apd  (MedChemExpress)

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: 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