Fig. 1.

Expression of CNN2 and SDK1 is androgen regulated in an SRF-dependent manner. (A) LNCaP cells were treated with 5nM of the synthetic androgen R1881 (+) or ethanol vehicle (−) for 48 h. Cells were harvested and protein and RNA were isolated. RNA was converted into cDNA and real-time RT–PCR was done using primers that target CNN2 and SDK1 expression. Expression values were normalized to glyceraldehyde 3-phosphate dehydrogenase expression levels and expressed as relative expression, where the value obtained from one of the vehicle-treated samples was taken as 1. Black columns, vehicle-treated samples; gray columns, R1881-treated samples. Columns, means of values obtained from three independent biological replicates; bars, standard error of the mean values (top panel). Western blotting was performed using antibodies directed against CNN2 and SDK1. Blots were reprobed for β-actin (β-act) to control for loading differences (bottom panel). (B) LNCaP cells were transfected with siRNA directed against AR or a non-targeting control (c) siRNA. One day later, medium was replaced by CSS-supplemented medium. The next day, medium was replaced and cells were treated with vehicle or R1881 (5nM). After 48 h, cells were harvested for RNA and protein isolation. Real-time RT–PCR using primers directed against CNN2 and SDK1 was performed as described (left panel). Western blotting was performed with antibodies directed against CNN2, SDK1, AR and β-actin (β-act) (right panel). (C) LNCaP cells were treated with ethanol vehicle, R1881 (1nM), Casodex (10 μM, csdx) or R1881 and an excess of Casodex for 48 h. Cells were harvested for RNA, and cDNA synthesis followed by real-time RT–PCR was performed (left panel). LNCaP cells were treated with R1881 or Casodex at various concentrations either alone or in combination, and whole-cell protein extracts were subjected to western blotting using antibodies directed against CNN2 or SDK1 as above (right panel). (D) LNCaP cells were treated with 5nM R1881 or vehicle and harvested for RNA isolation 4 or 16 h later. Real-time RT–PCR was performed using primers directed against CNN2, SDK1 orprostate-specific antigen as above (top panel). LNCaP cells were treated with vehicle or R1881 (5 nM) and harvested after 0, 4, 8, 16, 24, 48 and 72 h. Western blot analysis was performed using antibodies directed against CNN2, SDK1 and β-actin (β-act) (bottom panel). (E) LNCaP cells were transfected with non-targeting siRNAs or siRNAs directed against SRF. One day later, medium was replaced by CSS-supplemented medium. The next day, medium was replaced and cells were treated with vehicle or R1881 (5nM). After 48 h, cells were harvested for protein and RNA isolation, and western blotting (right panel) and real-time RT–PCR (left panel) were done to evaluate CNN2 and SDK1 expression levels. (F) LNCaP cells were treated with either vehicle or R1881 (5nM) for 16 h. ChIP assays were performed as before using an antibody directed against SRF or non-targeting IgG. CNN2, CArG box containing CNN2 promoter fragment; control, similarly sized non-CArG box-containing exonic CNN2 gene fragment. (G) Graphical representation of the structure of the CNN2 promoter-reporter constructs. CNN2wt-luc, wild-type construct containing a 982 bp CNN2 promoter fragment that harbors a CArG box 133 base pairs upstream of the transcriptional start site; CNN2mut-luc, mutant construct in which the CArG box has been mutated. (H) LNCaP cells were transfected with CNN2wt-luc or CNN2mut-luc and treated with vehicle, R1881 and/or Casodex (csdx) for 48 h (left panel and right panel), with vehicle or R1881 in the presence of non-targeting siRNA or SRF-directed siRNA (middle panel). The next day, cells were treated with ethanol vehicle or R1881 (5nM). After 48 h, a luciferase assay was done. Columns, means of values obtained from three independent biological replicates; bars, standard error of the mean.