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

Fig. 2.

Fig. 2.

Paclitaxel-induced BCSC enrichment is blocked by inhibition of glutathione synthesis. (A) MDA-MB-231 cells were sorted into aldehyde dehydrogenase negative [ALDH (-)] and positive [ALDH (+)] populations by flow cytometry. RT-qPCR was performed to analyze the expression of xCT (SLC7A11) and GCLM mRNA, and the results were normalized to ALDH (-) cells (mean ± SEM; n = 3). **P < 0.01 vs. ALDH (-). (B) MDA-MB-231 (Upper) and SUM-149 (Lower) cells were cultured on ultra-low adherence plates for 7 d to generate primary mammospheres (1° sphere). Half of the mammospheres were harvested for RT-qPCR analyses, and the other half of the mammospheres were dissociated and cultured for 7 d to generate secondary mammospheres (2° sphere), which were harvested for RT-qPCR analyses. Results were normalized to MDA-MB-231 and SUM-149 adherent cells (mean ± SEM; n = 3). *P < 0.05, **P < 0.01, ***P < 0.001 vs. adherent cells. (C and D) MDA-MB-231 subclones were treated without or with 10 nM paclitaxel (Pac) for 72 h. The number of 1° and 2° mammospheres per 1,000 cells initially seeded was calculated (mean ± SEM; n = 3). *P < 0.05, **P < 0.01, ***P < 0.001 vs. NTC Pac (-); ###P < 0.001 vs. NTC Pac (+). (E) MDA-MB-231 subclones were treated without or with 10 nM Pac for 4 d, and the percentage of ALDH (+) cells was determined (mean ± SEM; n = 3). *P < 0.05, **P < 0.01, ***P < 0.001 vs. NTC Pac (-); ###P < 0.001 vs. NTC Pac (+). (F and G) SUM-149 cells were treated without or with 5 nM Pac for 72 h, in combination with vehicle (V), 100 nM digoxin (Dig), 100 μM sulfasalazine (SSA), or 100 μM buthionine sulphoximine (BSO). Primary and secondary mammospheres were counted (mean ± SEM; n = 3). **P < 0.01, ***P < 0.001 vs. vehicle; ###P < 0.001 vs. Pac. (H) SUM-149 cells were treated with V, Dig, SSA, BSO, or Pac alone, or Pac in combination with Dig (P + D), SSA (P + S), or BSO (P + B) for 4 d, and the percentage of ALDH (+) cells was determined (mean ± SEM; n = 3). ***P < 0.001 vs. V; ###P < 0.001 vs. Pac. (I) MDA-MB-231 subclones were treated without or with 2 mM glutathione monoethyl ester (GSH-MEE) for 4 d and the percentage of ALDH (+) cells was determined (mean ± SEM; n = 3). **P < 0.01, ***P < 0.001 vs. GSH-MEE (-) in NTC group; ###P < 0.001 vs. GSH-MEE (-) in the same subclone. (Scale bars: 200 μm.)