SNX13 depletion redistributes cellular cholesterol in the absence of NPC1 function. (A–C) U2OS cells treated with U18666A (+U18) for 16 h labeled with GST-PFO* detected using anti-GST antibodies 72 h after transfection with indicated siRNAs. Scale bar, 10 µm. Insets, enlargements of boxed areas; scale bars, 2 µm. (D) Immunoblot (at top) of SNX13 siRNA-treated cells as in A and B; 50 µg cell extract was analyzed. Molecular weight marker mobility is shown in kD here and in all subsequent figures. Below are relative SNX14 mRNA levels measured by quantitative PCR from U2OS cells from two independent experiments; error bar, SD. (E and F) Quantitation of PFO*-positive vesicle numbers and vesicular PFO* fluorescence intensity. Colored dots reflect means from independent experiments; >860 cells analyzed for each condition; significance determined by unpaired t test: **, P < 0.01. (G–K) Analysis of HeLa cells as described for A–F; >950 cells analyzed in each condition; significance determined by unpaired t test: *, P < 0.05; ***, P < 0.001. Actual P values: (E) Ctrl siRNA versus SNX13 siRNA, P = 0.0079; Ctrl siRNA versus SNX14 siRNA, P = 0.16; (F) Ctrl siRNA⁺U18 versus SNX13 siRNA⁺U18, P = 0.0057; Ctrl siRNA⁺U18 versus SNX14 siRNA⁺U18, P = 0.0033; (J) Ctrl siRNA⁺U18 versus SNX13 siRNA⁺U18, P = 0.00035; Ctrl siRNA⁺U18 versus SNX14 siRNA⁺U18, P = 0.31; (K) Ctrl siRNA⁺U18 versus SNX13 siRNA⁺U18, P = 0.038; Ctrl siRNA⁺U18 versus SNX14 siRNA⁺U18, P = 0.39. AU, arbitrary unit.