Figure 1. Inhibition of V-ATPase blocks ABCA1 mediated cholesterol efflux.

ABCA1 induced (black bars) and non-induced (open bars) BHK cells were pretreated with 5 nM to 15 nM bafilomycin A1 for 16 hr (A), or 10 nM or 10 μM bafilomycin A1 for 1 hr (B), and % [³H]cholesterol efflux was determined after a 4 hr chase +/− 5 μg/ml apoA1 (N=3; mean ± SD; groups with different letters above the bars show P≤0.005 by ANOVA posttest). RAW264.7 cells were transfected with scramble or ATP6V₀C siRNA by Nucleofection and ATP6V₀C mRNA levels were measured by qPCR analysis normalized to β-actin mRNA (C, N=3; mean ± SD; P<0.0001), or % [³H]cholesterol efflux was calculated after a 4 hr incubation +/− 5 μg/ml apoA1 (D, N=3; mean ± SD; groups with different letters above the bars show P<0.001 by ANOVA posttest). E. BHK cells were incubated with fluorescein and tetramethylrhodamine (TMR) dual-labeled dextran for 16 hr, and chased in the absence (circles) or presence of 10 nM (triangles) or 10 μM (squares) bafilomycin A1 for 4 hr. Fluorescein/TMR ratio was measured by flow cytometry to show endosomal/lysosomal acidification. Plot shows frequency histogram of Fluorescein/TMR ratio; representative of 3 independent experiments. F. RAW264.7 cells were transfected with scramble (circles) or ATP6V₀C (triangles) siRNA by Nucleofection, or treated with 10 μM bafilomycin A1 (squares). The frequency histogram of Fluorescein/TMR ratio of endocytosed dual labeled dextran was plotted as in E; representative of 3 independent experiments.