Figure 4.

Targeted down-regulation of KLF6-SV1 increases the induction of apoptosis in combination with cisplatin. A, increasing doses of cisplatin result in increased KLF6-SV1 expression with no changes in wild-type KLF6 levels as measured by qRT-PCR, using wild-type KLF6– and SV1-specific primers (10). RNA and protein were harvested from adherent cells only after cisplatin treatment. B, Western blot of A549 cells treated with increasing doses of cisplatin probed with a KLF6 monoclonal antibody shows increased KLF6-SV1 expression with higher doses of cisplatin. C, targeted reduction of KLF6-SV1 in combination with subtherapeutic doses of cisplatin results in a significant increase in apoptosis in the A549 cell line (**, P < 0.001). Cells were first transfected with the indicated siRNA [nontargeting control (NTC) or SV1 (si-SV1)]; 24 h after transfection, 10 µm of cisplatin or vehicle control was added to the cells. FACS analysis was performed at the indicated time points (48, 72, or 96 h; *, P < 0.01). D, qRT-PCR revealed an additive effect in NOXA up-regulation after treatment of A549 cells with si-SV1 and cisplatin at all time points assayed (48, 72, and 96 h; **, P < 0.001). Numbers above the bars, fold up-regulation in NOXA expression for each condition. All experiments were repeated three independent times. Column, mean of all three experiments; bars, SD.