Volumetric analysis of endo/lysosomes during acute and chronic PIKfyve suppression. (A) RAW cells were pre-labelled with Lucifer Yellow and exposed to vehicle only or to 20 nM apilimod for the indicated times. Fluorescence micrographs are z-projections of 45-55 z-planes acquired by spinning disc confocal microscopy. Scale bar: 5 µm. (B-D) Quantification of individual endo/lysosome volume (B), endo/lysosome number per RAW macrophage (C) and total endo/lysosome volume (D). (E) Wild-type and Fig4−/− mouse embryonic fibroblasts labelled with Lucifer Yellow. Scale bar: 30 µm. (F-H) Analysis of individual endo/lysosome volume (F), endo/lysosome number per cell (G) and total endo/lysosome volume per cell (H) in wild-type and Fig4−/− mouse embryonic fibroblasts. In all cases, data shown are the mean±s.e.m. from three independent experiments, with at least 15-20 cells per condition per experiment. *P<0.05 compared with respective control conditions using one-way ANOVA and Tukey's post hoc test for B-D, and unpaired Student's t-test for F-H.