Fig 1.
Apilimod inhibits not only PIKfyve-catalyzed synthesis of PtdIns(3,5)P2 but also that of PtdIns5P.
Lysates, derived from HEK293 cells transduced with recombinant adenovirus expressing HA-PIKfyveWT and GFP from separate promoters, were immunoprecipitated with a polyclonal anti-PIKfyve (α-PIK) or preimmune (Pre) sera. Washed immunoprecipitates (IPs) were pretreated with various apilimod concentrations or with vehicle alone (0.1% DMSO) for 15 min at 37°C along with PI substrate and then subjected to a lipid kinase assay with 15 μM ATP and [γ-32P]ATP (30 μCi) in a 50-μl final volume. Lipid products were resolved by TLC n-propanol/2 M acetic acid solvent system (65:35 v:v). (A): Shown are autoradiograms from representative TLCs out of 6 independent experiments demonstrating that both PIKfyve lipid products, i.e., PtdIns5P and PtdIns(3,5)P2 (denoted by arrowheads) are inhibited significantly at low nanomolar concentrations of apilimod. (B): Quantification of the autoradiograms from six experiments using variable slope non-linear regression curve fitting option of ImageJ software (mean ± SEM). Note that the two lipids are inhibited with a similar efficiency.
Fig 2.
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.
Fig 3.
In intact podocytes apilimod reduces not only PtdIns(3,5)P2 but also PtdIns5P.
Podocytes cultured in complete media and grown to 90–100% confluence were labeled as described under Fig 2. 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) podocytes, 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.
Fig 4.
Apilimod-triggered cytoplasmic vacuoles are prevented or dissipated by BafA1 treatment.
(A): HEK293 cells grown to 70–80% confluence in complete DMEM medium were treated with various concentrations of apilimod in DMSO (0.1% final concentration) for 1–80 min at 37°C prior to monitoring vacuolation extent by light microscopy. Shown is a quantitative analysis of vacuolation responses, presented as percentage of the total cells, determined by counting at least 200 cells/condition from 10 or more random fields in 4 separate experiments (mean ± SEM). (B): HEK293 cells first pretreated with BafA1 (15 nM) or DMSO (0.1%) for 40 min at 37°C prior to further addition of apilimod (100 nM) or DMSO (0.1%) for 60 min. BafA1 precluded the appearance of any vacuoles. (C): HEK293 cells were pretreated with apilimod (100 nM) or DMSO (0.1%) for 60 min at 37°C. BafA1 (200 nM) or the DMSO vehicle (0.1%) was included for an additional 90 min. BafA1 dissipated apilimod-induced multiple vacuoles. (B and C): Presented are typical phase-contrast images of live cells out of 4 independent experiments with similar result. In each experiment at least 200 cells/condition from several random fields were inspected. Lack of vacuoles upon BafA1 treatment before or after apilimod was seen in ~98% of the monitored cells in each experiment. Bar, 10 μm.
Fig 5.
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-2xFYVEPIKfyve 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.
Fig 6.
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.
Fig 7.
BafA1 precludes EEA1 membrane recruitment induced by PIKfyve inhibition with apilimod.
HEK293 cells were treated with vehicle (0.1% DMSO, panels a and b) or BafA1 (15 nM) for 40 min followed by 100 nM apilimod (or 0.1% DMSO in the control) for an additional 60 min. Cells were then fixed, permeabilized, immunostained for EEA1 and observed by confocal microscope (40x objective). (A): Shown are typical immunofluorescence images for EEA1 (panels a—c) illustrating that fluorescence signals are markedly increased in cells with apilimod treatment (panels b vs. a) and dramatically diminished upon BafA1 pretreatment (panels b vs. c). (B): quantitation of the EEA1-associated immunofluorescence by ImageJ software based on randomly selected cells (30 cells/condition) from different fields in 2 separate experiments with similar results. Data are expressed as corrected integrated density of cell fluorescence (mean ± SEM) and analyzed by one-way Anova, *P<0.05; *** P<0.001. Bar, 10 μm.