ACSL3 regulates LD biogenesis and neutral lipid accumulation. (A) After transfection with the ACSL3 siRNA or an unrelated siRNA, the protein levels of ACSL3 were determined by Western blotting. (B) The number of HPos-positive structures in starved cells (pre-LDs) or in cells additionally incubated with OA (emerging LDs) was quantified. Red and white bars correspond to cells treated for 24 h with the ACSL3 siRNA (red bars) or the unrelated siRNA (white bars), transfected with HPos, and additionally starved for 24 h. In some of these experiments Triacsin C (slashed bars) was present during starvation and loading. Gray and black bars correspond to cells transfected with HPos (gray bars) or cotransfected with HPos and myc-ACSL3 (black bars) and starved for an additional 24 h. (C–E) Neutral lipids were quantified by flow cytometry after starvation or the indicated times of fatty acid addition in cells previously transfected with the ACSL3 siRNA (red bars) or the unrelated siRNA (white bars), nontransfected cells (gray bars), or cells transfected with myc-ACSL3 (black bars). In some experiments cells were additionally incubated with Triacsin C (slashed bars). (F–H) Cells treated as in C were loaded with OA for 12 h and stained with Nile red (red in F) and Hoechst to visualize the nucleus (blue). In these images the size (G) and number (H) of LDs were quantified. Bar, 20 µm. Error bars indicate the standard deviation of three independent experiments. *, P < 0.05; **, P < 0.01.