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. 2015 Jul 30;112(33):E4600–E4609. doi: 10.1073/pnas.1513433112

Fig. 3.

Fig. 3.

Paclitaxel-induced expression of pluripotency factors mediates BCSC enrichment. (A) MDA-MB-231 cells were sorted into ALDH (-) and ALDH (+) populations by flow cytometry. RT-qPCR was performed to analyze the expression of Nanog and Sox2 mRNA, and the results were normalized to ALDH (-) cells (mean ± SEM; n = 3). **P < 0.01 vs. ALDH (-). (B) Primary and secondary mammospheres from MDA-MB-231 (Upper) and SUM-149 (Lower) cells were harvested for RT-qPCR analyses. The results were normalized to adherent cells (mean ± SEM; n = 3). *P < 0.05, **P < 0.01, ***P < 0.001 vs. adherent. (C) MDA-MB-231 subclones were treated without or with 10 nM paclitaxel (Pac) for 72 h, and immunoblot assays were performed. (D) SUM-149 cells were treated with vehicle (V), 5 nM paclitaxel (Pac), 100 nM digoxin (Dig), 100 μM sulfasalazine (SSA), 100 μM buthionine sulphoximine (BSO), or Pac combined with Dig (P + D), SSA (P + S), or BSO (P + B) for 72 h, and immunoblot assays were performed. (E) MDA-MB-231 subclones were treated with 2 μM β-mercaptoethanol (β-ME) or 2 mM glutathione monoethyl ester (GSH) for 72 h and immunoblot assays were performed. (F) SUM-149 cells were treated with Dig, SSA, or BSO, in combination with vehicle, β-ME, or GSH for 72 h, and immunoblot assays were performed. (G and H) MDA-MB-231 subclones stably transfected with NTC or Nanog shRNA vector were treated without or with Pac (G), and without or with glutathione monoethyl ester (GSH-MEE) (H), and the percentage of ALDH (+) cells was determined (mean ± SEM; n = 3). ***P < 0.001 vs. NTC Pac (-) or GSH-MEE (-); ###P < 0.001 vs. NTC Pac (+) or GSH-MEE (+). (I) Kaplan–Meier analysis of relapse-free survival (RFS) was performed based on clinical and molecular data from 211 ER (-) breast cancer patients who received chemotherapy. The patients were stratified according to Nanog mRNA levels in the primary tumor. High Nanog, Nanog mRNA levels greater than the median; low Nanog, Nanog mRNA levels less than the median. The P value (log-rank test) and hazard ratio (HR) are shown.