Figure 2.

Evidence for lipolysis acting upstream of lipophagy on differently sized LDs. (A) Confocal micrographs of AML12 cells stained for LDs with Oil Red O show alterations in LD morphology following 72-h treatment with nontargeting control siRNA (siNT), siATGL, siLAL, or siATGL+siLAL. (B) ATGL knockdown resulted in a twofold increase in average LD size (μm²) and was mimicked by LAL+ATGL double knockdown. (C) Knockdown of LAL caused a greater than twofold increase in the number of LDs/cell, but this increase was blocked by ATGL+LAL knockdown. (D) Quantification of total LD area/cell under each knockdown condition. (E) Similar results were observed in AML12 cells preloaded for 2 h with 150 µM OA and then washed and chased for 48 h in an insulin-free medium containing 2% FBS and treated with DMSO, ATGLi (20 µM), LALi (20 µM), or ATGLi+LALi. (F) ATGLi caused a twofold increase in average LD size which was also observed in the presence of both inhibitors. (G) LALi caused a 2.5-fold increase in the number of LDs per cell, but this was reversed in cells treated with both inhibitors. (H) Quantification of total LD area per cell revealed significant increases by ATGLi, LALi, or both over DMSO control. Asterisks denote statistical significance by one-way ANOVA and Tukey’s post hoc test (*, P < 0.05; **, P < 0.01). Graphs depict mean and SEM. (A–D) n = 3 experiments. (E–H) n = 4 experiments.