Inhibition of the PI(3,5)P ₂ signal by apilimod or a selective Vps34 inhibitor (IN-1), based on the NES ^(2x) -mNG ^HO -(2x)DdSNXA ^PX construct expressed in HEK293-AT1. (A and B) Confocal images of HEK293-AT1 cells transfected with the high-avidity PI(3,5)P₂ probe, NES^(2x)-mNG^HO-(2x)DdSNXA^PX, and treated with either apilimod (1 µM; A) or IN-1 (300 nM; B). Integrated pixel intensities were calculated from orthogonal projections of z-stacks and normalized to the initial value as detailed under Materials and methods for three (A) and two (B) cells obtained in representative time-lapse recordings (mean ± SD). The bar diagram shows integrated pixel intensity values calculated for six cells before and after apilimod treatment recorded in two separate experiments (scale bars = 10 µm).