Figure 3

Cigarette smoke extract induces dendritic cell survival proteins. A) Human dendritic cells (1 × 10⁶/ml complete media) were incubated with 2% CSE and cellular lysates prepared following 4, 8, 22 and 45 hours. Equal amounts of protein from whole cell lysates were electrophoretically separated, transferred to nitrocellulose, and analyzed for HO-1 and Bcl-xL protein using relevant antibodies as described in methods. β-Actin protein levels were determined as an internal control to ensure equal protein loading. Representative of two independent experiments. B) Human dendritic cells (1 × 10⁶/ml) were incubated with complete media in the presence of absence of freshly-prepared 2% CSE, and in the presence or absence of either 2.5 mM NAC as an inhibitor of oxidative stress, or 10 μg/ml dexamethasone (NAC and dexamethasone where added 60 minutes prior to stimulation with CSE). Following 6 hours, whole cell lysates were prepared, and 50 μg of total protein were separated on a 12% gel and immunoblotting performed for HO-1. Representative of 3 independent experiments. C) Pooled data from 3 independent experiments performed as outlined in 3B is summarized in graph format. Actin protein levels were determined as an internal control and densitometry was performed [ratio of HO-1: Actin determined] using Image J software. The negative control samples (prepared from unstimulated dendritic cells) was arbitrarily assigned a 100% value. D) Human dendritic cells (1 × 10⁶/ml) were incubated with complete media in the presence of absence of freshly-prepared 2% CSE or 1000 ng/ml of nicotine. In other wells, 2.5 mM NAC was added as an inhibitor of oxidative stress. Following 48 hours, whole cell lysates were prepared, and 50 μg of total protein were separated on a 12% gel and immunoblotting performed for Bcl-xL. Representative of 2 independent experiments.