BafA1 attenuates PtdIns3P elevation but does not ameliorate PtdIns(3,5)P2 reduced by YM201636.
HEK293 cells cultured in complete media grown to 90–100% confluence were labeled with myo-[2-3H]inositol as described under Fig 2. Cells were then treated with vehicle (control, DMSO) or BafA1 (15 nM; in DMSO) for 40 min at 37°C when YM201636 (800 nM; in DMSO) or vehicle was added for an additional 60 min in the same labeling medium. Lipids were extracted and deacylated, followed by HPLC separation of deacylated GroPIns. (A): Shown are representative HPLC [3H]GroPInsP profiles from control (left panel), YM201636- (middle panel) and BafA1+YM201636-treated cells (right panel), demonstrating that BafA1 arrested PtdIns3P elevation induced by YM201636. (B): Quantification of YM201636- or BafA1-dependent changes in PtdIns3P, PtdIns4P, PtdIns5P, PtdIns(3,5)P2 and PtdIns(4,5)P2 from three independent experiments, presented as a percent of the corresponding control (mean ± SEM) and analyzed by one-way ANOVA. YM201636 decreased PtdIns5P and PtdIns(3,5)P2 and both remained similarly reduced by pretreatment with BafA1. PtdIns3P increased by ~1.6-fold above control levels by YM201636 but only ~1.15-fold after pretreating with BafA1. (*), P<0.05.
