Snx10 dictates ClC-7 activity by modulating cellular PI(3,5)P ₂ levels. (A and B) Phosphoinositide levels in WT and Snx10 KO RAW 264.7 macrophages determined by PRMC-MS: (A) PI(3,5)P₂; (B) PI(3)P. (C) Sucrosomal [Cl⁻] of WT and Snx10 KO RAW 264.7 macrophages treated with vehicle control or 100 nM YM201636 for 30 min [Cl⁻] was determined colorimetrically as outlined in Fig. 3 A; n = 3. (D) Representative fluorescence images of lysosomes in WT, ClC-7 KO, and Snx10 KO RAW 264.7 macrophages. Lysosomes were visualized by pulsing overnight with AlexaFluor-647–conjugated 10 kDa dextran and chasing for 1 h prior to imaging. Where indicated, the cells were treated with either 100 nM YM201636 or vehicle as control for 30 min immediately prior to imaging. Lower row shows five times (5×) zoom images of regions denoted by dashed lines. (E) Mean vacuole volumes determined from images like those in D and Fig. S1 C. n = 3. (F) WT and Snx10 KO RAW 264.7 macrophages were transiently transfected with either ClC-7-GFP or ClC-7^Y715C-GFP for 18 h prior to imaging GFP. (G) WT and ClC-7 KO HeLa (top 3 panels) and RAW 264.7 (bottom 3 panels) cells were imaged 16 h after being transiently transfected with Snx10-GFP (green). Where indicated, the cells were treated with either 500 nM CCA or vehicle only for 1 h prior to imaging. Images are representative of three independent experiments. PRMC-MS: phosphoinositide regioisomer measurement by chiral column chromatography and mass spectrometry.