Fig. 5.

HIF-1α silencing prevents protective effects of CoCl₂ against H₂O₂-induced barrier loss. HIF-1α was silenced in 16HBE14o− cells by lentiviral transfection of short-hairpin RNA (shRNA) against HIF-1α or nontarget (NT) shRNA as a control. Efficiency of HIF-1α silencing in transfected cells was evaluated by Western blot analysis of lysates of untreated or CoCl₂-treated cells (A). Effect of HIF-1α shRNA on protective effects of CoCl₂ preincubation against 3 h treatment with H₂O₂ was determined by analysis of TER using electric cell-substrate impedance sensing (ECIS), measured at a frequency of 4,000 Hz (B), or permeability to FITC-dextran of cells seeded in Transwell inserts. Ctl, nontransduced control; NT, nontarget shRNA; T1, HIF-1α shRNA target 1; T2, HIF-1α shRNA target 2. P < 0.05 compared with corresponding untreated control (*), with corresponding H₂O₂ group (**), and with NT H₂O₂ + CoCl₂ (#).