Figure 6

Determining the ability of nHMECs to undergo chemically induced and Rep78 induced apoptosis. (A) nHMECs were treated with either staurosporine (1 μM) to activate the instrinsic pathway, and a combination of cycloheximide (CHX) (10 μg/ml) and Tumor Necrosis Factor-Alpha (TNFα) (20 ng/ml) to activate the extrinsic pathway of apoptosis. Cells were treated for 24 h, harvested prepared for FACS analysis, and 10⁶ cells were analyzed by flow cytometry. Top panel denotes histograms of the DNA distribution representing percentages of cells in the G1, S and G2 phases of the cell cycle denoted in the bottom panel, as determined using the Cell Quest program of Becton Dickinson. Data were analyzed with the Mod Fit LT program. (B) Staurosporine treatment induced cleavage of caspase-9 and CHX/TNFα treatment induced cleavage of caspase-8 as determined from western blot analysis. (C) Calcium-phosphate transfection of the cloned Rep78 expression construct under CMV promoter control into nHMECs. Untransfected control, GFP-only controls (+GFP), and GFP vector and CMV-Rep78 construct co-transfected cells (+GFP/Rep78). Top panel denotes histograms of the DNA distribution representing percentages of cells in the G1, S and G2/M phases of the cell cycle denoted in the bottom panel, as determined using the Cell Quest program of Becton Dickinson. Data were analyzed with the Mod Fit LT program. Analysis of transfected cells analyzed at 48 h and 7 days post-transfection.