ATP uptake into LBs depends on VNUT. (A) Images illustrating loading of BODIPY FL ATP (green) into individual LBs (encircled). BODIPY FL ATP uptake was reduced in cells expressing VNUTshRNA. Cells expressing VNUTshRNA are identified by coexpression of nuclear BFP (blue). Bar, 10 µm. (B) Normalized BODIPY FL ATP fluorescence in individual LBs after a 3-h incubation period in the absence (untreated) or presence (treated) of VNUT inhibitor Evans Blue (100 µM), knockdown of VNUT expression by VNUT shRNA treatment for 72 h, inhibitor of H⁺-ATPase (100 nM bafilomycin A, 4 h), or treatment with NH₄Cl (10 mM). BODIPY FL ATP fluorescence was analyzed within regions encircling individual LBs (as illustrated by circles in A). n, number of cells analyzed. *, P < 0.05; ***, P < 0.001. (C) Quantification of ATP release from ATII cells after stimulation of LB exocytosis with either 100 µM UTP or 300 nM PMA for 10 min in control cells or cells treated with VNUT inhibitor Evans Blue (100 µM, 3 h), VNUTshRNA (72 h), bafilomycin A (100 nM, 4 h), or NH₄Cl (10 mM, 3 h). Data represent means ± SEM from ≥4 individual cell isolations. *, P < 0.05; **, P < 0.01.