Figure 4.

Combining NFV with BZ enhances ER stress, and ATF3 or CHOP silencing inhibits the combination-induced cell death. (a) Enhancement of ER stress. H157 cells were treated with either DMSO, 10 μM NFV, 12.5 nM BZ, or the combination for the indicated times, and RPMI8226 cells were treated with either DMSO, 10 μM NFV, 6.25 nM BZ, or the combination for the indicated times. Immunoblotting was performed for ER stress markers. C1 and C2 are controls that are H157 cell lysates treated with 6 μM Tunicamycin for 24 and 8 h, respectively. (b) PERK is involved in the combination-induced apoptosis. PERK−/− and WT MEFs were treated with either DMSO, 10 μM NFV, 12.5 nM BZ, or the combination for 24 h. Cells were harvested and analyzed by DNA fragmentation assays. Level of eIF2α (Ser51) phosphorylation was assessed by immunoblotting for evaluating PERK-deficiency in PERK−/− MEFs. (c and d) siRNA-mediated knockdown of ATF3 or CHOP inhibits the combination-induced cell death. Cells were pre-treated with either siRNA as described in Materials and Methods, and then H157 cells were treated with DMSO or the combination of 10 μM NFV and 12.5 nM BZ for 24 h (c) and for 17 h (d), and RPMI8226 cells were treated with DMSO or the combination of 10 μM NFV and 6.25 nM BZ for 24 h (c and d). Cells were harvested and analyzed by cell death assays. Columns, mean from at least three separate experiments; bars, S.D. *P<0.01, **P<0.001