pikfyve inhibitor apilimod  (MedChemExpress)

Journal: bioRxiv

Article Title: LRRK2 is activated by phosphatidylinositol 3-phosphate in conjunction with CASM

doi: 10.64898/2025.12.16.694573

Figure Lengend Snippet: (A) Schematic diagram of the PI3P metabolic pathway, where class III PI3K is responsible for the generation of PI3P from PI and PIKfyve is for the generation of PI(3,5)P 2 from PI3P. Compounds that inhibit each kinase are also shown. (B) Immunostaining of intracellular vacuoles formed by treatment with apilimod (Api) or YM201636 (YM) in RAW264.7 cells. Vacuoles were stained with anti-LAMP1 antibody (red) and nuclei were stained with DAPI (cyan). Scale bar = 10 μm. (C-F) Immunoblot analysis of Thr73-phosphorylated Rab10 (pRab10) and other indicated proteins in RAW264.7 cells treated with PIKfyve inhibitors as well as VPS34-IN1 (C, D) or Compound19 (Cmpd19) (E, F). Bar graphs in D and F show quantification of pRab10 relative to total Rab10. Data represent mean ± SEM, n = 6 (D) or 3 (F), and the difference was analyzed using two-way ANOVA with Tukey’s test. (G, H) Immunoblot analysis of pRab10 and other proteins in A549 cells treated with PIKfyve inhibitors as well as VPS34-IN1. A bar graph in H shows quantification of pRab10 relative to total Rab10. Data represent mean ± SEM, n = 4, two-way ANOVA with Tukey’s test. (I-J) Immunoblot analysis of pRab10 in HEK293 cells that overexpress GFP, LRRK2-wild type (WT) or pathogenic mutants (R1441C, Y1699C) and are treated with PIKfyve inhibitors. A bar graph in J shows quantification of pRab10/total Rab10. Data represent mean ± SEM, n = 4, two-way ANOVA with Tukey’s test. (K) Schematic illustration of the activation induction mechanism of PI3K by Tat-D11. (L-N) Immunoblot analysis of pRab10 and LC3B upon Tat-D11 and/or MLi-2 treatment in RAW264.7 cells. Bar graphs show quantifications of pRab10 relative to total Rab10 (M) and LC3B-II relative to α-tubulin (N). Data represent mean ± SEM, n = 6 (K) or 5 (L), two-way ANOVA with Tukey’s test.

Article Snippet: The following reagents were used at final concentrations as indicated: apilimod (100 nM, MedChemExpress), YM203616 (5 μM, AdooQ), VPS34-IN1 (10 μM, Cayman), Compound 19 (50 μM, Selleck), chloroquine (50-100 μM, Sigma), monensin (50 μM, Cayman), nigericin (10 μM, Sigma), diphenyleneiodonium chloride (DPI, 10 μM, Selleck), zymosan (Sigma), bafilomycin A 1 (30 nM, Wako).

Techniques: Immunostaining, Staining, Western Blot, Activation Assay

Journal: bioRxiv

Article Title: LRRK2 is activated by phosphatidylinositol 3-phosphate in conjunction with CASM

doi: 10.64898/2025.12.16.694573

Figure Lengend Snippet: (A) Immunoblot analysis of pRab10, LC3B and other indicated proteins in RAW264.7 cells treated with CQ as well as PI3K inhibitors. (B-E) Quantification of pRab10 relative to total Rab10 (B-C) or LC3B-II relative to α-tubulin (D-E), as shown in A, under treatment with or without CQ plus VPS34-IN1 (B, D) or Compound 19 (C, E). Data represent mean ± SEM, n = 4, and the difference was analyzed using two-way ANOVA with Tukey’s test. (F-G) Immunoblot analysis of pRab10 and LC3B in RAW264.7 cells treated with monensin (mon) (F) or nigericin (nig) (G) as well as PI3K inhibitors. (H) Mass spectrometry (MS)-based measurements of phosphoinositide isomer levels in RAW264.7 cells treated with apilimod, apilimod plus VPS34-IN1, or CQ. Data represent mean ± SEM, n = 4, and the difference was analyzed using one-way ANOVA with Tukey’s test.

Article Snippet: The following reagents were used at final concentrations as indicated: apilimod (100 nM, MedChemExpress), YM203616 (5 μM, AdooQ), VPS34-IN1 (10 μM, Cayman), Compound 19 (50 μM, Selleck), chloroquine (50-100 μM, Sigma), monensin (50 μM, Cayman), nigericin (10 μM, Sigma), diphenyleneiodonium chloride (DPI, 10 μM, Selleck), zymosan (Sigma), bafilomycin A 1 (30 nM, Wako).

Techniques: Western Blot, Mass Spectrometry

Journal: bioRxiv

Article Title: PIKfyve governs endoplasmic reticulum-lysosome contacts to modulate endoplasmic reticulum dynamics

doi: 10.1101/2025.07.15.664974

Figure Lengend Snippet: (A) Confocal images of HeLa cells immunostained for Ill-tubulin (gray) treated with 0.02% vehicle (DMSO), 240 nM apilimod or 1 µM YM-201636 for 2 h. (B-D) Quantification of microtubule morphology including number of branches per cell (B), number of junctions per cell (C), and average branch length (D). For B through D, all experiments were repeated four independent times. (E) Confocal images of COS-7A cells transiently transfected with EB1-GFP displayed as Rainbow RBG LUT and treated as in A. (F-H) Semi-quantification of microtubule dynamics including percentage of cells with >10 EB1-puncta (F), percentage of cells with mobile EB1-puncta (G), and percentage of cells with EB1-puncta moving away from the nucleus (H). For F-H, all experiments were repeated three independent times. For B-D and F-H, data was analyzed using an Ordinary one-way ANOVA test and Dunnett’s multiple comparisons test. (I) Quantification of the number of ER-hitchhiking events that occurred in a 100 µm 2 ROI over one minute, repeated three independent times. (J) Confocal images of COS-7A cells transiently transfected with ER-mNeonGreen (gray) and labeled with Dextran, Alexa Fluor 647 (magenta) and exposed to either 0.02% vehicle (DMSO) or 240 nM apilimod. Zoom inserts show an ER-hitchhiking event occurring over 2.5 seconds as indicated by the white arrow. Data were analyzed using a two-tailed paired Student’s t-test and p values are shown. Data points from matching independent experiments are color coded. Shown is the mean ∓ SEM. Scale bar: full size = 20 µm, zoom insert = 5 µm. P values are shown.

Article Snippet: PIKfyve inhibition was accomplished by treating cells with 80-240 nM apilimod (Selleck Chemicals LLC, Houston, TX) or 1 μM YM-201636 (Cayman Chemical Company, Ann Arbor, MI) for 1-2 h – specific concentrations and times are indicated in each figure legend.

Techniques: Transfection, Labeling, Two Tailed Test

Journal: bioRxiv

Article Title: PIKfyve governs endoplasmic reticulum-lysosome contacts to modulate endoplasmic reticulum dynamics

doi: 10.1101/2025.07.15.664974

Figure Lengend Snippet: (A) Confocal images of COS-7A cells labeled with Dextra, Alexa Fluor 647 (magenta) and exposed to either 0.02% vehicle (DMSO) or 240 nM apilimod for 2 h. Scale bar = 20 µm. (B) Schematic representation of the changes to lysosome spatial distribution between control and PIKfyve inhibited conditions. (C) Quantification of the distribution of lysosomes in the perinuclear region versus the peripheral region of the cell. A ratio of the number of lysosomes in the perinuclear region to total number of lysosomes in the cell was determined – the same was done for the peripheral region respectively. (D) Kymograph of lysosome movement along a linear ROI. The vertical axis represents time (s), and the horizontal axis represents distance along a selected path. Diagonal or segmented lines indicate motility; vertical lines represent stationary organelles. (E-G) Quantification of lysosome motility including track mean speed (E), total distance travelled (F), and track displacement (G). All experiments were repeated four independent times. Data points from matching independent experiments are colour coded. Shown is the mean ± SEM. Data were analyzed using a two-way ANOVA and Tukey’s multiple comparison test for (C) and two-tailed paired Student’s t-test for (E-G) and p values are shown.

Article Snippet: PIKfyve inhibition was accomplished by treating cells with 80-240 nM apilimod (Selleck Chemicals LLC, Houston, TX) or 1 μM YM-201636 (Cayman Chemical Company, Ann Arbor, MI) for 1-2 h – specific concentrations and times are indicated in each figure legend.

Techniques: Labeling, Control, Comparison, Two Tailed Test

Journal: bioRxiv

Article Title: PIKfyve governs endoplasmic reticulum-lysosome contacts to modulate endoplasmic reticulum dynamics

doi: 10.1101/2025.07.15.664974

Figure Lengend Snippet: (A-D) Confocal images of HeLa cells treated with 0.02% vehicle (DMSO), 240 nM apilimod, or 1 µM YM-201636 for 2 h followed by Proximity Ligation Assay between protrudin and VapA (A), or Rab7 and VapA (B), or Rab7 and protrudin (C), or Rab7 and Kinesin-1 (KIF5B; D). Images were generated by maximum projection of z-stacks of the PLA signal (magenta); DAPI-stained nucleus is shown in gray. Negative controls where one of the primary antibodies was missing are shown. Scale bar = 15 µm. (E-H) Quantification of the number of PLA dots per cell for each protein pair tested in A-D, as indicated in image. All experiments were repeated four independent times with matching independent experiments colour coded. Shown is the mean ± SEM. Data were analyzed using an Ordinary one-way ANOVA and Dunnett’s multiple comparisons test and p values are shown.

Article Snippet: PIKfyve inhibition was accomplished by treating cells with 80-240 nM apilimod (Selleck Chemicals LLC, Houston, TX) or 1 μM YM-201636 (Cayman Chemical Company, Ann Arbor, MI) for 1-2 h – specific concentrations and times are indicated in each figure legend.

Techniques: Proximity Ligation Assay, Generated, Staining