Figure 3.

AR knockdown suppresses the androgen-induced GNMT gene. (A) LNCaP cells were cultured in RPMI containing 10% DSS in the absence of antibiotics for 24 h. Cells were then transfected with siRNAs targeting AR or control. Twenty-four hours after transfection, cells were treated with 1 nM R1881 with an equal volume of ethanol being added to the vehicle control. RNA was prepared after a further 48 h and TaqMan RT-PCR for AR and GNMT was performed. Data have been normalised to GAPDH levels and AR or GNMT expression levels in the siRNA control treated with R1881 was set to one. Results are shown as mean values of three independent experiments performed in triplicates with error bars representing s.e.m. (B) LNCaP cells were transfected with siRNAs as for A. Whole cell lysates were prepared and immunoblotted for AR, GNMT (arrowed) and β-actin. Molecular weight markers are expressed in kDa. Suppression of the androgen-regulated genes PSA (C) and NDRG1 (D) following silencing of AR is also shown.