GW9662 treatment affects lipogenic and stem cell-related gene regulation in BT474 cells. (a) The expression of various lipogenic genes in BT474 cells with or without GW9662 treatment was examined by qRT–PCR. GAPDH gene was used as an internal control. Error bars indicate three individual experiments; *P<0.05, **P<0.001. (b) ALDH-positive or negative cells were isolated from BT474 cells; RNA was extracted and qRT–PCR analysis performed to examine the expression levels of various lipogenic genes (*P<0.05). (c) The expression of ALDH and KLF4 gene in BT474 cells. BT474 cells were treated with vehicle and GW9662 for 9 days, and qRT–PCR was performed to examine the expression level of ALDH and KLF4 mRNAs. GAPDH expression was used as an internal control (*P<0.05). BT474 cells were treated with vehicle, GW9662, rosiglitazone and C75. After 9 days, cell lysates were used for immunoblotting to examine the expression of ALDH and KLF4, GAPDH was used as loading control (bottom). (d) Effects of GW9662 on histone acetylation. BT474 or MCF-7 cells were treated with GW9662 for different times as indicated. Immunoblot analysis of total protein extracts from cells was conducted using anti-Ac-H3, lys9-H3, H3, Ac-H4 and H4 antibodies.