Figure 4.

Pharmacologic and genetic interruption of the JNK pathways significantly diminishes bortezomib/HA14-1 lethality in SUHDL16 cells. (A) SUDHL16 cells pretreated with the JNK inhibitor IB1 (ALX 159–600; 10 μM) for 2 hr were exposed to 3 nM bortezomib ± 3.0 μM HA14-1 for 36 hrs. At the end of drug exposure, apoptosis was monitored by 7 AAD staining and flow cytometry. ** = significantly less than values for cells treated in the absence of IB1; p < 0.01. (B) Cells were treated as above (A) for 14 hrs and western blot analysis was employed to monitor the effect of drugs on expression of the indicated proteins. Each lane was loaded with 30 μg of protein; blots were stripped and reprobed with antibodies directed against actin to ensure equivalent loading and transfer. The results of a representative study are shown; two additional studies yielded equivalent results. (C) SUDHL16 cells stably transfected with JNK shRNA or vectors encoding a scrambled sequence were exposed to 4.0 nM bortezomib + 4.0 μM HA14-1. After 36 hr of drug exposure, apoptotic cells were monitored by 7 AAD staining and flow cytometry. inset: relative expression of JNK protein in SUDHL16-scrambled sequence and shJNK clones (D) Following 14 hr of drug exposure as (C) above, western blot analysis was employed to monitor protein expression of phospho-JNK, caspase-4 and caspase 2. Blots were stripped and reprobed with anti-actin antibodies to ensure equal loading and transfer of protein For (A),** = significantly less than values for scrambled sequence clone; p < 0.01. For (C), ** = significantly less than values for empty-vector controls; p < 0.05.