Fig. 2. PI specificity modulates NOXA dependent synergistic cell death induction.
A–C SW982/WT cells were pre-incubated for 4 h with 5 nM or 100 nM of BTZ, CFZ or IXZ and Proteasome-GloTM cell based reagent was added. Luminescence indicating activity of the individual proteasomal subunits was detected after 5 min of incubation. D–F SW982/WT were transfected with PMAIP1 siRNA and subsequently incubated for 24 h in the presence or absence of 15 µM ABT-199 and/or 5 nM PI (BTZ, CFZ or IXZ). Apoptotic cell death was assessed flow cytometrically by detecting Annexin V-APC+ cells. Graphs represent mean values and individual data points. Statistical significance was calculated by an unpaired student´s t test. G–I After treatment with 15 µM ABT-199 and/or 5 nM PI (BTZ, CFZ, IXZ; + 10 µM Q-VD-OPh) for 8 h each 40 µg total protein per lane were separated on a 12–20% polyacrylamide gradient gel and knock-down of NOXA, with β-ACTIN as loading control, was verified by Western blot. BTZ bortezomib, CFZ carfilzomib, IXZ ixazomib, CTRL control.