Figure 3.

CTBP1-AS promotes tumour growth by activating AR signalling. (A) Immunoprecipitation by anti-CTBP1 antibody. LNCaP cells were treated with 10 nM R1881 or vehicle for 6 h. Lysates were immunoprecipitated by anti-CTBP1 antibody. The arrowhead indicates IgG heavy chain. (B) ChIP analysis of CTBP1 in ARBSs. Cells were treated with 10 nM R1881 for 1 h. Normalized to IgG-IP control. GAPDH, MB (myoglobin) locus: negative controls. (C) Western blot analysis of LNCaP cells transfected with siControl or siCTBP1. (D) PSA-LUC assay in LNCaP cells transfected with siControl or siCTBP1. (E) LNCaP cells transfected with siControl or siCTBP1 (10 or 50 nM) were treated with 10 nM R1881 for 4 h. Fold induction is shown. mRNA levels were measured by qRT–PCR. (F) MTS assay of LNCaP cells transfected with siCTBP1 (n=4). (G) CTBP1 knockdown accelerates cell proliferation. The numbers of LNCaP cells transfected with siControl or siCTBP1 were counted 3 days after transfection. (H) Cell proliferation assay of LNCaP cells ransfected with siControl or siCTBP1-AS (n=4). The number of viable cells is counted and shown. (I) MTS assay of LNCaP cells transfected with siControl or siCTBP1-AS (n=4). *P<0.05. (J, left) Tumour-growth curves in nude mice inoculated with LNCaP cells transfected with siRNA *P<0.05 (n=7). (J, right) Representative view of LNCaP-derived tumours in nude mice administered siRNA. (K) CTBP1-AS knockdown in tumours transfected with siCTBP1-AS (n=4). *P<0.05. (L) CTBP1 protein expression in tumours transfected with siCTBP1-AS. (M) PSA-LUC assay in LNCaP cells transfected with siControl or siCTBP1-AS (n=3). (N, left) Effect of siCTBP1-AS on androgen-upregulated gene expression (>2.0-fold change by R1881 versus vehicle, 198 genes) in LNCaP cells. (N, right) qRT–PCR analysis of androgen-regulated genes. At 2 days before androgen stimulation, siControl or siCTBP1-AS was transfected to LNCaP cells. LNCaP cells were treated with 10 nM R1881 or vehicle for 24 h. Bar: s.d.

Source data for this figure is available on the online supplementary information page.

Source Data for Figure 3 ^{(359.3KB, pdf)}