Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Inhibition of PIKfyve kinase prevents infection by Zaire ebolavirus and SARS-CoV-2

doi: 10.1073/pnas.2007837117

Figure Lengend Snippet: Endolysosomal traffic of VSV-MeGFP-ZEBOV in cells expressing TagRFP-Rab5c or TagRFP-Rab7a in the presence of Apilimod or Vacuolin-1 ( SI Appendix and Movies S1 and S2 ). ( A ) Schematic of live cell imaging experiment using SVG-A cells expressing fluorescently tagged TagRFP-Rab5c or TagRFP-Rab7a. Cells were infected with VSV-MeGFP, VSV-MeGFP-V269H, or VSV-MeGFP-ZEBOV (MOI = 4). Viruses trafficking (monitored with MeGFP) to the endolysosomal system (recognized by their labeling with TagRFP-Rab5c or TagRFP-Rab7a) and virus entry (established by MeGFP at the nuclear margin) were ascertained by live-cell florescence imaging using a spinning disk confocal microscope. ( B ) Visualization of VSV-MeGFP infection in TagRFP-Rab5c cells in the absence ( Left ) or presence ( Right , white arrows) of CHX using live-cell imaging. (Scale bar: 10 µm.) ( C ) Genomic PCR analysis of SVG-A cells showing biallelic integration of TagRFP into the RAB5C genomic locus by cotransfection of a plasmid coding for Cas9, a linear PCR product coding for the specific guide RNAstargeting a region near the ATG codon of Rab5c under the control of the U6 promoter, and a template plasmid containing the RFP sequence flanked by 800 base pairs upstream and downstream of the targeted region (see Materials and Methods for more details) to generate a clonal gene-edited cell line expressing TagRFP-Rab5c. ( D ) Quantification of VSV-MeGFP and VSV-MeGFP-ZEBOV colocalization with Rab5c containing endosomes in the presence of CHX together with absence or presence of 5 µM Apilimod depicted in E . Data show number of viruses that colocalized with endosomes containing or not containing Rab5c within the complete volume of the single cells depicted in E . ( E ) Representative examples of maximum-Z projection images from four optical sections spaced 0.35 µm apart of virus entry without or with IN1, Vacuolin, or Apilimod for VSV-MeGFP ( Top ), VSV-Me-GFP-V269H ( Middle ), and VSV-MeGFP-ZEBOV ( Bottom ). Each condition is in the presence of CHX. All viruses reach Rab5c-containing endosomes, but only VSV-MeGFP-ZEBOV fails to penetrate in the presence of IN1, Vacuolin-1, or Apilimod. (Scale bars: 10 µm.) Insets correspond to a single optical section. Insets (yellow boxes) correspond to a single optical section. (Scale bars: 3 µm.) ( F ) Visualization of VSV infection in TagRFP-Rab7a cells in the absence of CHX ( Left ) and entry in the presence of CHX ( Right , white arrows). (Scale bar: 10 µm.) ( G ) Genomic PCR analysis showing biallelic integration of TagRFP into the RAB7A genomic locus to generate a clonal gene-edited cell-line expressing TagRFP-Rab7a, using the same approach as used for RAB5C . ( H ) Quantification of VSV-MeGFP and VSV-MeGFP-ZEBOV colocalization with Rab7a containing endosomes in the presence of CHX with or without 5 µM Apilimod within the complete cell volumes in the images depicted in I . ( I ) Representative examples of maximum-Z projection images from four optical sections spaced 0.35 µm apart of virus entry without or with IN1, Vacuolin, or Apilimod for VSV-MeGFP ( Top ), VSV-Me-GFP-V269H ( Middle ), and VSV-MeGFP-ZEBOV ( Bottom ). All viruses reach Rab7a-containing endosomes, but only VSV-MeGFP-ZEBOV fails to penetrate in the presence of IN1, Vacuolin-1, or Apilimod. (Scale bars: 10 µm.) Insets correspond to a single optical section. Insets (yellow boxes) correspond to a single optical section. (Scale bars: 3 µm.)

Article Snippet: Vacuolin-1 ( ) was custom synthesized; Apilimod (HY-14644) was from MedChem Express, IN1 was a kind gift from N. Gray, Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA , U-18666A (10009085) and Filipin III (70440) were from Cayman Chemical, Bafilomycin A1 (B1793-2UG) was from Sigma-Aldrich, Cycloheximide (239764) was from Calbiochem, and wheat germ agglutinin conjugated with Alexa Fluor-647 (W32466) was from ThermoFisher.

Techniques: Expressing, Live Cell Imaging, Infection, Labeling, Virus, Imaging, Microscopy, Cotransfection, Plasmid Preparation, Control, Sequencing

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Inhibition of PIKfyve kinase prevents infection by Zaire ebolavirus and SARS-CoV-2

doi: 10.1073/pnas.2007837117

Figure Lengend Snippet: Apilimod and Vacuolin-1 inhibit VSV-MeGFP-ZEBOV infection. ( A ) Schematic of infectivity assay, where SVG-A cells were pretreated for 1 h with 5 μM Vacuolin, 5 μM Apilimod, 5 μM IN1, or 10 nM BAF A1 and subsequently infected with VSV-MeGFP (MOI = 2), VSV-MeGFP-V269H (MOI = 1), VSV-MeGFP-RABV (MOI = 0.6), VSV-MeGFP-LASV (MOI = 0.6), VSV-MeGFP-LCMV (MOI = 0.6), or VSV-MeGFP-ZEBOV (MOI = 0.6) for 1 h in the presence of drugs. The cells were then washed to remove unbound virus and incubated for the indicated times in the presence of drugs. The cells were then fixed, and the percentage of cells expressing viral MeGFP was measured by flow cytometry. ( B ) Representative flow cytometry results of an infection assay using VSV-MeGFP-ZEBOV. ( C ) Quantification of the infectivity is shown with averages from three independent experiments per condition, each determined as a duplicate measurement (error bars show SEM). The statistical significance was determined using a one-way ANOVA and Tukey post hoc test (* P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001).

Article Snippet: Vacuolin-1 ( ) was custom synthesized; Apilimod (HY-14644) was from MedChem Express, IN1 was a kind gift from N. Gray, Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA , U-18666A (10009085) and Filipin III (70440) were from Cayman Chemical, Bafilomycin A1 (B1793-2UG) was from Sigma-Aldrich, Cycloheximide (239764) was from Calbiochem, and wheat germ agglutinin conjugated with Alexa Fluor-647 (W32466) was from ThermoFisher.

Techniques: Infection, Virus, Incubation, Expressing, Flow Cytometry

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Inhibition of PIKfyve kinase prevents infection by Zaire ebolavirus and SARS-CoV-2

doi: 10.1073/pnas.2007837117

Figure Lengend Snippet: Apilimod and Vacuolin-1 inhibit VSV-MeGFP-ZEBOV. ( A ) Schematic of entry assay where SVG-A cells were infected with VSV-MeGFP (MOI = 4), VSV-MeGFP-V269H (MOI = 4), or VSV-MeGFP-ZEBOV (MOI = 4). Experiments were performed in the presence of 5 µg/mL cycloheximide (CHX) to prevent protein synthesis. Entry assay was based on the appearance of MeGFP fluorescence on the nuclear margin, on a per cell basis, of fixed infected cells visualized by fluorescence microscopy. Staining the fixed cells with Alexa647-labeled wheat germ agglutinin identified the plasma membrane of each cell (dashed outlines in C ). ( B ) Virus infection in the absence of CHX ( Left ) resulted in the appearance of MeGFP fluorescence throughout the cell volume. The presence of CHX resulted in virus entry being observed by MeGFP fluorescence at the nuclear margin, which was released from incoming viral particles ( Right , white arrows). (Scale bar: 10 µm.) ( C ) Representative examples of maximum-Z projections images from the whole-cell volume obtained with optical sections separated by 0.3 µm using spinning disk confocal microscopy. MeGFP fluorescence at the nuclear margin released from incoming viral particles is highlighted (white arrows). (Scale bar: 10 µm.) ( D ) Quantification of the number of cells with nuclear margin labeling from three independent experiments, each determined from fields containing 59 to 90 cells (error bars show SEM). The statistical significance of the entry data was analyzed for statistical significance by one-way ANOVA and Tukey post hoc test (*** P ≤ 0.001).

Article Snippet: Vacuolin-1 ( ) was custom synthesized; Apilimod (HY-14644) was from MedChem Express, IN1 was a kind gift from N. Gray, Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA , U-18666A (10009085) and Filipin III (70440) were from Cayman Chemical, Bafilomycin A1 (B1793-2UG) was from Sigma-Aldrich, Cycloheximide (239764) was from Calbiochem, and wheat germ agglutinin conjugated with Alexa Fluor-647 (W32466) was from ThermoFisher.

Techniques: Infection, Fluorescence, Microscopy, Staining, Labeling, Clinical Proteomics, Membrane, Virus, Confocal Microscopy

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Inhibition of PIKfyve kinase prevents infection by Zaire ebolavirus and SARS-CoV-2

doi: 10.1073/pnas.2007837117

Figure Lengend Snippet: Apilimod and Vacuolin-1 inhibit infection of VSV-eGFP-SARS-CoV-2. ( A ) Schematic of infectivity assay of VSV-eGFP, VSV-eGFP-ZEBOV, and VSV-eGFP-SARS-CoV-2 in MA104 cells. MA104 cells were pretreated for 1 h with the indicated concentration of Apilimod. Pretreated cells were inoculated with the indicated virus (MOI = 1) for 1 h at 37 °C. At 6 h postinfection cells were harvested, and the fraction of cell expressing eGFP cells was quantified by flow cytometry. ( B ) Quantification of the infectivity is shown with averages ± SEM from three independent experiments. Statistical significance was determined using a t test (* P ≤ 0.05; ** P ≤ 0.01).

Article Snippet: Vacuolin-1 ( ) was custom synthesized; Apilimod (HY-14644) was from MedChem Express, IN1 was a kind gift from N. Gray, Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA , U-18666A (10009085) and Filipin III (70440) were from Cayman Chemical, Bafilomycin A1 (B1793-2UG) was from Sigma-Aldrich, Cycloheximide (239764) was from Calbiochem, and wheat germ agglutinin conjugated with Alexa Fluor-647 (W32466) was from ThermoFisher.

Techniques: Infection, Concentration Assay, Virus, Expressing, Flow Cytometry

Journal: bioRxiv

Article Title: Damage-sensing recruitment of a lipid phosphatase couples lysosomal membrane repair to proteostatic adaptation

doi: 10.64898/2026.04.04.716461

Figure Lengend Snippet: (a) MTMR14 expression in human skeletal muscle inversely correlates with body weight. FPKM: fragments per kilobase per million mapped fragments, data were collected from 56 samples. (b) MTMR14 expression in human skeletal muscle. Linear plots representing the correlation of MTMR14 expression in human skeletal muscle samples and body mass index (BMI). FPKM: fragments per kilobase per million mapped fragments, data come from 56 samples. (c) LLOMe treatment decreases PI4K2A phosphorylation. Immunoprecipitation (IP) fractions of U2OS cells expressing HA-PI4K2A and treated with DMSO control or 30 minutes LLOMe were collected with anti-HA beads and immunoblotted with antibodies against the p-RXRXXs/t* motif. (d) Left: representative confocal images of U2OS cells treated with control (DMSO), 1 h 1 µM rapamycin, or 1 h 1 mM LLOMe stained with DAPI and antibodies against pS6 (Ser240/244). Scale bar, 20µM. Right: quantification of pS6 (Ser240/244) intensity. t test (n = 3 independent experiments, each datapoint represents 15 fields of view, one field of view containing 10-20 cells, with a size of 1664 × 1664 µm). (e) Representative confocal images of U2OS and MTMR14 KO cells treated with control (DMSO) or 1 h 1 mM LLOMe stained with DAPI and antibodies against pS6 (Ser240/244). Scale bar, 20µM. Quantification is shown in . (f) Left: Representative immunoblot of control and LLOMe treated WT and MTMR14 KO U2OS cell lysates. Right: quantitative analysis of pULK1 S757/ total ULK1 levels in control or LLOMe-treated (2 h) WT and MTMR14 KO U2OS cells. t test (n = 4 independent experiments). Dotted line denotes pULK1 S757/ total ULK1 levels in DMSO controls set to 1. (g) MTMR14 re-expression in KO cells rescues decreased 4E-BP1 phosphorylation upon LLOMe treatment. Left: representative immunoblot of U2OS WT, MTMR14 KO, and MTMR14 KO cells expressing mCherry-MTMR14 under doxycycline control subjected to 2 h DMSO control or LLOMe treatment. Right: densitometric quantification of 4E-BP1 S65 phosphorylation relative to total 4E-BP1. one-way ANOVA (n = 4 independent experiments). Dotted line denotes p4E-BP1 S65/ total 4E-BP1 in controls set to 1. (h) Left: representative immunoblot of U2OS WT and MTMR14 KO cells treated for 2 h with DMSO control or LLOMe. Right: quantification of pTFEB S211 relative to total TFEB from immunoblots as shown on the left. t test (n = 3 independent experiments). Dotted line denotes pTFEB S211/ total TFEB in controls set to 1. (i) Immunoblot of U2OS cells treated for 1 hr with DMSO control or 5 µM VPS34-IN1. (j) Immunoblot of U2OS cells treated for 1 hr with DMSO control or 200 nM Apilimod. (k) Representative confocal images of 1 h 1 mM LLOMe treated U2OS MTMR14 KO cells expressing mCherry-vector, mCherry-MTMR14 WT, or mCherry-MTMR14 C330S stained with DAPI and antibodies against pS6 (Ser 240/244). Scale bar, 10 µm. Quantification is shown in . (l) Representative confocal images of DMSO control or different time points of 1 mM LLOMe treated U2OS stained with DAPI and antibodies against pS6 (Ser 240/244). Quantification is shown in . (m) Immunoblot of U2OS cells treated with DMSO control or different time points of LLOMe. (n) Immunoblot of C2C12 cells treated with DMSO control or different time points of LLOMe. (o) Immunoblot of HeLa cells treated with DMSO control or different time points of LLOMe. (p) AZD8055 and S6Ki efficiency test. Immunoblot of S6 S240/S244 phosphorylation in U2OS cells treated for 1 h with DMSO control, 200 nM AZD8055, 2 µM S6Ki, or 5 µM S6Ki. Statistical analyses were performed using GraphPad Prism. Two-tailed unpaired t-test, paired t-test or one-sample t-tests were conducted using column statistics to compare the sample means to a hypothetical value of 1. All bar graphs represent mean ± SD unless otherwise stated. ***p < 0.001, **p < 0.01, *p < 0.05.

Article Snippet: L-Leucyl-L-Leucine methyl ester hydrobromide (LLOME, Sigma, Cat# L7393, 1mM, 2mM), Gly-Phe-β-naphthylamide (GPN, Cayman Chemical, Cat# 14634, 200μM), O-methyl-serine dodecylamide hydrochloride (MSDH, Avanti Polar Lipids, Cat# 850546, 50μM), Benzalkonium chloride (BAC, CAS# 63449-41-2, MP Biomedicals, Santa Ana, CA, USA, 5ug/mL), hydrogen peroxide (H 2 O 2 , Cat# ‘9681.2, Roth, 0.5mM), VPS34-IN1 (Selleckchem, Cat# S7980, 5μM), apilimod (Echelon Biosciences, Cat# B-0308, 50nM), rapamycin (Santa Cruz Biotechnology, Cat# sc-3504, 100nM), AZD8055 (AdooQ Bioscience/hölzel, Cat# A10114, 200nM), Anisomycin (Sigma, Cat# A9789, 30μM), LY2584702 (S6Ki, Selleckchem, Cat# S7704, 2μM, 5μM), MRT68921 dihydrochloride (MedChemExpress, Cat# 2080306-21-2, 1μM), bortezomib (MedChemExpress, Cat# 179324-69-7, 2μM), CCCP (Sigma, Cat# C2759-100MG, 10μM), Bafilomycin A1(Sigma, Cat# SML1661, 100nM or 200nM), nigericin (Invivogen, Cat# tlrl-nig,5μM), D-Mannitol (Sigma, Cat# M4125, 0.25M), Puromycin (InvivoGen, Cat# ant-pr-1, 2μM), Ionomycin (Calbiochem, Cat# 407952, 5μM), Doxycycline (Sigma, Cat# D-9891, 1μg/mL), Digitonin 5%(Thermo, Cat# BN20061, 20 mM).

Techniques: Expressing, Phospho-proteomics, Immunoprecipitation, Control, Staining, Western Blot, Plasmid Preparation, Two Tailed Test

Journal: bioRxiv

Article Title: Damage-sensing recruitment of a lipid phosphatase couples lysosomal membrane repair to proteostatic adaptation

doi: 10.64898/2026.04.04.716461

Figure Lengend Snippet: (a) Representative confocal live cell images of TMEM192-mKeima expressing HeLa cells treated with LLOMe for the indicated time points. Quantification is shown in . (b) Quantification of acidic TMEM192-mKeima puncta/cell area in U2OS cells after different durations of LLOMe treatment. n = 28-37 cells per datapoint. Data are mean ± s.e.m. (c) Quantification of acidic TMEM192-mKeima puncta/cell area after 5 h LLOMe treatment in U2OS WT and MTMR14 KO cells. t test (n = 4 independent experiments, total number of cells is 116 for WT and 113 for MTMR14 KO). Representative confocal images in . (d) Quantification of mean LysoTracker intensity/field of view fold change over control in BV2 cells after 5 h LLOMe plus control (DMSO), 5 µM VPS34-IN1, or 2µM MRT68921 treatment. one-way ANOVA (total number of fields of view is 30 each group, one field of view containing 35-50 cells, with a size of 3328 × 3328 µm). (e) Quantification of LDH cytotoxicity assay in BV2 cells treated for 5 h with 4mM LLOMe plus DMSO control or 5 µM VPS34-IN1. t test (n = 3 independent experiments, each datapoint represents triplicate measurements). (f) Quantification of mean LysoTracker intensity/field of view fold change over control in HeLa cells after 5 h LLOMe plus control (DMSO), 5 µM VPS34-IN1, or 200 nM Apilimod treatment. one-way ANOVA (total number of fields of view is 30 for LLOMe, 29 for LLOMe + VPS34-IN1, and 28 for LLOMe + Apilimod, one field of view containing 35-50 cells, with a size of 3328 × 3328 µm). Statistical analyses were performed using GraphPad Prism. Two-tailed unpaired t-test, paired t-test or one-sample t-tests were conducted using column statistics to compare the sample means to a hypothetical value of 1 or one-way ANOVA with Tukey’s multiple comparisons test. All bar graphs represent mean ± SD unless otherwise stated. ***p < 0.001, **p < 0.01, *p < 0.05.

Article Snippet: L-Leucyl-L-Leucine methyl ester hydrobromide (LLOME, Sigma, Cat# L7393, 1mM, 2mM), Gly-Phe-β-naphthylamide (GPN, Cayman Chemical, Cat# 14634, 200μM), O-methyl-serine dodecylamide hydrochloride (MSDH, Avanti Polar Lipids, Cat# 850546, 50μM), Benzalkonium chloride (BAC, CAS# 63449-41-2, MP Biomedicals, Santa Ana, CA, USA, 5ug/mL), hydrogen peroxide (H 2 O 2 , Cat# ‘9681.2, Roth, 0.5mM), VPS34-IN1 (Selleckchem, Cat# S7980, 5μM), apilimod (Echelon Biosciences, Cat# B-0308, 50nM), rapamycin (Santa Cruz Biotechnology, Cat# sc-3504, 100nM), AZD8055 (AdooQ Bioscience/hölzel, Cat# A10114, 200nM), Anisomycin (Sigma, Cat# A9789, 30μM), LY2584702 (S6Ki, Selleckchem, Cat# S7704, 2μM, 5μM), MRT68921 dihydrochloride (MedChemExpress, Cat# 2080306-21-2, 1μM), bortezomib (MedChemExpress, Cat# 179324-69-7, 2μM), CCCP (Sigma, Cat# C2759-100MG, 10μM), Bafilomycin A1(Sigma, Cat# SML1661, 100nM or 200nM), nigericin (Invivogen, Cat# tlrl-nig,5μM), D-Mannitol (Sigma, Cat# M4125, 0.25M), Puromycin (InvivoGen, Cat# ant-pr-1, 2μM), Ionomycin (Calbiochem, Cat# 407952, 5μM), Doxycycline (Sigma, Cat# D-9891, 1μg/mL), Digitonin 5%(Thermo, Cat# BN20061, 20 mM).

Techniques: Expressing, Control, LDH Cytotoxicity Assay, Two Tailed Test

Journal: Antimicrobial Agents and Chemotherapy

Article Title: Pan-antiviral effects of a PIKfyve inhibitor on respiratory virus infection in human nasal epithelium and mice

doi: 10.1128/aac.01050-23

Figure Lengend Snippet: Effects of intranasal apilimod (Apilimod base: AB, and Apilimod mesylate: AM) treatment on H1N1-infected BALB/c mice. Mice were inoculated intranasally with H1N1 PR8 (1.0 × 105 PFU/mouse), and the animals were sacrificed 5 days post-virus inoculation. Apilimod was treated once daily on day 0 (4 hours before infection), and then on Days 1, 2, and 3. Body weight loss triggered by virus infection (A, B), viral load in lung tissue (C) and nasal tissue (D), and neutrophil accumulation in BALF (E) were evaluated. For viral load, individual data have been plotted as geometric mean. For body weight. mean ± SEM was shown. *P < 0.05, ***P < 0.001 vs H1N1-infected control.

Article Snippet: Apilimod base (Santa Cruz Biotechnolgy, Heidelberg, Germany) was prepared as a suspension in 10% DMSO/90% isotonic saline and delivered using an intranasal installation of 40 μL/mouse at doses of 0.5, or 2 mg/mL on Day −1, again on Day 0 (1 hour before inoculation) and then once daily on Days 1, 2, and 3 post-inoculations.

Techniques: Infection, Virus, Control

Journal: Antimicrobial Agents and Chemotherapy

Article Title: Pan-antiviral effects of a PIKfyve inhibitor on respiratory virus infection in human nasal epithelium and mice

doi: 10.1128/aac.01050-23

Figure Lengend Snippet: Antiviral effects of PIKfyve inhibitors against influenza isolates in MDCK cells, evaluated by CPE a

Article Snippet: Materials Apilimod base, Apilimod mesylate, YM201636, oseltamivir phosphate, and oseltamivir carboxylate were purchased from MedChemExpress LLC (Monmouth Junction, NJ), ribavirin were purchased (confirmed by Pharmidex). .

Techniques:

Journal: Antimicrobial Agents and Chemotherapy

Article Title: Pan-antiviral effects of a PIKfyve inhibitor on respiratory virus infection in human nasal epithelium and mice

doi: 10.1128/aac.01050-23

Figure Lengend Snippet: Antiviral effects of PIKfyve inhibitors against influenza isolates. (A) Concentration-dependent cell toxicity and antiviral effects of apilimod mesylate (AM) in MDCK cells infected with H1N1 A/California/07/2009. (B) Concentration-dependent cell toxicity and antiviral effects of YM201636 (YM) in MDCK cells infected with H1N1 A/California/07/2009. (C) Antiviral effects of AM and YM in MDCK cells infected with H3N2, A/Ohio/88/2012 v. (D) Antiviral effects of AM and YM in MDCK cells infected with H5N1, Duck/MN/1525/81. Compounds were treated at the same time when the virus was inoculated, and CPE was determined on Day 3 post-inoculation by neutral red staining.

Article Snippet: Materials Apilimod base, Apilimod mesylate, YM201636, oseltamivir phosphate, and oseltamivir carboxylate were purchased from MedChemExpress LLC (Monmouth Junction, NJ), ribavirin were purchased (confirmed by Pharmidex). .

Techniques: Concentration Assay, Infection, Virus, Staining

Journal: Antimicrobial Agents and Chemotherapy

Article Title: Pan-antiviral effects of a PIKfyve inhibitor on respiratory virus infection in human nasal epithelium and mice

doi: 10.1128/aac.01050-23

Figure Lengend Snippet: Antiviral effects of PIKfyve inhibitors against other respiratory viruses a

Article Snippet: Materials Apilimod base, Apilimod mesylate, YM201636, oseltamivir phosphate, and oseltamivir carboxylate were purchased from MedChemExpress LLC (Monmouth Junction, NJ), ribavirin were purchased (confirmed by Pharmidex). .

Techniques:

Journal: Antimicrobial Agents and Chemotherapy

Article Title: Pan-antiviral effects of a PIKfyve inhibitor on respiratory virus infection in human nasal epithelium and mice

doi: 10.1128/aac.01050-23

Figure Lengend Snippet: Effects of Apilimod mesylate on H1N1(PR8) infected ALI-cultured nasal epithelium. (A) Kinetics of H1N1 viral load in apical washes post-virus inoculation. On Day 0, the virus was inoculated and removed after 60 min of absorption. Apical washes were collected after 1-hour incubation (and wash) on Day 0, and once daily from Day 1 to Day 3. (B) Viral load determined by TCID50 assay in apical washes from the inserts treated vehicle and AM, which were collected on Day 2 post-inoculation. (C) Kinetics of cell integrity determined as TEER post-virus inoculation. (D) TEER determined in epithelium inserts treated with vehicle and AM on Day 2 post-inoculation. (E) The kinetics of Rantes and CXCL8 release in apical washed from the epithelium inserts infected with H1N1, and the effects of AM on Rantes (F) and CXCL8 (G) in apical washes collected on Day 2 post-inoculation.

Article Snippet: Materials Apilimod base, Apilimod mesylate, YM201636, oseltamivir phosphate, and oseltamivir carboxylate were purchased from MedChemExpress LLC (Monmouth Junction, NJ), ribavirin were purchased (confirmed by Pharmidex). .

Techniques: Infection, Cell Culture, Virus, Incubation, TCID50 Assay

Journal: Antimicrobial Agents and Chemotherapy

Article Title: Pan-antiviral effects of a PIKfyve inhibitor on respiratory virus infection in human nasal epithelium and mice

doi: 10.1128/aac.01050-23

Figure Lengend Snippet: H1N1 viral load in apical wash from ALI nasal epithelium treated with Apilimod or oseltamivir

Article Snippet: Materials Apilimod base, Apilimod mesylate, YM201636, oseltamivir phosphate, and oseltamivir carboxylate were purchased from MedChemExpress LLC (Monmouth Junction, NJ), ribavirin were purchased (confirmed by Pharmidex). .

Techniques:

Journal: Antimicrobial Agents and Chemotherapy

Article Title: Pan-antiviral effects of a PIKfyve inhibitor on respiratory virus infection in human nasal epithelium and mice

doi: 10.1128/aac.01050-23

Figure Lengend Snippet: Effects of intranasal apilimod (Apilimod base: AB, and Apilimod mesylate: AM) treatment on H1N1-infected BALB/c mice. Mice were inoculated intranasally with H1N1 PR8 (1.0 × 105 PFU/mouse), and the animals were sacrificed 5 days post-virus inoculation. Apilimod was treated once daily on day 0 (4 hours before infection), and then on Days 1, 2, and 3. Body weight loss triggered by virus infection (A, B), viral load in lung tissue (C) and nasal tissue (D), and neutrophil accumulation in BALF (E) were evaluated. For viral load, individual data have been plotted as geometric mean. For body weight. mean ± SEM was shown. *P < 0.05, ***P < 0.001 vs H1N1-infected control.

Article Snippet: Materials Apilimod base, Apilimod mesylate, YM201636, oseltamivir phosphate, and oseltamivir carboxylate were purchased from MedChemExpress LLC (Monmouth Junction, NJ), ribavirin were purchased (confirmed by Pharmidex). .

Techniques: Infection, Virus, Control

Journal: Antimicrobial Agents and Chemotherapy

Article Title: Pan-antiviral effects of a PIKfyve inhibitor on respiratory virus infection in human nasal epithelium and mice

doi: 10.1128/aac.01050-23

Figure Lengend Snippet: Effects of Apilimod mesylate administered intranasally on H1N1-induced inflammation detected in bronchoalveolar lavage and nasal lavage of H1N1-infected mice a

Article Snippet: Materials Apilimod base, Apilimod mesylate, YM201636, oseltamivir phosphate, and oseltamivir carboxylate were purchased from MedChemExpress LLC (Monmouth Junction, NJ), ribavirin were purchased (confirmed by Pharmidex). .

Techniques: Inhibition, Saline, Virus

Journal: Antimicrobial Agents and Chemotherapy

Article Title: Pan-antiviral effects of a PIKfyve inhibitor on respiratory virus infection in human nasal epithelium and mice

doi: 10.1128/aac.01050-23

Figure Lengend Snippet: In vitro and in vivo antiviral effects of Apilimod against RSV infection. (A) Effects of Apilimod mesylate and ribavirin on viral load in apical washes collected from RSV A2-infected ALI-cultured nasal epithelium. Apilimod M was treated apically on Day 0 and Day 1 (and removed each time) or ribavirin was treated basally without removal. Viral load was determined by plaque assay. (B) Effects of intranasal Apilimod (Apilimod mesylate: AM) treatment on RSV A2-infected BALB/c mice. Mice were inoculated intranasally with RSV A2 (1.0 × 105 PFU/mouse), and the animals were sacrificed 4 days post-virus inoculation. AM was treated once daily on Day 0 (4 hours before infection), and then on Days 1 and 2. Body weight loss triggered by virus infection (B), viral load in lung tissue (C) and nasal tissue (D), and neutrophil accumulation in BALF (E) and nasal lavage (F) were evaluated. For viral load, individual data have been plotted and Geomean. For body weight and neutrophils, mean ± SEM were shown. *P < 0.05, **P<0.01, ***P < 0.001 vs RSV-infected control.

Article Snippet: Materials Apilimod base, Apilimod mesylate, YM201636, oseltamivir phosphate, and oseltamivir carboxylate were purchased from MedChemExpress LLC (Monmouth Junction, NJ), ribavirin were purchased (confirmed by Pharmidex). .

Techniques: In Vitro, In Vivo, Infection, Cell Culture, Plaque Assay, Virus, Control

Journal: bioRxiv

Article Title: Complete Protection from SARS-CoV-2 Lung Infection in Mice Through Combined Intranasal Delivery of PIKfyve Kinase and TMPRSS2 Protease Inhibitors

doi: 10.1101/2023.07.19.549731

Figure Lengend Snippet: A. Representative fluorescence images of VeroE6-TMPRSS2 and A549-AT cells pre-treated with DMSO (Ctrl) or 2 μM apilimod dimesylate and 25 μM nafamostat mesylate, and one hour later infected for 20 h with indicated SARS-CoV-2 variants. Cells were stained with nuclear DNA dye Hoechst (nuclei, cyan) and immunostained with an antibody against the viral N protein (N, magenta). Scale bar = 200 μm. B-C. Quantification of the experiment shown in A. The percentage of N positive cells was determined by automated image analysis. Values represent the mean of three independent experiments and data are normalized to the infection levels obtained in DMSO vehicle treated infected cells (indicated as 1) in each experiment. The error bars represent the standard deviation. D. Representative fluorescence images of cells treated as in B with indicated drugs that had been stored either at -20 °C or room temperature (r.t.), for three months. One hour after drug treatment, cells where infected with SARS-CoV-2 Wuh strain for 20 h before fixation and immunofluorescence analysis as described in A. Scale bar 200 μm. E. Quantification of the experiment shown in D. The percentage of viral N positive cells was determined by automated image analysis. Values represent the mean of three independent experiments and data are normalized to the infection levels obtained in DMSO vehicle treated infected cells (indicated as 1) in each experiment. The error bars represent standard deviation.

Article Snippet: Stocks of Apilimod dimesylate (Tocris, catalogue number 7283) and Nafamostat mesylate (Tocirs, catalogue number 3081) were diluted in the same medium and added at indicated times.

Techniques: Fluorescence, Infection, Staining, Standard Deviation, Immunofluorescence

Journal: bioRxiv

Article Title: Complete Protection from SARS-CoV-2 Lung Infection in Mice Through Combined Intranasal Delivery of PIKfyve Kinase and TMPRSS2 Protease Inhibitors

doi: 10.1101/2023.07.19.549731

Figure Lengend Snippet: A. Apilimod dimesylate (1 mg/ml) and nafamostat mesylate (2 mg/ml) were solubilized in DMEM, PBS, or de-ionized water (d-H 2 O). A visible cloudy precipitate formed when the drugs were solubilized in DMEM and PBS, but not in d-H 2 O. B. After centrifugation at 10.000xg for 1 minute, at room temperature, a visible pellet formed if the drugs were solubilized in DMEM or PBS (white arrows), but not in the drugs solubilized as in A precipitate d-H 2 O.

Article Snippet: Stocks of Apilimod dimesylate (Tocris, catalogue number 7283) and Nafamostat mesylate (Tocirs, catalogue number 3081) were diluted in the same medium and added at indicated times.

Techniques: Centrifugation

Journal: bioRxiv

Article Title: Complete Protection from SARS-CoV-2 Lung Infection in Mice Through Combined Intranasal Delivery of PIKfyve Kinase and TMPRSS2 Protease Inhibitors

doi: 10.1101/2023.07.19.549731

Figure Lengend Snippet: Stock solutions of apilimod dimesylate (1 mg/ml) and nafamostat mesylate (2 mg/ml) were prepared in DMEM, PBS, or de-ionized water (d-H 2 O). The drugs were further diluted in DMEM 2%FBS and added to Vero-E6 (apilimod dimesylate) or A549-AT cells (nafamostat mesylate) in 96-well plates at the indicated concentrations, 30 min before infection with SARS-CoV-2 Wuh (MOI= 0.5). Cells were fixed at 20 hours post infection and processed for immunofluorescence analysis using antibodies against the viral protein N to identify infected cells and Hoechst DNA staining to identify the cells nuclei. High-content imaging and automated image analysis were used to determine the number of infected cells in each sample. A-B. Number of infected cells for each drug treatment relative to the values obtained from infected cells treated with vehicle controls (indicated as 1). Values represent the mean and standard deviation of three independent experiments. C-D. Number of cells attached to the surface of the plates for each drug treatment relative to the values obtained from infected cells treated with vehicle controls (indicated as 1). Values represent the mean and standard deviation of three independent experiments.

Article Snippet: Stocks of Apilimod dimesylate (Tocris, catalogue number 7283) and Nafamostat mesylate (Tocirs, catalogue number 3081) were diluted in the same medium and added at indicated times.

Techniques: Infection, Immunofluorescence, Staining, Imaging, Standard Deviation

Journal: bioRxiv

Article Title: Complete Protection from SARS-CoV-2 Lung Infection in Mice Through Combined Intranasal Delivery of PIKfyve Kinase and TMPRSS2 Protease Inhibitors

doi: 10.1101/2023.07.19.549731

Figure Lengend Snippet: A. Schematic description of the intranasal drug treatment and SARS-CoV-2 beta infection. Anesthetized mice received drugs intranasally in aqueous solution (30 μl) 10-20 seconds prior intranasal inoculation of SARS-CoV-2 beta (5×10 5 plaque forming units in 20 μl DMEM). The drug treatment was repeated twice a day at 6 hours intervals at day 0 and day 1. At 48 hpi (day 2), mice were euthanized, and their right lung processed for real time quantitative PCR analysis to detect viral replication. The left lung and heads of the fixed animals were processed for immuno histology using anti N antibodies to monitor the integrity of the tissue and the distribution of viral antigens. B. PCR quantification of viral RNA in the lungs of mice treated with indicated drugs as described in A. For each mouse, the levels of viral RNA were detected using three non-overlapping primer sets, one targeting the viral genomic RNA dependent RNA polymerase gene (RdRp), and two sets against the viral gene E (E, subE). For each mouse, the obtained values were normalized first to the levels of actin in the same lung tissue and then to the mean viral RNA obtained in vehicle control treated infected mice (indicated as 100%). For each treatment, the mean (white bar) and standard deviation of the mean are indicated. The data were collected over two independent experiments each including vehicle controls. Each data point represents the RNA reads from one mouse. The concentration of apilimod dimesylate and nafamostat mesylate in mg/kg are indicated on the X axis. ***p<0.001.

Article Snippet: Stocks of Apilimod dimesylate (Tocris, catalogue number 7283) and Nafamostat mesylate (Tocirs, catalogue number 3081) were diluted in the same medium and added at indicated times.

Techniques: Infection, Real-time Polymerase Chain Reaction, Control, Standard Deviation, Concentration Assay

Journal: bioRxiv

Article Title: Complete Protection from SARS-CoV-2 Lung Infection in Mice Through Combined Intranasal Delivery of PIKfyve Kinase and TMPRSS2 Protease Inhibitors

doi: 10.1101/2023.07.19.549731

Figure Lengend Snippet: Immunohistology images of lungs and nasal mucosa from mice infected with the drugs as in . Virus infected cells were identified with an antibody against the viral NP protein, using the horseradish peroxidase method (brown) and haematoxylin counterstain. Insets depict magnified images of the areas indicated by the arrows. Apilimod dimesylate: 2 mg/kg; nafamostat mesylate: 4 mg/kg; Apilimod dimesylate + nafamostat mesylate: 0.2 mg/kg + 0.8 mg/kg. Scale bars = 500 µm. A. Lungs. B. Nasal mucosa. Non infected mice exhibit no viral antigen in lung and nasal mucosa, whereas vehicle treated infected mice exhibit widespread SARS-CoV-2 NP expression in the lungs, both in bronchiolar epithelial cells and in pneumocytes in large groups of alveoli. This is also seen in epithelial cells in the entire nasal mucosa. In mice treated with Apilimod alone, the viral antigen expression pattern is identical, but its extent slightly reduced in the lung. After Nafamostat treatment, it is further reduced and only seen in small patches of alveolar epithelial cells. After combined Nafamostat and apilimod treatment, there is no evidence of viral antigen expression in the lung. In the nasal mucosa, positive cells are mainly seen in caudodorsal areas, in olfactory epithelial cells.

Article Snippet: Stocks of Apilimod dimesylate (Tocris, catalogue number 7283) and Nafamostat mesylate (Tocirs, catalogue number 3081) were diluted in the same medium and added at indicated times.

Techniques: Infection, Virus, Expressing

Journal: bioRxiv

Article Title: Complete Protection from SARS-CoV-2 Lung Infection in Mice Through Combined Intranasal Delivery of PIKfyve Kinase and TMPRSS2 Protease Inhibitors

doi: 10.1101/2023.07.19.549731

Figure Lengend Snippet: A. PCR quantification of viral RNA in the lungs of mice treated intranasally with indicated drugs at 0 h (i.e., 10-20 seconds prior infection), 3 h and 6 h post SARS-CoV-2 beta infection. For each mouse, the levels of viral RNA were detected using two primer sets, targeting the viral genomic RNA dependent RNA polymerase gene (RdRp) and the sub-genomic viral gene E (subE), respectively. For each mouse, the obtained values were normalized first to the levels of actin in the same lung tissue and then to the mean viral RNA value obtained in vehicle control-treated infected mice (indicated as 100%). For each treatment, the mean (white bar) and standard deviation of the mean are indicated. Each data point represents the RNA reads from one mouse. The concentration of apilimod dimesylate and nafamostat mesylate in mg/kg, and the time of drug administration are indicated on the X axis. B, C. Immunohistology images of lungs from mice infected and treated as described in A. Virus infected cells were identified with an antibody against the viral N protein (B, C) and apoptotic cells were visualised with an antibody against cleaved caspase 3 (C), using the horseradish peroxidase method (brown) and haematoxylin counterstain. B. Mice treated with vehicle or with nafamostat (4 mg/kg) and apilimod (2 mg/kg), starting at 3 h.p.i. or 6 h.p.i. Vehicle treated mice exhibit widespread SARS-CoV-2 NP expression in epithelial cells of bronchi (arrows) and in groups of alveoli (arrowheads). With onset of treatment at 3 h.p.i., lung infection is seen, but is less widespread than in the vehicle treated animals. With onset of treatment at 6 h.p.i., viral antigen expression is also less extensive than in the vehicle treated animals, but the reduction is less marked. Scale bars = 500 µm. C. Mice treated with vehicle or with nafamostat (4 mg/kg) and apilimod (2 mg/kg), starting at 6 h.p.i. Consecutive sections of a bronchiole stained with hematoxylin-eosin (HE), for SARS-CoV-2 NP and for cleaved caspase-3. Insets represent higher magnifications of areas indicated by the arrows in the overview images. In the vehicle treated mouse, abundant degenerate cells are present in the lumen of the bronchiole, the epithelium exhibits several degenerating cells (arrowheads in inset). There is extensive viral NP expression in epithelial cells, including degenerate cells in the lumen (arrowheads). Staining for cleaved caspase-3 shows that infected epithelial cells die via apoptosis. Inset: apoptotic epithelial cells (arrowheads); arrow: sloughed off apoptotic epithelial cell). In the nafamostat and apilimod treated animals, the bronchiolar lumina are free of degenerate cells and the epithelium appears intact, although the majority of cells are virus infected as shown by the expression of viral NP. The extreme rarity of cleaved caspase 3-positive apoptotic cells (arrow; inset: arrowhead) confirms that infected cells are viable. Scale bars = 25 μm

Article Snippet: Stocks of Apilimod dimesylate (Tocris, catalogue number 7283) and Nafamostat mesylate (Tocirs, catalogue number 3081) were diluted in the same medium and added at indicated times.

Techniques: Infection, Control, Standard Deviation, Concentration Assay, Virus, Expressing, Staining

Journal: bioRxiv

Article Title: Complete Protection from SARS-CoV-2 Lung Infection in Mice Through Combined Intranasal Delivery of PIKfyve Kinase and TMPRSS2 Protease Inhibitors

doi: 10.1101/2023.07.19.549731

Figure Lengend Snippet: A. Schematic description of the intranasal drug treatment and SARS-CoV-2 beta infection in mice. The drug treatment was repeated twice a day at 6 hours intervals at day 0 and day 1. At 48 hpi (day 2) and 96 hpi (day 4) mice were euthanized and lungs processed for PCR analysis and Immunohystochemistry to detect viral RNA and the tissue distribution of infection, respectively. B. PCR quantification of viral RNA in the lungs of mice treated intranasally with indicated drugs and infected with SARS-CoV-2 beta. For each mouse, the levels of viral RNA were detected using two primer sets, one targeting the viral genomic RNA dependent RNA polymerase (RdRp) gene and other the sub-genomic viral gene E (subE). For each mouse, the obtained values were normalized first to the levels of actin in the same lung tissue and then to the mean viral RNA value obtained in vehicle control-treated infected mice (indicated as 100%). For each treatment, the mean (white bar) and standard deviation of the mean are indicated. Each data point represents the RNA reads from one mouse. For each treatment group, the day of euthanasia is indicated on the X axis. Apilimod dimesylate 2 mg/kg, nafamostat mesylate 4 mg/kg. C. Immunohistology for SARS-CoV-2 NP in the lungs at 2 and 4 days post infection with SARS-CoV-2 beta and treated as described above. Virus infected cells were identified with an antibody against the viral N protein, using the horseradish peroxidase method (brown) and haematoxylin counterstain. Bars = 500 µm. At 2 dpi, vehicle treated mice exhibit widespread SARS-CoV-2 N expression both in bronchiolar epithelial cells and in pneumocytes in groups of alveoli. In nafamostat and apilimod treated mice, there is no evidence of viral antigen expression. At 4 dpi, the infection has been cleared in the vehicle treated mouse, i.e. there is no evidence of viral antigen expression. In mice treated with nafamostat and apilimod for the first two days after intranasal virus challenge, there are a few small groups of alveoli with viral antigen expression in pneumocytes (inset: arrowhead; the inset is a higher magnification of the area highlighted by the arrowhead in the overview image).

Article Snippet: Stocks of Apilimod dimesylate (Tocris, catalogue number 7283) and Nafamostat mesylate (Tocirs, catalogue number 3081) were diluted in the same medium and added at indicated times.

Techniques: Infection, Control, Standard Deviation, Virus, Expressing

Journal: Nature Metabolism

Article Title: Lysosomal phosphoinositide turnover acts upstream of RagGTPase–mTORC1 and controls muscle growth

doi: 10.1038/s42255-026-01484-1

Figure Lengend Snippet: a , A schematic of the lysosomal phosphoinositide conversion pathway linking MTM1 phosphatase activity to RagGTPase–mTORC1 signalling. PI3P and PI(3,5)P 2 are dynamically regulated by PI3KC2β/Vps34, MTM1 and PIKfyve at the lysosome. b , c , MTM1 phosphatase activity is required to repress mTORC1 signalling and support myogenic differentiation. Representative immunoblots of mTORC1 activity (p-S6K/S6K) ( b ) and representative images with quantification of fusion index and myotube area ( c ) in MTM1 -KO Cas9 myotubes expressing wild-type MTM1 (FL) or phosphatase-dead MTM1 (C375S). Data are shown as mean ± s.d.; n = 20 myotubes (fusion index) and n = 22 myotubes (area) from three biologically independent experiments; one-way ANOVA with Dunnett’s multiple-comparisons test; scale bar, 100 µm. d , MTM1 phosphatase activity is necessary for normal lysosomal abundance of the LAMTOR–RagGTPase complex. Representative immunoblots showing that MTM1-C375S does not restore lysosomal LAMTOR and RagA levels in MTM1 -KO Cas9 myotubes (three biologically independent experiments). e , The quantification of total cellular and lysosomal phosphoinositide species (PIPx) in CTRL and MTM1 -KO Cas9 myotubes. Data are shown as mean ± s.d. from three biologically independent experiments for whole-cell extracts and six biologically independent Lyso-IP preparations for lysosomal fractions. Two-sided Welch’s t- test. f , The lipidomic profiling of PI, PI3P, PI5P and PI(3,5)P 2 species in total cell and lysosomal fractions from CTRL and MTM1 -KO Cas9 myotubes. Data are shown as log 2 FC relative to CTRL; circle size reflects −log 10 P values. Two-sided Welch’s t- test; P < 0.01. g , PI3KC2β contributes to mTORC1 hyperactivation in MTM1 -KO Cas9 myotubes. Representative immunoblots showing reduced p-S6K/S6K following PI3KC2β knockdown using two independent shRNAs. Data are shown as mean ± s.d. from three biologically independent experiments. One-way ANOVA with Dunnett’s multiple-comparisons test. h , The pharmacological inhibition of PIKfyve partially suppresses mTORC1 signalling in MTM1 -KO Cas9 myotubes. Representative immunoblots of p-S6K/S6K following Apilm treatment. Data are shown as mean ± s.d. from three biologically independent experiments; one-way ANOVA with Dunnett’s multiple-comparisons test. i , Combined PI3KC2β knockdown and PIKfyve inhibition normalize lysosomal PI3P and PI(3,5)P 2 levels in MTM1 -KO Cas9 myotubes. Data are shown as mean ± s.d. of six biologically independent Lyso-IP preparations; one-way ANOVA with Dunnett’s multiple-comparisons test. j , The reduced lysosomal recruitment of the LAMTOR–Rag–mTORC1 complex following PI3KC2β depletion, with partial restoration upon PIKfyve inhibition. Representative Lyso-IP immunoblots showing LAMTORs, RagA, Raptor and mTOR in MTM1 -KO Cas9 myotubes. Two biologically independent experiments. For b , d , g , h and j , protein molecular weight is indicated in kDa. Illustration in a created in BioRender; Karim, H. https://biorender.com/n6ucxpz (2026).

Article Snippet: The following drugs were diluted in DMSO to obtain the appropriate concentration: the pan-PI3Ks inhibitors wortmannin (5 mM; W1628, Sigma) and LY294002 (5 mM; L9908, Sigma), the mTORC1-ATP competitors torin 1 (200 nM; 475991, Sigma) and AZD8055 (200 nM; S1555, Selleckchem), the p70 ribosomal S6 kinase (S6K1) inhibitor PF-4708671 (10 mM; PZ0143, Sigma), the eIF2α inhibitor salubrinal (10 mM; SML0951, Sigma), the Vps34 inhibitor IN1 (0.25–1 mM; S7980, Selleck) and the PikFyve inhibitor Apilm (0.05–1 mM; S0785, Selleck).

Techniques: Activity Assay, Cell Characterization, Western Blot, Expressing, Knockdown, Inhibition, Molecular Weight

Journal: Nature Metabolism

Article Title: Lysosomal phosphoinositide turnover acts upstream of RagGTPase–mTORC1 and controls muscle growth

doi: 10.1038/s42255-026-01484-1

Figure Lengend Snippet: a , Experimental strategy used to assess the impact of MTM1 loss of phosphatase activity (catalytically inactive mutant) on muscle cell differentiation in vitro and on muscle regeneration in vivo following injury. b , Representative images showing the effect of ectopic expression of the catalytically inactive MTM1 mutant (MTM1-C375S) on protein synthesis, assessed by HPG incorporation, in Mtm1-KO primary muscle cells. Scale bar, 100 µm. c , Representative immunoblots showing expression of MTM1 full-length (MTM1-FL) or catalytically inactive MTM1-C375S in Mtm1-KO primary cells and the corresponding impact on mTORC1 activity, assessed by the p-S6K/S6K ratio. d , Representative immunoblots showing expression of AAV-delivered GFP–MTM1-FL or GFP–MTM1-C375S in skeletal muscle following injury-induced regeneration and the associated effects on mTORC1 activity (p-S6K/S6K). e , Representative images of regenerating tibialis anterior (TA) muscle sections stained with hematoxylin and eosin (H&E) and GFP–MTM1 (FL or C375S). Scale bar, 200 µm. Left, quantification of nuclear position and muscle fiber diameter distribution in regenerating Mtm1-KO muscle expressing GFP–MTM1-FL or GFP–MTM1-C375S. f , Representative images of PI(3,5)P 2 detection in myotubes using an anti-PI(3,5)P 2 antibody. Increasing concentrations of the PIKfyve inhibitor apilimod were used to validate antibody specificity. Scale bar, 20 µm. g , Representative structured illumination microscopy (SIM) images showing colocalization of PI3P (2×FYVE probe) and PI(3,5)P 2 (eGFP–SNX-A probe or anti-PI(3,5)P 2 antibody) with Lyso-Tag (HA) or LAMP2 in CTRL and MTM1-KO cas9 myotubes at day 7 of differentiation. Scale bar, 20 µm. All graphs show mean ± s.d. Individual data points represent individual myotube areas, fibers, or cells analyzed across biologically independent samples. For b, data are from n = 20 myotube areas per condition; for f, n = 10 myotube areas per condition; for g, n = 29 (2×FYVE/HA), n = 80 (eGFP–SNX-A/LAMP2), and n = 47 (anti-PI(3,5)P 2 antibody) myotubes, all from three biologically independent experiments. For c – e , data represent three biologically independent experiments or mice, as indicated. One-way ANOVA followed by Dunnett’s multiple-comparisons test was used ( b – e ), and two-sided Mann–Whitney U tests were used ( g ). Molecular weights in c and d are indicated in kDa. Exact P values are reported in the figures, except when P < 0.0001. Illustration in a created in BioRender; Karim, H. https://biorender.com/n6ucxpz (2026).

Article Snippet: The following drugs were diluted in DMSO to obtain the appropriate concentration: the pan-PI3Ks inhibitors wortmannin (5 mM; W1628, Sigma) and LY294002 (5 mM; L9908, Sigma), the mTORC1-ATP competitors torin 1 (200 nM; 475991, Sigma) and AZD8055 (200 nM; S1555, Selleckchem), the p70 ribosomal S6 kinase (S6K1) inhibitor PF-4708671 (10 mM; PZ0143, Sigma), the eIF2α inhibitor salubrinal (10 mM; SML0951, Sigma), the Vps34 inhibitor IN1 (0.25–1 mM; S7980, Selleck) and the PikFyve inhibitor Apilm (0.05–1 mM; S0785, Selleck).

Techniques: Activity Assay, Mutagenesis, Cell Differentiation, In Vitro, In Vivo, Expressing, Western Blot, Staining, Microscopy, MANN-WHITNEY

Journal: Nature Metabolism

Article Title: Lysosomal phosphoinositide turnover acts upstream of RagGTPase–mTORC1 and controls muscle growth

doi: 10.1038/s42255-026-01484-1

Figure Lengend Snippet: a , Representative immunoblots showing the effect of VPS34 inhibition (IN1) on mTORC1 downstream signaling, assessed by p-S6K levels. b , Representative images showing the impact of VPS34 inhibition (IN1) on the differentiation capacity of MTM1-KO cas9 myotubes, quantified as fusion index. Scale bar, 100 µm. c , d , Representative images of MHC-labelled myotubes illustrating the effects of PI3KC2β knockdown (shRNA Sh1 and Sh2) and apilimod (Apilm) treatment on differentiation capacity of MTM1-KO cas9 myotubes, quantified as fusion index ( c ) and myotube area ( d ). Scale bar, 100 µm. e , Relative abundance of individual phosphoinositide (PIPₓ) species in lysosome immunoprecipitation (Lyso-IP) samples from MTM1-KO cas9 cells following PI3KC2β knockdown (shRNA-Sh2), apilimod treatment, or the combined condition. Lipid levels are expressed as FC relative to CTRL cells and normalized to Lyso-Tag (HA) content. All graphs show mean ± s.d. Individual data points represent individual myotubes or independent Lyso-IP preparations. For b , n = 15 myotubes per condition; for c , d , n = 24 myotubes per condition; all derived from three biologically independent experiments. For a and e, data represent three biologically independent experiments (Lyso-IP preparations). One-way ANOVA followed by Dunnett’s multiple-comparisons test was used throughout ( a – e ). Molecular weights in a are indicated in kDa. Exact P values are reported in the figures, except when P < 0.0001.

Article Snippet: The following drugs were diluted in DMSO to obtain the appropriate concentration: the pan-PI3Ks inhibitors wortmannin (5 mM; W1628, Sigma) and LY294002 (5 mM; L9908, Sigma), the mTORC1-ATP competitors torin 1 (200 nM; 475991, Sigma) and AZD8055 (200 nM; S1555, Selleckchem), the p70 ribosomal S6 kinase (S6K1) inhibitor PF-4708671 (10 mM; PZ0143, Sigma), the eIF2α inhibitor salubrinal (10 mM; SML0951, Sigma), the Vps34 inhibitor IN1 (0.25–1 mM; S7980, Selleck) and the PikFyve inhibitor Apilm (0.05–1 mM; S0785, Selleck).

Techniques: Western Blot, Inhibition, Knockdown, shRNA, Immunoprecipitation, Derivative Assay

Journal: PLoS Genetics

Article Title: PIKfyve regulates melanosome biogenesis

doi: 10.1371/journal.pgen.1007290

Figure Lengend Snippet: A) MNT-1 cells were treated with PIKfyve inhibitors YM-201636 (YM), apilimod or vehicle control for five days. A spectrophotometric melanin quantitation assay was used to measure the amount of accumulated melanin. (n = 6 as indicated by error bars). B) Equal numbers of MNT-1 cells treated with the indicated doses of YM or Apilimod were pelleted. Photographs of the cell pellets were obtained to document that YM and Apilimod treatment inhibited the accumulation of melanin. Normal human melanocytes (NHM) were treated with 1000 nM YM-201636 or vehicle without (Control) or with DOPA histochemistry (DOPA Tx) and (C,D) observed by electron microscopy Scale bar, C = 5 μm and D = 2 μm. (E) Pie graphs representing quantification of melanosome stages as percentage in NHM treated with 100, 500, or 1000 nM YM-021636 or vehicle. Each experiment was performed three times in triplicate. For all experiments, data shown are mean ± S.D *, p < 0.05; **, p < 0.01; or ***, p < 0.001 using a Student’s paired T test versus vehicle treated control.

Article Snippet: Cells were then incubated with varying concentrations of YM-201636 (Cayman Chemical), Apilimod (US Biological), or vehicle control (DMSO) in normal MNT-1 media.

Techniques: Control, Quantitation Assay, Electron Microscopy

Journal: PLoS Genetics

Article Title: PIKfyve regulates melanosome biogenesis

doi: 10.1371/journal.pgen.1007290

Figure Lengend Snippet: A) MNT-1 cells were treated with 1000 nM YM-201636 (YM), 100 nM apilimod (AP), or vehicle (VEH) control for 16 hours. Cells were fixed and stained with anti- MART-1 or anti-TYRP1 antibodies and imaged by confocal microscopy depicted as GFP positive puncta. Scale bar , 10 μm. B) The number of GFP positive puncta in each MNT-1 cell was counted, and at least 20 cells were included for each group. The data are presented as the mean ± S.D. based on three independent experiments, data shown as percent signal relative to vehicle. For all experiments, data shown are mean ± S.D *, p < 0.05; **, p < 0.01; or ***, p < 0.001 using a Student’s paired T test versus vehicle treated control. C) MNT-1 cells were treated with PIKfyve inhibitors YM-201636, apilimod or vehicle control for 72 hours. The relative accumulation of TYR, and unprocessed PMEL (100 kD) and Cathepsin D was measured by immunoblotting. Protein accumulation relative to GAPDH levels was quantified by densitometry. Pre pro cathepsin D and pro cathepsin D was quantified relative to GAPDH level (black) as well as relative to mature Cathepsin D (gray). Representative experiment of three independent experiments is shown.

Article Snippet: Cells were then incubated with varying concentrations of YM-201636 (Cayman Chemical), Apilimod (US Biological), or vehicle control (DMSO) in normal MNT-1 media.

Techniques: Control, Staining, Confocal Microscopy, Western Blot