in triplicate and data are shown as mean ± SD. h, Cleaved caspase 3 ELISA assays on CD44^high/CD24^low or CD44^low/CD24^high cells isolated from TAM treated MCF10A-ER-Src cells infected with control shRNA or XBP1 shRNA encoding lentivirus (72h after infection). Experiments were performed in triplicate and data are shown as mean ± SD.