Characterization of PI(3)P and its effectors on endosomes. (A) WT and Snx10 KO RAW 264.7 macrophages were co-transfected with PX-GFP and mRFP-Rab5. The percentage of organelles found to be positive for both markers is shown in B; n = 3. (C–F) WT and Snx10 KO RAW 264.7 macrophages were co-transfected with EEA1-GFP and (C) mRFP-Rab5 or (E) mRFP-Rab7. The percentage of (C) Rab5 or (E) Rab7 compartments found to be positive for EEA1 is indicated in D and F, respectively; n = 3. (G) HeLa cells were co-transfected with mRFP-Rab5 and EGFP-Rab7 and treated with either vehicle alone (control) or with 100 nM YM201636 for 30 min prior to imaging. Shown is the percentage of Rab5-positive puncta that were also Rab7 positive. n = 3. (H) WT RAW 264.7 macrophages pulsed overnight with AlexaFluor-647–conjugated 10 kDa dextran and chased for 1 h with fresh medium were treated for 20 min with either vehicle control (left), 100 nM YM201636 (middle), or 50 nM SAR405, a selective VPS34 inhibitor (right) prior to imaging. (I and J) (I) WT and (J) Snx10 KO RAW 264.7 macrophages were transiently transfected with mRFP-FKBP-PIKfyve^CC, iRFP-FRB-Rab5, and PX-GFP 16 h prior to imaging. Representative fluorescence images of cells prior to (top 3 panels) and 1 min after the addition of 100 nM rapamycin (bottom 3 panels).