The role of MAPK signaling in the anti-proliferative effects of CO. Fibroblasts were stimulated with serum and exposed to CO (250 ppm) or control incubator air. A: Cells were subsequently analyzed for MAPK phosphorylation by Western blotting at time points ranging from 5 minutes to 1 hour. B: Densitometry for the same experiment is shown. Bar graphs represent an average of two to three blots for each time point and all values were normalized to RA control for each blot (*, P < 0.05 compared with RA control for same time point). To test whether deletion of select genes contributing to the MAPK pathways would affect fibroblast proliferation, lung fibroblasts were extracted from lungs of Jnk1−/−, p38β−/−, and MKK3−/− mice. Early passage serum-starved fibroblasts were stimulated with serum in the presence or absence of CO. C: Deletion of these select MAPK genes did not affect the anti-proliferative effect of CO on these fibroblasts (*, P < 0.005 compared with corresponding room air control). D: To confirm this finding, the experiment was repeated using chemical inhibitors of the MAPK pathways. The black bars represent ambient air controls and white bars represent CO-exposed fibroblasts. Dimethyl sulfoxide, vehicle control; SB, p38 MAPK inhibitor SB203580; SP, JNK inhibitor SP600125; UO126, MEK1/2 inhibitor UO126; mix, all three inhibitors combined. JNK and ERK inhibition slowed fibroblast proliferation at baseline, but none of the inhibitors abrogated the effect of CO (*, P < 0.05 compared with corresponding room air control).