Figure 4.

Differential involvement of NOXA after single-agent and combination therapy. (a) Total JURKAT cell lysates from Figure 2b were analyzed for the expression of NOXA, PUMA and Mcl-1. (b) Parental JURKAT cells, JURKAT cells stably transfected with shRNA against BIM (shBIM), BID (shBID), PUMA (shPUMA), NOXA (shNOXA) or NOXA and simultaneously PUMA (shNOXA+shPUMA) were stimulated with doxorubicin and BA. (c) Parental JURKAT cells (upper panel) and JURKAT cells stably transfected with shRNA against NOXA (lower panel) were stimulated with several drug combinations for doxo (0.01, 0.03, 0.06 μM) and BA (2, 6, 12 μM). Synergism was evaluated using isobolograms. (d and e) JURKAT cells were pretreated with cycloheximide (CHX, 0.3 ng/ml, d) or acetylcysteine (ACC, 500 μg/ml, e) for 6 h followed by stimulation with doxorubicin and BA. (f) Parental JURKAT cells and JURKAT cells stably transfected with shRNA against Mcl-1 (shMcl-1) were stimulated with doxorubicin and BA. Drug concentrations, experimental procedure, measurement of apoptosis induction, statistical analysis and presentation of data were identical as in Figure 1a and Supplementary Figure S1 if not stated differently. Statistical significance is indicated with an asterix, whenever the P-value was <0.05. NS, statistically not significant