Pharmacologic and genetic interruption of the JNK pathways significantly diminishes CFZ/vorinostat lethality in SUHDL16 cells. (A) SUDHL16 cells stably transfected with JNK shRNA or vectors encoding a scrambled sequence were exposed to CFZ (3.0nM) plus vorinostat (0.75μM). After 36 hours of drug exposure, cell death was monitored by 7-AAD staining and flow cytometry. Inset: Relative expression of JNK protein in SUDHL16-scrambled sequence and shJNK clones. (B) After 14 hours of drug exposure as in panel A, Western blot analysis was used to monitor protein expression of phospho-JNK and the cleavage fragment of caspase-3 (CF caspase-3). (C) SUDHL16 cells stably transfected with JNK-DN cDNA or empty vector (pcDNA3.1) were exposed to CFZ (3.0nM) plus vorinostat (0.75μM). After 36 hours of drug exposure, cell death was monitored by 7-AAD staining and flow cytometry. Inset: Expression of JNK protein in SUDHL16 empty vector and JNK-DN clones. (D) After 14 hours of drug exposure as in panel C, Western blot analysis was used to monitor protein expression of phospho-JNK and cleaved PARP. (E) SUDHL16 cells pretreated with the selective JNK inhibitor IB1 (ALX 159-600; 10μM) for 2 hours were exposed to CFZ (3.0nM) plus vorinostat (0.75μM) for 36 hours. At the end of drug exposure, cell death was monitored by 7-AAD staining and flow cytometry. (F) After 14 hours of drug exposure as described in panel E, Western blot analysis was used to monitor protein expression of phospho-JNK, JNK, and cleaved PARP. For all studies, blots were stripped and reprobed with antitubulin antibodies to ensure equal loading and transfer of proteins. Each lane was loaded with 20 μg of protein. All values represent the means of triplicate experiments performed on 3 separate occasions plus or minus SD. (A,E) **Significantly less than values for scrambled sequence clone or control (P < .01). (C) *Significantly less than values for empty-vector controls (P < .05).