In intact HEK293 cells apilimod reduces not only PtdIns(3,5)P2 but also PtdIns5P.
HEK293 cells cultured in complete media and grown to 90–100% confluence were incubated for 24 h at 37°C in glucose- and inositol-free “starvation” medium prior to labeling for 25 h with 25 μCi/ml myo-[2-3H]inositol. Cells were then treated with vehicle (control, 0.1% DMSO) or 100 nM apilimod for 60 min at 37°C in the same labeling medium prior to lipid extraction, deacylation and HPLC separation of deacylated GroPIns. Fractions were analyzed for [3H] radioactivity. (A): Representative HPLC [3H]GroPInsP profiles from control (left panels) and apilimod treated (right panels) HEK293 cells showing the large reduction in PtdIns5P or PtdIns(3,5)P2 as well as a significant rise in PtdIns3P induced by apilimod. (B): Quantification of apilimod-induced changes in PtdIns3P, PtdIns4P, PtdIns5P, PtdIns(3,5)P2 and PtdIns(4,5)P2 levels from 3 independent experiments (mean ± SEM) (*), P<0.05.
