Effect of V-ATPase subunit a1 and B2 knockdown on surfactant secretion by alveolar type II cells.
(A) Freshly isolated type II cells were transduced without (Blank Con) and with adenoviruses (MOI: 100) containing irrevelant siRNA sequences (AdCon), or siRNAs targeted to a1 (AdVoa1) and B2 (AdV1B2) subunits for 5 days. Cell lysates were immunoblotted for a1 and B2 proteins. The blots were reprobed for GAPDH for confirming equal loading of protein. (B) Type II cells were cultured on air-liquid cultures system. The cells were transduced with adenoviruses at a MOI of 100 on day 2 and cultured for 5 more days. The cells were labeled with [3H]-choline on day 6 overnight, then stimulated with 1 mM ATP for 2 hrs and assayed for surfactant secretion. The results were expressed as a percentage of unstimulated cells transduced with AdCon. Data shown are means ± SE (n = 4 independent cell preparations).
