Fig. 2.

PIKfyve dysfunction in Vps34 KO podocytes underlies the aberrant vacuolation. On day 4 post-Ad-Cre-treatment, Vps34 KO podocytes (panels a–d) and Pikfyve KO MEFs (panels e and f) were transfected with pTag-YFP-Vps34^wt cDNA. Twenty-four h post-transfection, cells were treated with vehicle (panels a, b, e and f) or 1 μm YM201636 for 1 hour (panels c, d and not shown). A, Live cells were observed by fluorescence microscopy and phase-contrast. Shown are representative images of transfected cells (panels a, c and e) and the overlay with the phase-contrast images (panels b, d and f). Rescue of the vacuolation phenotype occurs in Vps34 KO podocytes upon YFP-Vps34^wt expression (panels a and b) in practically all transfected cells (see B). If endogenous PIKfyve activity is inhibited by YM201636 such rescue is not observed (panels c and d). As expected, YFP-Vps34^wt expression in Pikfyve KO MEFs did not prevent the cytoplasmic vacuolation (panels e and f). Bar, 10 μm. B, Quantification of vacuolated Vps34^wt-expressing cells in the presence or absence of the inhibitor was obtained by inspecting >20 transfected cells per condition per experiment in three separate experiments and presented as percentage of the total number of transfected cells. *, different vs. (−) inhibitor, P<0.001