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. Author manuscript; available in PMC: 2016 Mar 1.
Published in final edited form as: Biochim Biophys Acta. 2015 Jan 21;1849(3):317–327. doi: 10.1016/j.bbagrm.2015.01.003

Figure 8.

Figure 8

Figure 8

Effects of CREB and the CREB co-activator CRTC2 on BCRP mRNA and protein expression in human LNCaP prostate cancer cells. (A) p-AKT, p-CREB and BCRP protein expression in LNCaP cells cultured in medium containing either 10% FBS or 10% charcoal stripped (CSS) medium for 7 days, as determined by Western blot. A Western blot for actin is used as loading control. (B) BCRP mRNA expression in LNCaP or LNCaP/CSS cells (LNCaP cells were cultured in medium containing 10% CSS for 7 days) as measured by real time qPCR, normalized for expression of β-actin mRNA. (C) Distribution of CRTC2 between cytosol and nucleus analyzed by Western blot, as described in Methods. (D-F) Effects of CREB or CRTC2 silencing on CREB, CRTC2 and BCRP mRNA expression in LNCaP cells. Twenty four hours after transfection of LNCaP cells with CREB siRNA, CRTC2 siRNA, or negative control siRNA were cultured in medium containing 10% FBS or 10% CSS respectively for 7 days, then total RNA was isolated and mRNA expression was determined by real-time qPCR for CREB (D), CRTC2 (E) or BCRP (F); the mRNA expression data shown were normalized for the mRNA expression by the normal control (NC) siRNA transfected cells, which was given a value of 1.0. The data shown are the mean and standard deviation of 3 different experiments, done on different days. Each individual assay was run in duplicate. *, P< 0.05 compared to untreated control group. a, P< 0.05 compared to negative control siRNA transfected FBS group. b, P< 0.05 compared to control siRNA transfected CSS group. (G) Effects of CREB or CRTC2 silencing on BCRP protein expression in LNCaP cells. LNCaP cells in complete medium were transfected with CREB siRNA or CRTC2 siRNA, or negative control siRNA as described in Materials and Methods; after 72 hours, cells were lysed in RIPA buffer and CREB, CRTC2 and BCRP protein expression was determined by Western blot. A Western blot for actin is used as loading control. The Western blot results shown are representative of the results of three independent replicate experiments, done on different days.