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. 2005 Jul;25(13):5429–5444. doi: 10.1128/MCB.25.13.5429-5444.2005

FIG. 8.

FIG. 8.

JNK inhibition attenuates the lethality of the Bay 11-7082/HDAC inhibitor regimen without affecting ROS production or NF-κB inactivation. (A) U937 cells were exposed for 24 h to 3 μM Bay 11-7082 (B) plus 2 μM MS-275 (MS) or 1 μM SAHA in the absence or presence of 10 μM SP600125 (SP), after which the percentages of cells exhibiting positive DCF fluorescence and annexin V-FITC were determined by flow cytometry. Values represent the means ± SD for three separate experiments performed in triplicate. *, significantly lower than the value for treatment with Bay 11-7082 with MS-275 or SAHA in the absence of SP600125 (P < 0.02). Ctrl, control. (B) Alternatively, cells were lysed and subjected to either EMSA (upper panels) or Western blot analysis (lower panels). Abbreviations are the same as those for panel A. (C) U937 cells were transiently transfected with JNK1 siRNA oligonucleotides as described in Materials and Methods. After 24 h, the levels of JNK1 and JNK2 were monitored by Western blot analysis (inset). Following a 6-h posttransfection recovery interval, JNK1 siRNA-transfected cells were exposed to 3 μM Bay 11-7082 plus 2 μM MS-275 (B+MS) or 1 μM SAHA (B+SAHA) for 24 h, after which the percentage of viable cells was determined by ViaCount assay. (D) U937 cells stably transfected with a TAM67 construct (inset, Western blot) or its empty vector (pMM) were treated with 3 μM Bay 11-7082 plus 2 μM MS-275 (B+MS) or 1 μM SAHA (B+SAHA), as well as 50 ng/ml anisomycin (Aniso) for 24 h, after which the percentage of cells exhibiting apoptotic morphology was determined by evaluating Wright-Giemsa-stained cytospin slides. The results represent the means ± SD for three separate experiments performed in triplicate. Ctrl, control. Asterisks signify values that are significantly lower than the values for cells transfected with empty vector (pMM) (**, P < 0.01; *, P < 0.02). For Western blot analyses in panels B through D, 30 μg of protein was loaded in each lane; blots were subsequently stripped and reprobed for the expression of β-actin, as indicated, to ensure equivalent loading and transfer of protein. CF, cleavage fragment. Two additional studies yielded equivalent results.