Characterization of Snx10 localization and effect on endolysosomal morphodynamics. (A) RAW 264.7 macrophages transiently transfected with ClC-7-GFP (green) for 18 h in the absence (control) or presence of 50 mM sucrose. (B) Relative DQ-BSA signal in sucrosomes generated in WT, ClC-7 KO, and Snx10 KO RAW 264.7 macrophages by overnight incubation with 50 mM sucrose. Cells were acutely incubated with DQ-BSA for 30 min prior to imaging. (C) Representative fluorescence images of lysosomes in WT, ClC-7 KO, and Snx10 KO RAW 264.7 macrophages. Lysosomes were visualized by pulsing overnight with TMR-conjugated 10 kDa dextran and chasing for 1 h prior to imaging. Lower row shows four times (4×) zoom images of regions denoted by dashed lines. (D) Lysosomes were visualized by pulsing overnight with TMR-conjugated 10 kDa dextran and chasing for 1 h prior to receiving a secondary pulse of AlexaFluor-647–conjugated 10 kDa dextran for 10–30 min prior to imaging, as indicated. (E) HeLa cells were transiently transfected with Snx10-GFP and PX-RFP 18 h prior to imaging. (F) HeLa cells were transiently transfected with Snx10-GFP for 18 h, then treated acutely with either DMSO only (control), 100 nM VPS34-IN-1, or 100 nM YM201636 for 1 h prior to imaging. (G) WT RAW 264.7 macrophages were transiently transfected with either Snx10-GFP or Snx10^R51Q-GFP, a variant incapable of binding PI(3)P (see Barnea-Zohar et al., 2021) for 16 h prior to imaging. (H) HeLa cells were transiently transfected with either PX-RFP or 2xFYVE-RFP for 18 h, then subsequently treated with either control (DMSO) or 100 nM SAR405 for 30 min prior to imaging. TMR, tetramethylrhodamine.