Figure 6

Supraphysiological androgen levels mediate cellular senescence in LNCaP cells partially through rapid signaling. The induction of cellular senescence in LNCaP cells induced by androgens was determined after indicated treatment times with solvent control, 1 pM (low androgen levels = LAL) or 1 nM R1881 (supraphysiological androgen level = SAL). After the treatment times cells were washed to remove the compounds and cultured for total of 3 days. Afterwards, cells were fixed and analyzed for SA β-Gal activity using a light microscopy at 200x magnification and 3x 200 cells were counted and as means of the triplets plotted in percent. A. LNCaP cells were treated between 1 for up to 72 h. Thereby at shorter incubation times, indicated compounds were removed and cells were cultivated until 72 h in total. B. LNCaP cells were incubated for 72 h with R1881 and followed by detection of Akt and Src as well as their phosphorylated forms by specific antibodies by Western blot analysis. For quantification LabImage D1 was used. C. Detection of SA β-gal staining of LNCaP cells incubated for 72 h with the Src tyrosine kinase inhibitor PP2 (1 μM) in combination with LAL and SAL. D. The PI3-kinase inhibitor 3-MA was added to LNCaP cell culture at the indicated concentrations with and without R1881 for 3 d and analyzed using light microscopy and 3x 200 cells were counted and the mean of the triplets is diagrammed in percent. E. Detection of SA β-Gal staining of LNCaP cells incubated for 72 h with an Akt-inhibitor (Akti; 1 μM) in combination with SAL treatment. F. Detection of SA β-Gal staining of LNCaP cells incubated for 72 h with the mTOR inhibitor rapamycin (1 nM) in combination with LAL or SAL treatment.