Low dose gemcitabine cannot efficiently induce cell death in lung cancer cells, and leads to NF-κB activation and Bfl-1 overexpression. A and B, A549, H157, PC9 and H460 cells were treated with 0, 0.4, 4, 40, 400, or 4000 ng/ml of gemcitabine for 72 h. Cell viabilities were determined using CCK-8 assays ( A ), and subG1 populations were quantified by PI staining and flow cytometry ( B ). The values shown are mean percentages of cells relative to untreated cells in three independent experiments performed in triplicate; error bars represent SDs. C , A549, H157, PC9 and H460 cells were incubated in the presence of 0, 4, 40, 400, or 4000 ng/ml of gemcitabine for 24 h. Total RNAs were extracted and the mRNA levels of Bfl-1, Bcl-xL, Bcl-2, and β-actin were determined by RT-PCR. D , A549 and H157 cells were transiently transfected with pNF-κB-Luc (firefly) plasmid for 16 h, and subsequently treated with 0, 4, 40, 400, or 4000 ng/ml of gemcitabine for an additional 6 h. Cell were then lysed and analyzed for firefly luciferase activity. Data are mean luciferase activities normalized versus β-gal expression obtained from three independent experiments; error bars represent SDs. Results are presented as means ± SDs of three independent experiments performed in triplicate. *p < 0.001 by the Student's t test for difference in tumor volumes between experimental groups.