Activation of caspases and mitochondria.

(A) Processing of caspase-3, -8 and -9 is shown in Mel-2a cells at 24 h and at 48 h after transduction with AdV-AK (MOI=50). Expression of Bcl-x_AK was switched on in the absence of doxycycline (ON) or shut off with doxycycline (OFF). Equal protein loading (20 µg/lane) was confirmed by GAPDH. The whole experiment was performed twice. (B) Inhibition of apoptosis by preincubation with the pancaspase inhibitor zVAD-fmk (1 h, 10 µM) is shown. SK-Mel-13 cells had been transduced with AdV-AK (MOI=100, 48 h). Means and SDs of triplicate values of a representative experiment (one of two) are shown. (C) Decrease of the mitochondrial membrane potential (Δψ_m) is shown for Mel-2a cells at 48 h after transduction of AdV-AK, as determined by flow cytometry after JC-1 or TMRM⁺ staining. Cultures with doxycycline (OFF, grey) are compared to cultures grown in the absence of doxycycline (ON, open graphs). The experiment was performed three times, resulting in highly comparable results. (D) ROS levels were determined in Mel-2a cells at 24 h and 48 h after transduction with AdV-AK under ON and OFF conditions (flow cytometry after H₂DCFDA staining). Below, parallel cultures were pre-treated for 1 h with 200 µM NAC before transduction. (E) Relative DNA-fragmentation rates (apoptosis) at 48 h with or without NAC were determined in parallel. Non-transduced cells (−/+NAC) are shown as additional controls (open bars). Values had been normalized with regard to non-treated controls, set to 1. Means and SDs of triplicate values of a representative experiment are shown (two independent experiments). (F) Expression levels of Bcl-2 proteins, of p53 and Survivin were determined by Western blot analysis in Mel-2a cells at 24 h and 48 h after transduction with AdV-AK (ON and OFF conditions). Equal protein loading (20 µg/lane) was confirmed by GAPDH.