Fig. 5.

Glutathione activates FoxO3 and induces pluripotency factor expression through inhibition of MEK1 activity by chelating copper. (A) MDA-MB-231 cells were treated with GSH-MEE or MnTMPyP for 72 h. The percentage of cells positive for MitoSox Red staining was determined by flow cytometry, and the results were normalized to vehicle control (C) (mean ± SEM; n = 3). **P < 0.01, ***P < 0.001 vs. GSH-MEE; ns, not significant. (B) MDA-MB-231 cells were treated with GSH-MEE or MnTMPyP for 72 h. Glutathione levels were measured, and the results were normalized to control (mean ± SEM; n = 3). ***P < 0.001 vs. control; ns, not significant. (C) Cytosolic and nuclear lysates were prepared from MDA-MB-231 cells treated with GSH-MEE or MnTMPyP, and immunoblot assays were performed. (D) MDA-MB-231 cells were treated with GSH-MEE or MnTMPyP, and immunoblot assays were performed. (E) MDA-MB-231 subclones were treated without or with GSH-MEE, and immunoblot assays were performed. (F and G) MDA-MB-231 cells were treated with 0.625 μM U0126 (+) or vehicle (-) for 72 h and immunoblot assays were performed (F), or the percentage of ALDH (+) cells was determined by flow cytometry (G; mean ± SEM; n = 3). ***P < 0.001 vs. control. (H) MDA-MB-231 cells were treated with 2 mM GSH-MEE, either alone or in combination with CuSO₄ for 72 h, and immunoblot assays were performed. (I) MDA-MB-231 cells were treated with GSH-MEE, either alone or in combination with CuSO₄, and the percentage of ALDH (+) cells was determined (mean ± SEM; n = 3). ***P < 0.001 vs. control; ^###P < 0.001 vs. GSH-MEE alone. (J) MDA-MB-231 cells were treated with tetrathiomolybdate (TTM), paclitaxel (Pac), or GSH-MEE (GSH) for 72 h, and immunoblot assays were performed. (K) MDA-MB-231 cells were treated with trientine for 72 h, and immunoblot assays were performed. (L) MDA-MB-231 cells were treated with TTM, trientine, or GSH-MEE, and the percentage of ALDH (+) cells was determined (mean ± SEM; n = 3). ***P < 0.001 vs. control.