Lysosomal V-ATPase inhibitor bafilomycin blocks the effect of EUE on lysosomal BAX location and cell death.

Abstract

<p>Cells were treated with 500 µM palmitate in the presence or absence of 100 µg/mL EUE after pretreatment with 1 µM bafilomycin for 24 hours. Lysosomal V-ATPase activity was measured (A). Acridine orange solution and valinomycin were added to cell monolayers and intravesicular H⁺ uptake was initiated by the addition of Mg-ATP (B); the fluorescence was quantified at 24 hours (B; right). Cell viability assay (C) and caspase-3 activity analysis (D) were performed. Immunostaining was performed with anti-BAX or LAMP-1 antibody and the co-localized BAX was quantified as the percent of lysosomal-translocated BAX (E). Immunoblot analysis of lysosome fractions with antibody against BAX, t-Bid, PDI, COX II, or LAMP-1 (F). ^*<i>p</i><0.05, significantly different from EUE-treated condition in the presence of palmitate. Con; control, Pal.; palmitate, EUE<i>; Eucommia ulmoides</i> Oliver extract, Bafi<i>; Bafilomycin.</i></p