Figure 3. Apilimod inhibits PIKfyve kinase activity in cells.
(A) HeLa cells metabolically labeled with [3H] inositol for 72 hrs and treated with two doses of apilimod or the inactive analog API09 for 120 mins. Lipids were extracted, deacetylated and analysised by HPLC. Data were normalized with cells treated with DMSO vehicle control, and analyzed using Student’s t-Test (*, P<0.05, **, P<0.01), showing a significant difference of the indicated phosphoinositide levels between API09 and apilimod-treated samples. (B) Images of RAW264.7 cells treated with DMSO, apilimod (10 nM) or API09 (10 nM) for 3 hrs. The results are representive from three independent experiments. (C) Electron microscope images of RAW264.7 cells treated with apilimod (200 nM) for 6 hrs. (D) RAW264.7 cells stably expressing GFP-FYVE (early endosome marker) or mCherry-CD63 (endolysosome marker) were treated with DMSO or apilimod (100 nM) for 60 mins. Images were acquired using a Zeiss LSM510 confocal microscope with 63X lens. Scale bar, 5 µm. (E) A549 cells transfected with GFP-PIKfyve or GFP-PIKfyve K1831E mutant were treated with 10 nM apilimod for 4 hrs and imaged using a Zeiss Axiovert microscope. Arrow indicates GFP positive cells. The results are representive from three independent experiments.