Fig. 4

AR antagonist-induced cellular senescent PCa LNCaP cells are sensitive to apoptosis induction by MK2206. LNCaP cells were treated for 72 h with 1 nM R1881, 10 μM ENZ, or 0.1% DMSO as solvent control. After that, the AR ligands were removed. Fresh medium with 0.1% DMSO or 1 μM MK2206 was added and further incubated for additional 72 h. a Growth of LNCaP cells was analysed by crystal violet staining and OD 590 nm measurement. Values obtained from day 0 were set arbitrarily as 1. Line graphs are shown as mean ± standard deviation (n = 2). Red circles indicate the time point of protein extractions. b The protein extraction was performed after 24 h treatment with MK2206. Detection of full-length PARP (PARP FL), cleaved PARP (c-PARP), RIP3, and phosphorylated RIP3 (p-RIP3) proteins was performed by Western blotting and normalized to β-Actin levels. Upper and middle numbers indicate normalized p-RIP3 and RIP3 band intensities. Lower numbers indicate the ratios of p-RIP3 versus RIP3 levels. c Quantification of fold c-PARP levels normalized to β-Actin from Western blotting data. Values obtained from DMSO + MK were set arbitrarily as 1. Bar graphs are shown as mean ± SEM (n = 3). d Percentage of SA-β-Gal positive stained cells after 24 h treatment with MK2206. Bar graphs are shown as mean ± SEM (n = 3). e A schematic figure illustrates an unchanged/compensated percentage level of SA-β-Gal positive cells under MK2206 treatment of ENZ treated cell. Numbers represent the calculated percentage of SA-β-Gal positive cells