monensin  (Tocris)

 
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  • 93
    Name:
    Monensin sodium salt
    Description:
    Sodium ionophore antibacterial agent
    Catalog Number:
    5223
    Price:
    None
    Category:
    Ionophores Ion Channels Pharmacology
    Formula:
    2-[5-Ethyltetrahydro-5-[tetrahydro-3-methyl-5-[tetrahydro-6-hydroxy-6-(hydroxymethyl)-3,5-dimethyl-2H-pyran-2-yl]-2-furyl]-2-furyl]-9-hydroxy-β-methoxy-α,γ,2,8-tetramethyl-1,6-Dioxaspiro[4.5]decane-7-butyric acid sodium salt
    Buy from Supplier


    Structured Review

    Tocris monensin
    Monensin sodium salt
    Sodium ionophore antibacterial agent
    https://www.bioz.com/result/monensin/product/Tocris
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    monensin - by Bioz Stars, 2021-03
    93/100 stars

    Images

    1) Product Images from "Lgl1 controls NG2 endocytic pathway to regulate oligodendrocyte differentiation and asymmetric cell division and gliomagenesis"

    Article Title: Lgl1 controls NG2 endocytic pathway to regulate oligodendrocyte differentiation and asymmetric cell division and gliomagenesis

    Journal: Nature Communications

    doi: 10.1038/s41467-018-05099-3

    Aberrant NG2 recycling is linked to differentiation defects. a Steady-state images of two single Lgl1 cKO cells monitored for NG2-EC (white dots) by TIRF, treated (bottom panel) or not treated (upper panel) with monensin. Pictures are acquired each 30 seconds. Note that NG2 is recycled to the membrane in non-treated cells (yellow arrow), whereas no recycling is observed in treated cells. Scale bar: 10 μm. b Fluorescent pictures illustrating Lgl1 cKO cells (Tom) monensin -treated or untreated and labeled for NG2 (blue) and O4 (red) after 5 days in differentiation conditions. Scale bar: 10 μm. c Graph representing quantification of O4+ and NG2+ cells in monensin-treated or untreated Lgl1 cKO cells. Untreated cells are mostly NG2+ and O4−, whereas treated cells show a reverse phenotype with a higher number of cells expressing O4. Data are depicted as mean ± s.e.m., n = 20 cells per genotype, three independent experiments (** p
    Figure Legend Snippet: Aberrant NG2 recycling is linked to differentiation defects. a Steady-state images of two single Lgl1 cKO cells monitored for NG2-EC (white dots) by TIRF, treated (bottom panel) or not treated (upper panel) with monensin. Pictures are acquired each 30 seconds. Note that NG2 is recycled to the membrane in non-treated cells (yellow arrow), whereas no recycling is observed in treated cells. Scale bar: 10 μm. b Fluorescent pictures illustrating Lgl1 cKO cells (Tom) monensin -treated or untreated and labeled for NG2 (blue) and O4 (red) after 5 days in differentiation conditions. Scale bar: 10 μm. c Graph representing quantification of O4+ and NG2+ cells in monensin-treated or untreated Lgl1 cKO cells. Untreated cells are mostly NG2+ and O4−, whereas treated cells show a reverse phenotype with a higher number of cells expressing O4. Data are depicted as mean ± s.e.m., n = 20 cells per genotype, three independent experiments (** p

    Techniques Used: Labeling, Expressing

    2) Product Images from "Lgl1 controls NG2 endocytic pathway to regulate oligodendrocyte differentiation and asymmetric cell division and gliomagenesis"

    Article Title: Lgl1 controls NG2 endocytic pathway to regulate oligodendrocyte differentiation and asymmetric cell division and gliomagenesis

    Journal: Nature Communications

    doi: 10.1038/s41467-018-05099-3

    Aberrant NG2 recycling is linked to differentiation defects. a Steady-state images of two single Lgl1 cKO cells monitored for NG2-EC (white dots) by TIRF, treated (bottom panel) or not treated (upper panel) with monensin. Pictures are acquired each 30 seconds. Note that NG2 is recycled to the membrane in non-treated cells (yellow arrow), whereas no recycling is observed in treated cells. Scale bar: 10 μm. b Fluorescent pictures illustrating Lgl1 cKO cells (Tom) monensin -treated or untreated and labeled for NG2 (blue) and O4 (red) after 5 days in differentiation conditions. Scale bar: 10 μm. c Graph representing quantification of O4+ and NG2+ cells in monensin-treated or untreated Lgl1 cKO cells. Untreated cells are mostly NG2+ and O4−, whereas treated cells show a reverse phenotype with a higher number of cells expressing O4. Data are depicted as mean ± s.e.m., n = 20 cells per genotype, three independent experiments (** p
    Figure Legend Snippet: Aberrant NG2 recycling is linked to differentiation defects. a Steady-state images of two single Lgl1 cKO cells monitored for NG2-EC (white dots) by TIRF, treated (bottom panel) or not treated (upper panel) with monensin. Pictures are acquired each 30 seconds. Note that NG2 is recycled to the membrane in non-treated cells (yellow arrow), whereas no recycling is observed in treated cells. Scale bar: 10 μm. b Fluorescent pictures illustrating Lgl1 cKO cells (Tom) monensin -treated or untreated and labeled for NG2 (blue) and O4 (red) after 5 days in differentiation conditions. Scale bar: 10 μm. c Graph representing quantification of O4+ and NG2+ cells in monensin-treated or untreated Lgl1 cKO cells. Untreated cells are mostly NG2+ and O4−, whereas treated cells show a reverse phenotype with a higher number of cells expressing O4. Data are depicted as mean ± s.e.m., n = 20 cells per genotype, three independent experiments (** p

    Techniques Used: Labeling, Expressing

    3) Product Images from "The metalloproteinase ADAM10 requires its activity to sustain surface expression"

    Article Title: The metalloproteinase ADAM10 requires its activity to sustain surface expression

    Journal: Cellular and Molecular Life Sciences

    doi: 10.1007/s00018-020-03507-w

    Recovery from ADAM10 depletion by de novo synthesis. a THP-1 cells were treated with 10 μM GI or vehicle control for 24 h. After washing, 2 μg/ml monensin or vehicle, control was added and cells were incubated for the indicated periods of recovery time before they were analyzed for surface expression of ADAM10 by flow cytometry. b THP-1 cells were treated with 10 μM GI or vehicle control for 24 h. After washing, 100 μM cycloheximide (CHX) or vehicle control was added and cells were incubated for the indicated periods of recovery time before they were analyzed for ADAM10 surface expression. *** p
    Figure Legend Snippet: Recovery from ADAM10 depletion by de novo synthesis. a THP-1 cells were treated with 10 μM GI or vehicle control for 24 h. After washing, 2 μg/ml monensin or vehicle, control was added and cells were incubated for the indicated periods of recovery time before they were analyzed for surface expression of ADAM10 by flow cytometry. b THP-1 cells were treated with 10 μM GI or vehicle control for 24 h. After washing, 100 μM cycloheximide (CHX) or vehicle control was added and cells were incubated for the indicated periods of recovery time before they were analyzed for ADAM10 surface expression. *** p

    Techniques Used: Incubation, Expressing, Flow Cytometry

    4) Product Images from "Lgl1 controls NG2 endocytic pathway to regulate oligodendrocyte differentiation and asymmetric cell division and gliomagenesis"

    Article Title: Lgl1 controls NG2 endocytic pathway to regulate oligodendrocyte differentiation and asymmetric cell division and gliomagenesis

    Journal: Nature Communications

    doi: 10.1038/s41467-018-05099-3

    Aberrant NG2 recycling is linked to differentiation defects. a Steady-state images of two single Lgl1 cKO cells monitored for NG2-EC (white dots) by TIRF, treated (bottom panel) or not treated (upper panel) with monensin. Pictures are acquired each 30 seconds. Note that NG2 is recycled to the membrane in non-treated cells (yellow arrow), whereas no recycling is observed in treated cells. Scale bar: 10 μm. b Fluorescent pictures illustrating Lgl1 cKO cells (Tom) monensin -treated or untreated and labeled for NG2 (blue) and O4 (red) after 5 days in differentiation conditions. Scale bar: 10 μm. c Graph representing quantification of O4+ and NG2+ cells in monensin-treated or untreated Lgl1 cKO cells. Untreated cells are mostly NG2+ and O4−, whereas treated cells show a reverse phenotype with a higher number of cells expressing O4. Data are depicted as mean ± s.e.m., n = 20 cells per genotype, three independent experiments (** p
    Figure Legend Snippet: Aberrant NG2 recycling is linked to differentiation defects. a Steady-state images of two single Lgl1 cKO cells monitored for NG2-EC (white dots) by TIRF, treated (bottom panel) or not treated (upper panel) with monensin. Pictures are acquired each 30 seconds. Note that NG2 is recycled to the membrane in non-treated cells (yellow arrow), whereas no recycling is observed in treated cells. Scale bar: 10 μm. b Fluorescent pictures illustrating Lgl1 cKO cells (Tom) monensin -treated or untreated and labeled for NG2 (blue) and O4 (red) after 5 days in differentiation conditions. Scale bar: 10 μm. c Graph representing quantification of O4+ and NG2+ cells in monensin-treated or untreated Lgl1 cKO cells. Untreated cells are mostly NG2+ and O4−, whereas treated cells show a reverse phenotype with a higher number of cells expressing O4. Data are depicted as mean ± s.e.m., n = 20 cells per genotype, three independent experiments (** p

    Techniques Used: Labeling, Expressing

    5) Product Images from "Rapid Clathrin-Mediated Uptake of Recombinant α-Gal-A to Lysosome Activates Autophagy"

    Article Title: Rapid Clathrin-Mediated Uptake of Recombinant α-Gal-A to Lysosome Activates Autophagy

    Journal: Biomolecules

    doi: 10.3390/biom10060837

    The rapid uptake of rh-α-Gal-A is achieved by clathrin and caveolae-mediated endocytosis. HEK293 ( A ), HUVEC ( B ), fibroblasts ( C ), and UKEC ( D ) cells were co-treated with the indicated concentration of rh-α-Gal A and inhibitors genistein, chloroquine, and monensin for 1 h. The enzyme assay was performed to determine the relative α-Gal A enzyme level. ( E ) HEK293, HUVEC, fibroblast, and UKEC cells were treated with the microtubule inhibitor, nocodazole. The enzyme assay was performed to determine the relative α-Gal A enzyme level. Values are average ±STDEV of minimum three experiments. * p
    Figure Legend Snippet: The rapid uptake of rh-α-Gal-A is achieved by clathrin and caveolae-mediated endocytosis. HEK293 ( A ), HUVEC ( B ), fibroblasts ( C ), and UKEC ( D ) cells were co-treated with the indicated concentration of rh-α-Gal A and inhibitors genistein, chloroquine, and monensin for 1 h. The enzyme assay was performed to determine the relative α-Gal A enzyme level. ( E ) HEK293, HUVEC, fibroblast, and UKEC cells were treated with the microtubule inhibitor, nocodazole. The enzyme assay was performed to determine the relative α-Gal A enzyme level. Values are average ±STDEV of minimum three experiments. * p

    Techniques Used: Concentration Assay, Enzymatic Assay

    The trafficking of the recombinant enzyme is inhibited in the presence of chloroquine, monensin, and nocodazole. Control fibroblasts were co-treated with fluorescence-labeled rh-α-Gal-A and inhibitors genistein, chloroquine, monensin, and nocodazole. ( A ) Immunofluorescence images of control fibroblasts treated with fluorescence-labeled rh-α-Gal-A (red). Bars: 200 μm. ( B ) Immunofluorescence images of control fibroblasts treated with fluorescence-labeled rh-α-Gal-A (red) and stained with LysoTracker (green). Bars: 200 μm. ( C ). The magnified merge image of cells with Alexa-Fluor-α-Gal-A (red) and LysoTracker (green) after nocodazole treatment.
    Figure Legend Snippet: The trafficking of the recombinant enzyme is inhibited in the presence of chloroquine, monensin, and nocodazole. Control fibroblasts were co-treated with fluorescence-labeled rh-α-Gal-A and inhibitors genistein, chloroquine, monensin, and nocodazole. ( A ) Immunofluorescence images of control fibroblasts treated with fluorescence-labeled rh-α-Gal-A (red). Bars: 200 μm. ( B ) Immunofluorescence images of control fibroblasts treated with fluorescence-labeled rh-α-Gal-A (red) and stained with LysoTracker (green). Bars: 200 μm. ( C ). The magnified merge image of cells with Alexa-Fluor-α-Gal-A (red) and LysoTracker (green) after nocodazole treatment.

    Techniques Used: Recombinant, Fluorescence, Labeling, Immunofluorescence, Staining

    Related Articles

    Synthesized:

    Article Title: Astrocytes Modulate Neural Network Activity by Ca2+-Dependent Uptake of Extracellular K+
    Article Snippet: The peak fluorescence intensity was calculated as percentage of baseline intensity before stimulation (Δ F / F ). .. Pharmacological agents used in cultures included adenosine (100 μM), ATP (100 μM), CPA (20 μM), glutamate (100 μM), H89 (10 μM), ionomycin (0.01 to 0.05 μM), monensin (5 nM to 3 μM), t -ACPD (100 μM), 2MeSADP (100 μM, Tocris), N 6 ,2′- O -dibutyryladenosine cAMP (DBcAMP, 1 mM), PGE2 (20 μM, Tocris), ouabain (1 mM), FMRF (15 μM), rhod-2 AM (4.5 μM, Invitrogen), phorbol 12-myristate 13-acetate (PMA, 1 μM), SEA0400 (10 μM, synthesized by Taisyo Pharmaceutical Co. Ltd.), potassium chloride (KCl, 5, 10, 20 mM), 1-[2-(4-methoxyphenyl)-2-[3-(4-methoxyphenyl)propoxy]ethyl]imidazole ( , 25 μM), (2-[[4-[(4nitrophenyl) methoxy]phenyl]methyl]-4-thiazolidinecarboxylic acid ethyl ester (SN6, 10 μM, Tocris), staurosporine (2 μM), UTP (100 μM), TFLLR-NH2 (30 μM, Tocris), MPEP (50 μM), (100 μM), and DL- threo -β-benzyloxyaspartic acid (TBOA, 100 μM, Tocris). .. To detect K+ changes in the extracellular space, we fabricated ion-sensitive microelectrodes from double-barreled pipette glass (PB150F-6, WPI) pulled to a tip of < 3 μm with a puller (Sutter P-97).

    other:

    Article Title: Non-canonical autophagy drives alternative ATG8 conjugation to phosphatidylserine
    Article Snippet: ReagentsBafilomycin A1 (#1334) and PP242 (#4257) were purchased from Tocris; Monensin (M5273), DAPI (D9542) and human IgG (I4506) were from Sigma.

    Article Title: Immunomodulation of the NLRP3 Inflammasome through Structure-Based Activator Design and Functional Regulation via Lysosomal Rupture
    Article Snippet: APG-4 was purchased from TEFLabs, monensin from Tocris, and filipin-III from Sigma-Aldrich.

    Concentration Assay:

    Article Title: Lgl1 controls NG2 endocytic pathway to regulate oligodendrocyte differentiation and asymmetric cell division and gliomagenesis
    Article Snippet: After live imaging, cells were fixed 10 min in PFA4%, stained with the appropriate antibodies and imaged with a Nikon Ti Yokogawa CSU-22 spinning disk confocal equipped with a Plan Apo VC 60×/1.4 oil objective. .. Endocytosis inhibitor Monensin (TOCRIS, 5223), a selective recycling endocytosis , was used and was not toxic at a working concentration of 10 µM. .. Univariate Cox model The prognostic significance of high Lgl1 expression (≥median) on overall survival (defined as the time between diagnosis and death due to any cause) was retrospectively tested.

    Article Title: Lgl1 controls NG2 endocytic pathway to regulate oligodendrocyte differentiation and asymmetric cell division and gliomagenesis
    Article Snippet: After live imaging, cells were fixed 10 min in PFA4%, stained with the appropriate antibodies and imaged with a Nikon Ti Yokogawa CSU-22 spinning disk confocal equipped with a Plan Apo VC 60×/1.4 oil objective. .. Monensin (TOCRIS, 5223), a selective recycling endocytosis , was used and was not toxic at a working concentration of 10 µM. .. The prognostic significance of high Lgl1 expression (≥median) on overall survival (defined as the time between diagnosis and death due to any cause) was retrospectively tested.

    Inhibition:

    Article Title: Lgl1 controls NG2 endocytic pathway to regulate oligodendrocyte differentiation and asymmetric cell division and gliomagenesis
    Article Snippet: Different quantities of NG2 recycling over time were represented as total corrected fluorescence measured using ImageJ. .. Recycling inhibition Lgl1 cKO OPC were treated with differentiation medium containing 10 μM Monensin (TOCRIS, 5223) for 4 h. Cells were monitored under TIRF for NG2 recycling monitoring during the first hour of treatment or were switched back to normal differentiation medium for 5 days before being fixed and immunostained for O4 and NG2. ..

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  • 93
    Tocris monensin
    The rapid uptake of rh-α-Gal-A is achieved by clathrin and caveolae-mediated endocytosis. HEK293 ( A ), HUVEC ( B ), fibroblasts ( C ), and UKEC ( D ) cells were co-treated with the indicated concentration of rh-α-Gal A and inhibitors genistein, chloroquine, and <t>monensin</t> for 1 h. The enzyme assay was performed to determine the relative α-Gal A enzyme level. ( E ) HEK293, HUVEC, fibroblast, and UKEC cells were treated with the microtubule inhibitor, nocodazole. The enzyme assay was performed to determine the relative α-Gal A enzyme level. Values are average ±STDEV of minimum three experiments. * p
    Monensin, supplied by Tocris, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/monensin/product/Tocris
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    monensin - by Bioz Stars, 2021-03
    93/100 stars
      Buy from Supplier

    Image Search Results


    The rapid uptake of rh-α-Gal-A is achieved by clathrin and caveolae-mediated endocytosis. HEK293 ( A ), HUVEC ( B ), fibroblasts ( C ), and UKEC ( D ) cells were co-treated with the indicated concentration of rh-α-Gal A and inhibitors genistein, chloroquine, and monensin for 1 h. The enzyme assay was performed to determine the relative α-Gal A enzyme level. ( E ) HEK293, HUVEC, fibroblast, and UKEC cells were treated with the microtubule inhibitor, nocodazole. The enzyme assay was performed to determine the relative α-Gal A enzyme level. Values are average ±STDEV of minimum three experiments. * p

    Journal: Biomolecules

    Article Title: Rapid Clathrin-Mediated Uptake of Recombinant α-Gal-A to Lysosome Activates Autophagy

    doi: 10.3390/biom10060837

    Figure Lengend Snippet: The rapid uptake of rh-α-Gal-A is achieved by clathrin and caveolae-mediated endocytosis. HEK293 ( A ), HUVEC ( B ), fibroblasts ( C ), and UKEC ( D ) cells were co-treated with the indicated concentration of rh-α-Gal A and inhibitors genistein, chloroquine, and monensin for 1 h. The enzyme assay was performed to determine the relative α-Gal A enzyme level. ( E ) HEK293, HUVEC, fibroblast, and UKEC cells were treated with the microtubule inhibitor, nocodazole. The enzyme assay was performed to determine the relative α-Gal A enzyme level. Values are average ±STDEV of minimum three experiments. * p

    Article Snippet: ChemicalsGenistein, chloroquine, monensin, nocodazole (cat no. 1228) were purchased from Tocris Bioscience (Bristol, UK).

    Techniques: Concentration Assay, Enzymatic Assay

    The trafficking of the recombinant enzyme is inhibited in the presence of chloroquine, monensin, and nocodazole. Control fibroblasts were co-treated with fluorescence-labeled rh-α-Gal-A and inhibitors genistein, chloroquine, monensin, and nocodazole. ( A ) Immunofluorescence images of control fibroblasts treated with fluorescence-labeled rh-α-Gal-A (red). Bars: 200 μm. ( B ) Immunofluorescence images of control fibroblasts treated with fluorescence-labeled rh-α-Gal-A (red) and stained with LysoTracker (green). Bars: 200 μm. ( C ). The magnified merge image of cells with Alexa-Fluor-α-Gal-A (red) and LysoTracker (green) after nocodazole treatment.

    Journal: Biomolecules

    Article Title: Rapid Clathrin-Mediated Uptake of Recombinant α-Gal-A to Lysosome Activates Autophagy

    doi: 10.3390/biom10060837

    Figure Lengend Snippet: The trafficking of the recombinant enzyme is inhibited in the presence of chloroquine, monensin, and nocodazole. Control fibroblasts were co-treated with fluorescence-labeled rh-α-Gal-A and inhibitors genistein, chloroquine, monensin, and nocodazole. ( A ) Immunofluorescence images of control fibroblasts treated with fluorescence-labeled rh-α-Gal-A (red). Bars: 200 μm. ( B ) Immunofluorescence images of control fibroblasts treated with fluorescence-labeled rh-α-Gal-A (red) and stained with LysoTracker (green). Bars: 200 μm. ( C ). The magnified merge image of cells with Alexa-Fluor-α-Gal-A (red) and LysoTracker (green) after nocodazole treatment.

    Article Snippet: ChemicalsGenistein, chloroquine, monensin, nocodazole (cat no. 1228) were purchased from Tocris Bioscience (Bristol, UK).

    Techniques: Recombinant, Fluorescence, Labeling, Immunofluorescence, Staining

    Aberrant NG2 recycling is linked to differentiation defects. a Steady-state images of two single Lgl1 cKO cells monitored for NG2-EC (white dots) by TIRF, treated (bottom panel) or not treated (upper panel) with monensin. Pictures are acquired each 30 seconds. Note that NG2 is recycled to the membrane in non-treated cells (yellow arrow), whereas no recycling is observed in treated cells. Scale bar: 10 μm. b Fluorescent pictures illustrating Lgl1 cKO cells (Tom) monensin -treated or untreated and labeled for NG2 (blue) and O4 (red) after 5 days in differentiation conditions. Scale bar: 10 μm. c Graph representing quantification of O4+ and NG2+ cells in monensin-treated or untreated Lgl1 cKO cells. Untreated cells are mostly NG2+ and O4−, whereas treated cells show a reverse phenotype with a higher number of cells expressing O4. Data are depicted as mean ± s.e.m., n = 20 cells per genotype, three independent experiments (** p

    Journal: Nature Communications

    Article Title: Lgl1 controls NG2 endocytic pathway to regulate oligodendrocyte differentiation and asymmetric cell division and gliomagenesis

    doi: 10.1038/s41467-018-05099-3

    Figure Lengend Snippet: Aberrant NG2 recycling is linked to differentiation defects. a Steady-state images of two single Lgl1 cKO cells monitored for NG2-EC (white dots) by TIRF, treated (bottom panel) or not treated (upper panel) with monensin. Pictures are acquired each 30 seconds. Note that NG2 is recycled to the membrane in non-treated cells (yellow arrow), whereas no recycling is observed in treated cells. Scale bar: 10 μm. b Fluorescent pictures illustrating Lgl1 cKO cells (Tom) monensin -treated or untreated and labeled for NG2 (blue) and O4 (red) after 5 days in differentiation conditions. Scale bar: 10 μm. c Graph representing quantification of O4+ and NG2+ cells in monensin-treated or untreated Lgl1 cKO cells. Untreated cells are mostly NG2+ and O4−, whereas treated cells show a reverse phenotype with a higher number of cells expressing O4. Data are depicted as mean ± s.e.m., n = 20 cells per genotype, three independent experiments (** p

    Article Snippet: Endocytosis inhibitor Monensin (TOCRIS, 5223), a selective recycling endocytosis , was used and was not toxic at a working concentration of 10 µM.

    Techniques: Labeling, Expressing

    The ATG16L1 WD40 domain supports alternative LC3 lipidation, at PS enriched membranes. HCT116 ATG16L1 -/- cells, re-expressing WT or K490A ATG16L1, and GFP-LC3B, were treated -/+ 100 μM monensin (Mon) for 40 mins. Cells were analysed by a , western blot, b , confocal microscopy (scale bar: 20 μm) and c, d , Mass spectrometry for normalised LC3B-PE or PS. Data represent 3-4 independent experiments, ***p

    Journal: bioRxiv

    Article Title: Non-canonical autophagy drives alternative ATG8 conjugation to phosphatidylserine

    doi: 10.1101/2020.05.14.096115

    Figure Lengend Snippet: The ATG16L1 WD40 domain supports alternative LC3 lipidation, at PS enriched membranes. HCT116 ATG16L1 -/- cells, re-expressing WT or K490A ATG16L1, and GFP-LC3B, were treated -/+ 100 μM monensin (Mon) for 40 mins. Cells were analysed by a , western blot, b , confocal microscopy (scale bar: 20 μm) and c, d , Mass spectrometry for normalised LC3B-PE or PS. Data represent 3-4 independent experiments, ***p

    Article Snippet: Reagents Bafilomycin A1 (#1334) and PP242 (#4257) were purchased from Tocris; Monensin (M5273), DAPI (D9542) and human IgG (I4506) were from Sigma.

    Techniques: Expressing, Western Blot, Confocal Microscopy, Mass Spectrometry

    Pharmacological activation of non-canonical autophagy promotes ATG8-PS lipidation in cells. Wild type MCF10A GFP-LC3A cells were treated with 1 μM PP242/100 nM BafA1 for 60 mins. ATG13 -/- cells were treated with 100 μM monensin (Mon) for 40mins. a , Confocal images of GFP-LC3A and DAPI. Scale bar: 20 μm. b , Western blotting for ATG13 or GAPDH, GFP-IPs were visualised by Coomassie staining. c , C-terminal peptides of LC3A conjugated to either the PE or PS headgroup. Predicted MWs are indicated. d , Representative CID mass spectra of unmodified, PE or PS modified LC3A peptides. y-ions (C-terminal) undergo monoisotopic mass shift upon modification: 197.05, glycerophosphoethanolamine (from PE); 241.04, glycerophosphoserine (from PS); y8 peaks are highlighted (arrow heads) as examples. Some y-ions give a secondary fragment consistent with neutral loss of phosphoglycerol (172). As expected, b-ions (N-terminal) do not shift. b14 is characteristically absent from the unmodified peptide, but can be observed in the modified peptides, along with b* (cleavage between Gly and the head group), confirming the C-terminal amide linked modification. e-h , Normalized mass spectrometry analysis of LC3-PE and LC3A-PS in WT e, f , and ATG13 -/- g, h , cells. Data represent means from 3 independent experiments, **p

    Journal: bioRxiv

    Article Title: Non-canonical autophagy drives alternative ATG8 conjugation to phosphatidylserine

    doi: 10.1101/2020.05.14.096115

    Figure Lengend Snippet: Pharmacological activation of non-canonical autophagy promotes ATG8-PS lipidation in cells. Wild type MCF10A GFP-LC3A cells were treated with 1 μM PP242/100 nM BafA1 for 60 mins. ATG13 -/- cells were treated with 100 μM monensin (Mon) for 40mins. a , Confocal images of GFP-LC3A and DAPI. Scale bar: 20 μm. b , Western blotting for ATG13 or GAPDH, GFP-IPs were visualised by Coomassie staining. c , C-terminal peptides of LC3A conjugated to either the PE or PS headgroup. Predicted MWs are indicated. d , Representative CID mass spectra of unmodified, PE or PS modified LC3A peptides. y-ions (C-terminal) undergo monoisotopic mass shift upon modification: 197.05, glycerophosphoethanolamine (from PE); 241.04, glycerophosphoserine (from PS); y8 peaks are highlighted (arrow heads) as examples. Some y-ions give a secondary fragment consistent with neutral loss of phosphoglycerol (172). As expected, b-ions (N-terminal) do not shift. b14 is characteristically absent from the unmodified peptide, but can be observed in the modified peptides, along with b* (cleavage between Gly and the head group), confirming the C-terminal amide linked modification. e-h , Normalized mass spectrometry analysis of LC3-PE and LC3A-PS in WT e, f , and ATG13 -/- g, h , cells. Data represent means from 3 independent experiments, **p

    Article Snippet: Reagents Bafilomycin A1 (#1334) and PP242 (#4257) were purchased from Tocris; Monensin (M5273), DAPI (D9542) and human IgG (I4506) were from Sigma.

    Techniques: Activation Assay, Western Blot, Staining, Modification, Mass Spectrometry

    GABARAP proteins are conjugated to PS during non-canonical autophagy. a , C-terminal peptides of GABARAP proteins conjugated to either the PE or PS headgroup. Predicted MWs are indicated. b, c , MCF10A cells expressing different GFP-tagged GABARAP proteins were treated with 100 μM monensin for 60 mins to activate SMAC and analysed for lipidation to PE and PS. Data represent means from 3 independent experiments, ****p

    Journal: bioRxiv

    Article Title: Non-canonical autophagy drives alternative ATG8 conjugation to phosphatidylserine

    doi: 10.1101/2020.05.14.096115

    Figure Lengend Snippet: GABARAP proteins are conjugated to PS during non-canonical autophagy. a , C-terminal peptides of GABARAP proteins conjugated to either the PE or PS headgroup. Predicted MWs are indicated. b, c , MCF10A cells expressing different GFP-tagged GABARAP proteins were treated with 100 μM monensin for 60 mins to activate SMAC and analysed for lipidation to PE and PS. Data represent means from 3 independent experiments, ****p

    Article Snippet: Reagents Bafilomycin A1 (#1334) and PP242 (#4257) were purchased from Tocris; Monensin (M5273), DAPI (D9542) and human IgG (I4506) were from Sigma.

    Techniques: Expressing

    LC3A-PS and LC3A-PE undergo differential delipidation by the ATG4 family. a , Molecular modelling of LC3-PE and LC3-PS in complex with ATG4B (based on 2Z0D.pdb), with critical ATG4B catalytic residues marked. b , Coomassie staining of GFP-LC3A immunoprecipitated from monensin (mon) treated MCF10A ATG13 -/- cells, then incubated -/+ recombinant ATG4B for 60 mins. c , Mass spectrometry analysis of LC3-PE and LC3-PS from monensin treated cells following a time course of ATG4B incubation and d , 3 independent experiments at 60 mins. Data represent means normalised to time 0, *p

    Journal: bioRxiv

    Article Title: Non-canonical autophagy drives alternative ATG8 conjugation to phosphatidylserine

    doi: 10.1101/2020.05.14.096115

    Figure Lengend Snippet: LC3A-PS and LC3A-PE undergo differential delipidation by the ATG4 family. a , Molecular modelling of LC3-PE and LC3-PS in complex with ATG4B (based on 2Z0D.pdb), with critical ATG4B catalytic residues marked. b , Coomassie staining of GFP-LC3A immunoprecipitated from monensin (mon) treated MCF10A ATG13 -/- cells, then incubated -/+ recombinant ATG4B for 60 mins. c , Mass spectrometry analysis of LC3-PE and LC3-PS from monensin treated cells following a time course of ATG4B incubation and d , 3 independent experiments at 60 mins. Data represent means normalised to time 0, *p

    Article Snippet: Reagents Bafilomycin A1 (#1334) and PP242 (#4257) were purchased from Tocris; Monensin (M5273), DAPI (D9542) and human IgG (I4506) were from Sigma.

    Techniques: Staining, Immunoprecipitation, Incubation, Recombinant, Mass Spectrometry

    Lipidomic analysis of monensin treated cells. Global lipids from ATG13 -/- MCF10A cells +/-100 μM monensin for 60 mins were analysed for a , phosphoethanolamine (PE) or b , phosphatidylserine (PS) molecular species. Data represent means from 6 biological replicates.

    Journal: bioRxiv

    Article Title: Non-canonical autophagy drives alternative ATG8 conjugation to phosphatidylserine

    doi: 10.1101/2020.05.14.096115

    Figure Lengend Snippet: Lipidomic analysis of monensin treated cells. Global lipids from ATG13 -/- MCF10A cells +/-100 μM monensin for 60 mins were analysed for a , phosphoethanolamine (PE) or b , phosphatidylserine (PS) molecular species. Data represent means from 6 biological replicates.

    Article Snippet: Reagents Bafilomycin A1 (#1334) and PP242 (#4257) were purchased from Tocris; Monensin (M5273), DAPI (D9542) and human IgG (I4506) were from Sigma.

    Techniques:

    Recovery from ADAM10 depletion by de novo synthesis. a THP-1 cells were treated with 10 μM GI or vehicle control for 24 h. After washing, 2 μg/ml monensin or vehicle, control was added and cells were incubated for the indicated periods of recovery time before they were analyzed for surface expression of ADAM10 by flow cytometry. b THP-1 cells were treated with 10 μM GI or vehicle control for 24 h. After washing, 100 μM cycloheximide (CHX) or vehicle control was added and cells were incubated for the indicated periods of recovery time before they were analyzed for ADAM10 surface expression. *** p

    Journal: Cellular and Molecular Life Sciences

    Article Title: The metalloproteinase ADAM10 requires its activity to sustain surface expression

    doi: 10.1007/s00018-020-03507-w

    Figure Lengend Snippet: Recovery from ADAM10 depletion by de novo synthesis. a THP-1 cells were treated with 10 μM GI or vehicle control for 24 h. After washing, 2 μg/ml monensin or vehicle, control was added and cells were incubated for the indicated periods of recovery time before they were analyzed for surface expression of ADAM10 by flow cytometry. b THP-1 cells were treated with 10 μM GI or vehicle control for 24 h. After washing, 100 μM cycloheximide (CHX) or vehicle control was added and cells were incubated for the indicated periods of recovery time before they were analyzed for ADAM10 surface expression. *** p

    Article Snippet: NH4 Cl and saponin were from Carl Roth (Karlsruhe, Germany), monensin was from Tocris Bioscience (Bristol, UK), cycloheximide, ionomycin, and bafilomycin A1 were from Sigma-Aldrich (St. Louis, USA), ikarugamycin was from biomol (Hamburg, Germany), recombinant human TIMP-1 (970-TM-010) was from R & D Systems (Minneapolis, USA), E. coli pHrodo (P35366), EZ-Link™ Sulfo-NHS-LC-Biotin, and Aldehyde/Sulfate latex beads were from Thermo Fisher Scientific (Waltham, USA), Streptavidin-Sepharose High Performance was from GE Healthcare (Chicago, USA) and the ADAM10 substrate peptide (PRYEAYKMG) was from peptides & elephants (Hennigsdorf, Germany).

    Techniques: Incubation, Expressing, Flow Cytometry