Figure 2: REST directly targets and inhibits MAOA promoter. | Scientific Reports

Figure 2: REST directly targets and inhibits MAOA promoter.

From: MAOA-a novel decision maker of apoptosis and autophagy in hormone refractory neuroendocrine prostate cancer cells

Figure 2

(A) Schematic representation of REST binding site (RE-1 site) identified by ChIP-seq analysis and JASPAR prediction (upper panel) in the promoter region of MAOA. The locations of qPCR primers (short bars) are indicated. ChIP was performed using anti-REST antibody and chromatin prepared from LNCaP cells. REST binding to MAOA promoter region was analyzed by real-time qPCR. Data represent mean ± S.D. (n = 3); ***p < 0.001 by Student’s-t test. (B) LNCaP cells were cultured in phenol-red free medium supplied with 10% FBS, 10% or 2.5% CDT for 72 and 96 hours. ChIP-qPCR assay shows that REST binding on MAOA promoter, and this binding is reduced by CDT treatment. The enrichment of amplicon was normalized to input. Data represent mean ± S.D. (n = 3); ***p < 0.001 by Student’s-t test. (C) Luciferase reporter assays were performed by transfecting LNCaP cells with a reporter construct containing the MAOA promoter. At 24 hours post-transfection, cells were incubated with 10% or 2.5% CDT for 96 hours and the luciferase activities were measured. Data represent mean ± S.D. (n = 3); *p < 0.05, ***p < 0.001 by Student’s-t test. (D) Immunoblotting of REST and MAOA in LNCaP cells treated as described in (C). β-actin was used as loading control. Uncropped images are presented in Supplementary Figure S10A. (E) The mRNA level of MAOA in LNCaP-TR-REST cells treated as described in (C) for 96 hours in the presence ( + ) or absence (−) of Dox was analyzed by RT-qPCR and normalized with β-actin. ***p < 0.001 by Student’s-t test. (F) Immunoblotting of REST and MAOA in cells treated as in (F). β-actin was used as loading control. Uncropped images are presented in Supplementary Figure S10B. (G and H) Immunohistochemistry (IHC) analysis of REST and MAOA in six primary and six relapsed PCa specimens. Images are representative (magnification, x40) (G) and quantification based on their H-score. Data are average with S.D. from 6 specimens in each group (H). *p < 0.05, **p < 0.01 by Student’s-t test.

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