Figure 3

Effect of CS on ATF4 pathway. Panel A: NHBE cells were exposed to air (M = mock treatment) or 2R4F cigarette smoke with 35 cc puffs diluted in 250 cc air (CS = smoke treatment) for 20 minutes. Cells were then placed in fresh medium and returned to the incubator for the time periods specified. Nuclear extracts were prepared as described in the Methods section. Western blots of nuclear fractions were probed with antibodies to ATF4 and Lamin A/C, a nuclear antigen, as a loading control. Panel B: NHBE cells were exposed to air (M = mock treatment) or 2R4F cigarette smoke with 35 cc puffs diluted in 250 cc air (CS = smoke treatment) for 20 minutes. Cells were then placed in fresh medium and returned to the incubator for the time periods specified. Western blots of whole cell lysates were probed with antibodies to GADD34 and α-tubulin as a loading control. Panel C:A549 cells were exposed to air (M = mock treatment) or 2R4F cigarette smoke (CS = smoke treatment) for 20 minutes with 35 cc puffs diluted in 250 cc air. Cells were then placed in fresh medium and returned to the incubator for the time periods specified. Western blots of whole cell lysates were probed with antibodies to ATF3 and GAPDH.