Androgen-induced cellular senescence is mediated through the tumor suppressors p16-pRb. To examine the signaling pathways involved in the induction of cellular senescence, Western blotting, 3D-FISH of interphase nuclei, qRT-PCRs and transient transfections with siRNA were performed. LNCaP cells were incubated for 72 h with solvent control or different R1881 concentrations (1 pM = LAL; 1 nM = SAL). C, solvent control (DMSO). A. qRT-PCR was performed to analyze mRNA expression of p14. Gene expression was normalized to β-actin and the values for untreated samples (C). Error bars indicate the standard deviation of the mean of doublets. B. Protein levels of p53 as a target of p14 were detected with immunoblotting using antibodies against p53 and acetylated p53 (ac p53) known to be involved in cellular senescence pathway. Quantification of the bands was realized via Labimage D1 and the expression levels of the target proteins were normalized and given as band intensity to the loading control α-tubulin, untreated sample. C. Changes of the p16-pRb pathway by androgen treatment were analyzed by Western blotting. D-G) Quantitative qRT-PCR was performed to analyze mRNA expression with primers directed against indicated mRNAs. Gene expression was normalized to β-actin. Error bars indicate the standard deviation of the mean of doublets. C: solvent control (DMSO). H. Transient transfection of small interference RNA (siRNA) and subsequent SA β-gal assays were used to analyze the effects of p16 on the androgen-induced senescence. LNCaP cells were transfected via electroporation with srambled control (SCR) siRNA as negative control or p16 siRNA and without any siRNA (-) 24 h prior R1881 treatment. Thereafter, cells were fixed and analyzed for SA β-Gal activity using a light microscopy at 200x magnification and 3× 200 cells were counted and as means of the triplets in percent diagramed.