GFP-2x-TgPH1 retains membrane association in several mammalian cells inhibited for PIKfyve.

<p>Human PC3 cells and RPE cells were transfected with GFP or GFP-2x-TgPH1, labelled with fluorescent dextran to identify lysosomes and then treated with vehicle or apilimod to inhibit PIKfyve for 1 h, followed by spinning disc confocal imaging. A, B. PC3 cells (A) and RPE (B) expressing GFP treated with vehicle or apilimod. In all cases, GFP appeared cytosolic. C, D: PC3 cells (C) and RPE cells (D) expressing GFP-2x-TgPH1 treated with vehicle or apilimod. In all cases, GFP-2x-TgPH1 retained membrane association. E, F. Quantification of GFP and GFP-2x-TgPH1 membrane association in PC3 (E) and RPE cells (F). Data shown are mean F^H/F_L ± STD from N = 3 from 25–40 cells per experiment per condition. Using a one-way ANOVA and Tukey's post-hoc test, there was a significance difference in the FH/FL for GFP versus GFP-2x-TgPH1 for the respective cells and treatments (* p<0.01) but there was no difference in the membrane distribution of GFP-2x-TgPH1 between vehicle and apilimod in PC3 or RPE cells. Scale bar represent 10 μm.