SNX13 depletion increases LD abundance in the absence of NPC1 function. (A) U2OS cells treated with U18666A for 16 h and labeled with GST-PFO* and anti-GST primary antibodies 72 h after transfection with the indicated siRNAs without oleic acid addition. Cells were also stained with LipidTOX. One set was also treated with ACAT1 inhibitor Sandoz 53-035. Scale bar, 10 µm. (B) Number of LDs and area determined as in A. Colored dots reflect means from independent experiments; >650 cells analyzed in each condition. Significance was determined by multiple t tests and Holm-Sidak method with α = 0.05. (C) TLC of lipids as in A. At left is mobility of indicated marker lipids. (D) Quantitation of triacylglycerol (TAG) and free fatty acid (FFA) levels determined by TLC. P values were determined by multiple t tests corrected by the Holm-Sidak method; *, P < 0.05; **, P < 0.01. Actual P values: (B) Top: Ctrl siRNA⁺U18 versus SNX13 siRNA⁺U18, P = 0.0052; Ctrl siRNA⁺U18 versus SNX13 siRNA⁺U18 + Sandoz, P = 0.0039; SNX13 siRNA versus SNX13 siRNA + Sandoz, P = 0.85; (B) bottom: Ctrl siRNA versus SNX13 siRNA, P = 0.0451; Ctrl siRNA versus SNX13 siRNA + Sandoz, P = 0.0144; SNX13 siRNA versus SNX13 siRNA + Sandoz, P = 0.382; (D) left: Ctrl siRNA versus SNX13 siRNA, P = 0.00683; Ctrl siRNA + U18 versus SNX13 siRNA + U18, P = 0.312; (D) right: Ctrl siRNA versus SNX13 siRNA, P = 0.0198; Ctrl siRNA + U18 versus SNX13 siRNA + U18, P = 0.351.