2xFYVE vesicular pattern is rendered diffuse in Vps34 KO podocytes. On day 4 post-Ad-Cre-treatment, Vps34 KO podocytes and Pikfyve KO MEFs were transfected with pEGFP-2xFYVEPIKfyve cDNA for visualizing PtdIns3P-enriched endosomal structures. Twenty-four h post-transfection cells were fixed for fluorescence microscopy (panels a and c) and phase-contrast inspection (panels b and d). White arrows denote transfected cells. The 2xFYVEPIKfyve fluorescence signals in the transfected cells (and hence, the PtdIns3P-stained vesicles) were diffuse in Vps34 KO podocytes (panels a and b), which was observed in 77 ± 4% of transfected Vps34 KO podocytes. The appearance of the 2xFYVEPIKfyve in Pikfyve KO MEFs was vesicular (panels c and d), seen in 93 ± 3% of transfected cells. Quantitation was obtained by counting >20 transfected cells per condition per experiment in 3 separate experiments. Bar, 10 μm.