BafA1 suppresses PtdIns3P elevation but does not mitigate PtdIns(3,5)P₂ reduced by apilimod.

(A and B): HEK293 cells, cultured in complete media and grown to 90–100% confluence, were incubated for 24 h at 37°C in “starvation” medium prior to labeling with myo-[2-³H]inositol as described under Fig 2. Cells were treated at 37°C for 40 min with vehicle (control, 0.1% DMSO) or BafA1 (15 nM) followed by 100 nM apilimod (in DMSO) or vehicle (0.1% DMSO) for an additional 60 min in the same labeling medium. Lipids were extracted, deacylated and GroPIns were separated by HPLC. Shown are representative HPLC [³H]GroPInsP profiles from apilimod (left panel) and BafA1+apilimod treated HEK293 cells (right panel) (A) and quantification of BafA1-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 (B). Note that BafA1 reduces the PtdIns3P elevation by apilimod without ameliorating reduced PtdIns(3,5)P₂ levels. (C): Confocal microscopy analysis in transfected HEK293 cells expressing PtdIns3P-binding reporter GFP-2xFYVE^PIKfyve at low levels. Fluorescence signals associated with GFP-2xFYVE are markedly increased in cells with apilimod (panels a vs. c) and drastically reduced by BafA1 pretreatment (panels a vs. b), resembling those in transfected control cells receiving only vehicle (panel c) or only BafA1 (panel d). Shown are typical confocal images (60x objective) out of inspected 100 transfected cells/condition from several randomly selected fields. Bar, 10 μm.