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

Fig. 4.

Fig. 4.

Paclitaxel promotes nuclear translocation of FoxO3 to activate NANOG transcription. (A and B) MDA-MB-231 cells were treated without or with 2 mM GSH-MEE (A), or 10 nM paclitaxel (B), for 72 h. Cytosolic and nuclear lysates were prepared, and immunoblot assays were performed. (C and D) MDA-MB-231 subclones expressing NTC or a FoxO3 shRNA vector were treated without or with paclitaxel (C), or GSH-MEE (D) for 72 h, and immunoblot assays were performed. (E and F) MDA-MB-231 NTC and FoxO3 shRNA subclones were treated without or with paclitaxel (Pac, E), or GSH-MEE (F), and the percentage of ALDH (+) cells was determined (mean ± SEM; n = 3). **P < 0.01, ***P < 0.001 vs. NTC Pac (-) or GSH-MEE (-); ###P < 0.001 vs. NTC Pac (+) or GSH-MEE (+). (G) Chromatin immunoprecipitation (ChIP) was performed by using IgG or FoxO3 antibodies in NTC and FoxO3 shRNA subclones. qPCR was performed using primers that flanked the candidate binding site, and the results were normalized to NTC ChIP with IgG (mean ± SEM; n = 3). The nucleotide sequence of the FoxO3 binding site, which is located 3.6 kb 5′ to the NANOG transcription start site, is shown. ***P < 0.001 vs. NTC with IgG; ##P < 0.01, ###P < 0.001 vs. NTC with FoxO3 antibody.