gal3  (Galectin Therapeutics)

Journal: Biomolecules

Article Title: Galectin-3 Binds to the Allosteric Site and Activates Integrins αvβ3, αIIbβ3, and α5β1, and Lactose Inhibits This Activation

doi: 10.3390/biom16040586

Figure Lengend Snippet: Gal3 binding to soluble integrins αvβ3 and αIIbβ3 in 1 mM Mn 2+ . ( a , b ) Gal3 binding to soluble integrins was measured as described in Methods. Briefly, Gal3 was immobilized to wells and incubated with soluble αvβ3 or αIIbβ3. Bound integrins were measured using anti-β3 and HRP-conjugated anti-mouse IgG. Data is shown as means +/− SD of triplicate experiments. ( c , d ) Effect of integrin inhibitors RGDfV (1 mM) and eptifibatide (0.65 μg/mL) on Gal3 binding to soluble αvβ3 and αIIbβ3, respectively, was measured. ( e , f ) Effect of lactose (up to 50 mM) on Gal3 binding to soluble αvβ3 and αIIbβ3, respectively, was measured. There was no significant effect of lactose on Gal3 binding to integrins. Data is shown as means +/− SD of triplicate experiments.

Article Snippet: Galectin-3 (Gal3): cDNA encoding the carbohydrate-binding domain of Gal3 (residues 114–250) [LIVPYNLPLPGGVVPRMLITILGTVKPNANRIALDFQRGNDVAFHFNPRFNENNRRVIVCNTKLDNNWGREERQSVFPFESGKPFKIQVLVEPDHFKVAVNDAHLLQYNHRVKKLNEISKLGISGDIDLTSASYTMI] were synthesized and subcloned into the BamHI/EcoRI site of pET28a vector.

Techniques: Binding Assay, Incubation

Journal: Biomolecules

Article Title: Galectin-3 Binds to the Allosteric Site and Activates Integrins αvβ3, αIIbβ3, and α5β1, and Lactose Inhibits This Activation

doi: 10.3390/biom16040586

Figure Lengend Snippet: Docking simulation of Gal3 binding to αvβ3. Docking simulation between Gal3 and open headpiece αvβ3 (1L5G.pdb) was performed using autodock3 as described in the . ( a ) Clustering analysis of docked poses. The first cluster with docking energy −23 kcal/mol represents the most likely poses when Gal3 binds to site 1. ( b ) Docking model of Gal3 binding to site 1 of αvβ3 (1L5G.pdb). ( c ) Binding of Gal3 mutations to αvβ3 in 1 mM Mn 2+ . Point mutations of amino acid residues of Gal3 in the predicted binding interface effectively suppressed Gal3 binding to soluble αvβ3. ( d ) Binding of Gal3 mutations to αIIbβ3 in 1 mM Mn 2+ . Point mutations of amino acid residues of Gal3 in the predicted binding interface effectively suppressed Gal3 binding to soluble αIIbβ3. Data is shown as means +/− SD of triplicate experiments.

Article Snippet: Galectin-3 (Gal3): cDNA encoding the carbohydrate-binding domain of Gal3 (residues 114–250) [LIVPYNLPLPGGVVPRMLITILGTVKPNANRIALDFQRGNDVAFHFNPRFNENNRRVIVCNTKLDNNWGREERQSVFPFESGKPFKIQVLVEPDHFKVAVNDAHLLQYNHRVKKLNEISKLGISGDIDLTSASYTMI] were synthesized and subcloned into the BamHI/EcoRI site of pET28a vector.

Techniques: Binding Assay

Journal: Biomolecules

Article Title: Galectin-3 Binds to the Allosteric Site and Activates Integrins αvβ3, αIIbβ3, and α5β1, and Lactose Inhibits This Activation

doi: 10.3390/biom16040586

Figure Lengend Snippet: Docking simulation of galectin-3 to site 2 of αvβ3 (1JV2.pdb). Docking simulation between Gal3 and closed-headpiece αvβ3 (1JV2) was performed using autodock3 as described in the . ( a ) Docking model of galectin-3-αvβ3 interaction. The positions of amino acid residues in the predicted site 2 binding site. Amino acid residues involved in the interaction are shown in . ( b ) The clustering analysis of docked poses. The first cluster with docking energy −21 kcal/mol represents the most likely poses when Gal3 binds to site 2. ( c ) Binding of cyclic site 2 peptide and β3 SDL peptide to Gal3. Peptide binding was performed as described in the . Cyclic site 2 peptide, β3 SDL peptide and β3 SDL peptide bind significantly better to Gal3 than scramble peptide. Data is shown as means +/− SD of triplicate experiments.

Article Snippet: Galectin-3 (Gal3): cDNA encoding the carbohydrate-binding domain of Gal3 (residues 114–250) [LIVPYNLPLPGGVVPRMLITILGTVKPNANRIALDFQRGNDVAFHFNPRFNENNRRVIVCNTKLDNNWGREERQSVFPFESGKPFKIQVLVEPDHFKVAVNDAHLLQYNHRVKKLNEISKLGISGDIDLTSASYTMI] were synthesized and subcloned into the BamHI/EcoRI site of pET28a vector.

Techniques: Binding Assay

Journal: Biomolecules

Article Title: Galectin-3 Binds to the Allosteric Site and Activates Integrins αvβ3, αIIbβ3, and α5β1, and Lactose Inhibits This Activation

doi: 10.3390/biom16040586

Figure Lengend Snippet: Gal3 binds to the allosteric binding site (site 2) and induces allosteric activation of soluble integrins. ( a , b ) Activation of soluble integrins. Gal3 was incubated with soluble αvβ3 ( a ) or αIIbβ3 ( b ) in 1 mM Ca 2+ . Bound integrins were measured using anti-human β3 and HRP-conjugated anti-mouse IgG. ( c – f ). Mutations of the site 2 binding interface of Gal3 inhibit activation of soluble integrin αvβ3 ( c ) and soluble αIIbβ3 ( d ). ( e , f ) Integrin activation by Gal3 mutations (at 50 μg/mL). Data is shown as means +/− SD of triplicate experiments.

Article Snippet: Galectin-3 (Gal3): cDNA encoding the carbohydrate-binding domain of Gal3 (residues 114–250) [LIVPYNLPLPGGVVPRMLITILGTVKPNANRIALDFQRGNDVAFHFNPRFNENNRRVIVCNTKLDNNWGREERQSVFPFESGKPFKIQVLVEPDHFKVAVNDAHLLQYNHRVKKLNEISKLGISGDIDLTSASYTMI] were synthesized and subcloned into the BamHI/EcoRI site of pET28a vector.

Techniques: Binding Assay, Activation Assay, Incubation

Journal: Biomolecules

Article Title: Galectin-3 Binds to the Allosteric Site and Activates Integrins αvβ3, αIIbβ3, and α5β1, and Lactose Inhibits This Activation

doi: 10.3390/biom16040586

Figure Lengend Snippet: Gal3 activates α5β1 by binding to site 2. Docking simulation of α5β1-Gal3 interaction. Docking simulation of α5β1 (4wjk.pdb) and Gal3 (1A3K.pdb) was carried out as described in the . ( a ) Activation of soluble α5β1 by Gal3 in 1 mM Ca 2+ in a dose-dependent manner. α5β1 activation was assayed as described for αvβ3 except that His-tagged soluble α5β1 was used. α5β1 was quantified using anti-His antibody conjugated with HRP. ( b ) Clustering analysis. The first cluster with docking energy −24.26 Kcal/mol represents the poses that bind to site 2 and activate α5β1. Docking simulation was performed as described in the . ( c ) Docking model of Gal3 binding to α5β1 in the first cluster. ( d ) Effect of mutations in the predicted site 2 binding interface of Gal3 to α5β1. Data is shown as means +/− SD of triplicate experiments. ( e ) Activation of cell-surface integrin α5β1 by Gal3. CHO cells (α5β1+) were incubated with Gal3 (50 μg/mL) and/or lactose (50 mM) in 1 mM Ca 2+ and FITC FN8-11. Bound FN8-11 was measured in flow cytometry. Data is shown as mean fluorescent intensity +/− SD of triplicate experiments.

Article Snippet: Galectin-3 (Gal3): cDNA encoding the carbohydrate-binding domain of Gal3 (residues 114–250) [LIVPYNLPLPGGVVPRMLITILGTVKPNANRIALDFQRGNDVAFHFNPRFNENNRRVIVCNTKLDNNWGREERQSVFPFESGKPFKIQVLVEPDHFKVAVNDAHLLQYNHRVKKLNEISKLGISGDIDLTSASYTMI] were synthesized and subcloned into the BamHI/EcoRI site of pET28a vector.

Techniques: Binding Assay, Activation Assay, Incubation, Flow Cytometry

Journal: Biomolecules

Article Title: Galectin-3 Binds to the Allosteric Site and Activates Integrins αvβ3, αIIbβ3, and α5β1, and Lactose Inhibits This Activation

doi: 10.3390/biom16040586

Figure Lengend Snippet: Anti-inflammatory FGF1, NRG1, and IVM suppress Gal3-induced integrin activation of αvβ3 ( a ) and αIIbβ3 ( b ). Activation of soluble αvβ3 ( a ) or αIIbβ3 ( b ) by Gal3 (25 μg/mL) in 1 mM Ca 2+ was measured as described in the . The effect of FGF1, IVM, or NRG1 (up to 100 μg/mL) was studied. Data is shown as means +/− SD of triplicate experiments. The statistical analysis of the effect of FGF1, NRG1, and IVM at 25 μg/mL was performed.

Article Snippet: Galectin-3 (Gal3): cDNA encoding the carbohydrate-binding domain of Gal3 (residues 114–250) [LIVPYNLPLPGGVVPRMLITILGTVKPNANRIALDFQRGNDVAFHFNPRFNENNRRVIVCNTKLDNNWGREERQSVFPFESGKPFKIQVLVEPDHFKVAVNDAHLLQYNHRVKKLNEISKLGISGDIDLTSASYTMI] were synthesized and subcloned into the BamHI/EcoRI site of pET28a vector.

Techniques: Activation Assay

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) Schematic illustration of putative roles of PI(4)P and PI(3)P in lysosome repair and removal pathways. ESCRT, endosomal sorting complex required for transport. PITT, phosphoinositide-initiated membrane tethering and lipid transport pathway. ER, endoplasmic reticulum. mTORC1, mechanistic target of rapamycin complex 1. (b) Biphasic PI(3)P dynamics in 0.5mM LLOMe-treated cells. The same HeLa cell expressing eGFP-2xFYVE was imaged before and after different durations of LLOMe treatment. Scale bar, 10 µm. (c) Left: quantification of (b), number of PI(3)P puncta/cell area before and after 30 minutes of 0.5mM LLOMe was compared by t test (n = 39 cells). Right: time course of PI(3)P change in 0.5mM LLOMe treated HeLa cells expressing eGFP-2xFYVE. Quantification of the number of PI(3)P puncta/cell area, fold change over control. (n = 39 cells). Data are mean ± s.e.m. (d) LLOMe decreases PI(3)P on lysosomes. Left: magnification of the perinuclear region of a U2OS cell co-expressing eGFP-2xFYVE and mCherry-LAMP1 imaged after different durations of 0.5mM LLOMe treatment. Capital letters indicate same vesicles before and after 8 minutes of LLOMe treatment. Scale bar, 5 µm. Middle: time-resolved changes of GFP-2xFYVE on the same mCherry-LAMP1 vesicles as indicated in the left panels by capital letters. Right: Representative time course (mean±s.e.m.) of LMP-induced PI(3)P decline on lysosomes from ten cells. See also and for quantifications. (e) Volcano plot illustrating the enrichment of phosphoinositide kinases, phosphatases, phosphoinositide-binding proteins, and lipid-transport proteins in Lyso-IP samples from LLOMe-treated cells. Lysosomes were immunoprecipitated from control or 30 minutes 1mM LLOMe-treated HEK293 cells expressing TMEM192-3xHA and subjected to quantitative MS/MS analysis. (f) Representative confocal images of mScarlet-MTMR14 CRISPR-Cas9 knockin U2OS cells treated with control (DMSO) or 15 minutes 1mM LLOMe. Cells were co-stained with antibodies specific for mScarlet (MTMR14) and LAMP1. Scale bar, 10 µm. (g) Representative confocal images of HeLa cells co-expressing YFP-MTMR14 with mCherry-Galectin3 imaged live before and after 5 and 15 minutes of 1mM LLOMe treatment. Scale bar, 10 µm. (h) Quantification of the number of MTMR14 puncta/cell from control and 1 h 1mM LLOMe treated HeLa cells. t test (n = 29 cells). (i) Expression of EGFP-Tau P301L recruits MTMR14 to lysosomes. Shown are magnifications of confocal images of HeLa cells co-expressing EGFP-Tau P301L with mCherry-MTMR14 and stained with antibodies against Lamp2a. Scale bar, 2 µm. (j) Left: representative confocal live cell images of wild type and MTMR14 KO U2OS cells co-expressing GFP-2xFYVE with mCherry-LAMP1 imaged before and after 20 minutes of 0.5mM LLOMe treatment. Right: quantification of the Pearson’s Coefficient of GFP-2xFYVE and mCherry-LAMP1 from LLOMe treated U2OS WT and MTMR14 KO cells before and after 20 minutes LLOMe treatment. Dotted line denotes Pearson’s Coefficient of GFP-2xFYVE and mCherry-LAMP1 before LLOMe set to 1. Scale bar, 20 µm. t test, n=43 cells in U2OS WT and 41 cells in U2OS MTMR14 KO cells. 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. See also and .

Article Snippet: At 24 or 48 hours post-transfection, lysosomal integrity was assessed by monitoring Galectin-3 (Gal3) recruitment, a marker of lysosomal membrane damage.

Techniques: Membrane, Expressing, Control, Binding Assay, Immunoprecipitation, Tandem Mass Spectroscopy, CRISPR, Knock-In, Staining, 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) Left: eGFP-2xFYVE cytosolic intensity before and after 24 minutes LLOMe treatment in U2OS cells. Right: eGFP-2xFYVE intensity in the cytosol at different times post-induction of LMP by LLOMe. Scale bar, 10 µm. Data represent mean of 12 cytoplasmic ROIs in 12 cells ± s.e.m. See also . (b) Quantification of the Pearson’s Coefficient of GFP-2xFYVE and mCherry-LAMP1 from U2OS cells before and after 23 minutes treatment with control (DMSO), 0.5mM LLOMe and 0.5mM LLOMe plus 5µM VPS34-IN1. Dotted line denotes Pearson’s Coefficient of GFP-2xFYVE and mCherry-LAMP1 before treatment set to 1. One-way ANOVA, n=10 cells analysed for each condition. (c) Left: time-lapse of single lysosome in U2OS cells co-expressing GFP-OSBP-PH and mCherry-LAMP1 imaged after different durations of LLOMe treatment. Scale bar, 2 µm. Right: quantification of the eGFP-OSBP-PH intensity on lysosomes. n = 30 lysosomes quantified from 14 cells. Data are mean ± s.e.m. (d) PI(3,5)P 2 dynamics after LLOMe treatment. Top: representative U2OS cell expressing GFP-SNXA imaged after different durations of LLOMe treatment. Scale bar, 10 µm. Bottom: the number of SNXA puncta/cell area quantified from images as shown in the top panel. Data represent mean of 3 independent experiment ± s.e.m from 33 cells. (e) Enrichment of myotubularin phosphatases in Lyso-IP fractions of 30 minutes 1mM LLOMe-treated over control (DMSO)-treated HEK293-TMEM192-3×HA cells determined by mass spectrometry. (f) Representative U2OS cells expressing GFP-MTM1, GFP-MTMR1, GFP-MTMR2, GFP-MTMR6, GFP-MTMR7, GFP-MTMR8 or YFP-MTMR14 imaged before and after 2 h LLOMe treatment. Scale bar, 10 µm. (g) Quantification of the number of MTMR14 puncta/cell from control and 1 h 1mM LLOMe treated U2OS cells. t test (n = 40 cells). (h) C2C12 cells co-expressing YFP-MTMR14 and mCherry-Galectin3 imaged after 2 h control (DMSO) or LLOMe treatment. Scale bar, 10 µm. (i) BV2 cells co-expressing YFP-MTMR14 and mCherry-Galectin3 imaged after 1 h LLOMe treatment. Scale bar, 10 µm. (j) HMC3 cells co-expressing YFP-MTMR14 and mCherry-Galectin3 imaged before and after LLOMe treatment. Scale bar, 10 µm. (k) Left: representative HeLa cells expressing YFP-MTMR14 imaged after 30 minutes treatment with different concentrations of LLOMe. Scale bar, 10 µm. Right: quantification of MTMR14 foci per cell in images as shown in the top panel. one-way ANOVA (34 cells in control, 41 cells in 100µM LLOMe, 36 cells in 250µM LLOMe, 32 cells in 500µM LLOMe, 30 cells in 1mM LLOMe). 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: At 24 or 48 hours post-transfection, lysosomal integrity was assessed by monitoring Galectin-3 (Gal3) recruitment, a marker of lysosomal membrane damage.

Techniques: Control, Expressing, Mass Spectrometry, 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 U2OS cell expressing YFP-MTMR14 stained with LysoTracker and imaged before and after 30 minutes 50 µM MSDH treatment. Scale bar, 10 µm. (b) Representative U2OS cell expressing YFP-MTMR14 stained with LysoTracker and imaged before and after 30 minutes 200 nM GPN treatment. Scale bar, 10 µm. (c) Representative U2OS cells expressing YFP-MTMR14 imaged before and after 5 µg/mL BAC, 0.5 mM H 2 O 2 , 0.25 M D-Mannitol, 50% H 2 O, 5 µM CCCP, 100 nM Bafilomycin A1 or 5 µM Nigericin treatment. Scale bar, 10 µm. (d) Left: representative U2OS cells co-expressing GFP-Tau P301L and mCherry-Galectin3 for 24 h or 48 h. Scale bar, 10 µm. Right: quantification of Galectin3 puncta/cell area after 24 h or 48 h GFP-Tau P301L expression in U2OS cells as shown in the left panel. t test (n = 3 independent experiments, total number of cells is 86 in 24 h and 107 in 48 h conditions). (e) Top: immunoblot of U2OS WT and MTMR14 KO #1 cells. Bottom: immunoblot of U2OS WT and MTMR14 KO #2 cells. (f) Left: WT and MTMR14 KO #1 U2OS cells expressing GFP-2xFYVE imaged before and after 1 h 1mM LLOMe treatment. Scale bar, 10 µm. Right: quantification of PI(3)P puncta/cell area in WT and MTMR14 KO #1 U2OS cells as shown in the left panel. t test (n = 4 independent experiments, total number of cells is 230 for WT and 190 for MTMR14 KO). (g) Impaired LMP-induced PI(3)P dynamics in MTMR14 knockout cells. Quantification of GFP 2xFYVE intensity from confocal images of U2OS WT and U2OS MTMR14 KO cells. Cells were treated with LLOMe for the indicated time points, fixed, permeabilized, and stained with purified GFP-2xFYVE as overlay probe. One-way ANOVA (n = 5 independent experiments, each datapoint represents 15 fields of view, one field of view containing 10-20 cells, with a size of 1664 × 1664 µm). Data are mean ± s.e.m. 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: At 24 or 48 hours post-transfection, lysosomal integrity was assessed by monitoring Galectin-3 (Gal3) recruitment, a marker of lysosomal membrane damage.

Techniques: Expressing, Staining, Western Blot, Knock-Out, Purification, 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) Left: defective lysosomal recovery in MTMR14 KO cells. Representative confocal images of LysoTracker loaded WT and MTMR14 KO clone #1 U2OS cells imaged before LLOMe treatment, after 30 minutes LLOMe treatment, and 4.5 h after washout of LLOMe. Scale bar, 20 µm. Right: quantification of mean LysoTracker intensity/field of view, fold change over control. t test (n = 6 independent experiments, each datapoint represents 10 fields of view, one field of view containing 35-50 cells, with a size of 3328 × 3328 µm). (b) Quantification of mean LysoTracker intensity/field of view after 5 h chronic LLOMe treatment in U2OS MTMR14 KO clone #1 fold change over WT U2OS cells, t test (n = 5 independent experiments, each datapoint represents 10 fields of view, one field of view containing 35-50 cells, with a size of 3328 × 3328 µm). (c) Left: Loss of MTMR14 causes elevated lysosomal membrane damage monitored by mCherry-Galectin3 in U2OS cells co-expressing mutant eGFP-Tau (P301L) for 24 hours. Confocal images of representative cells. Scale bar, 10 µm. Right: Quantification of mCherry-Galectin3 puncta/cell area in U2OS WT and MTMR14 KO cells after co-expressing mutant eGFP-Tau (P301L) for 24 hours. t test (n = 4 independent experiments, total number of cells is 107 for both WT and MTMR14 KO). (d) Quantification of cytotoxicity using LDH assay in 5 h 4mM LLOMe-treated WT and MTMR14 KO U2OS cells. t test (n = 4 independent experiments, each datapoint represents triplicate measurements). (e) MTMR14 regulates PI(4)P generation in response to LMP. Left: representative confocal images of WT and MTMR14 KO U2OS cells stained with DAPI and antibodies against PI(4)P and Lamp2a in control conditions (DMSO) and after 30 minutes LLOMe treatment. Right: quantification of PI(4)P intensity on Lamp2a detections in images as shown in the left panel. t test (n = 4 independent experiments, total number of fields of view is 109 for WT and 114 for MTMR14 KO, one field of view containing 10-15 cells, with a size of 1664 × 1664 µm). (f) Left: representative colored electron micrographs of lysosomes in WT and MTMR14 KO U2OS cells treated with LLOMe, ER is colored in blue and lysosomes in pink. Scale bar, 200 nm. Right: length of lysosome-ER MCS relative to lysosomal perimeter in WT and MTMR14 KO U2OS cells. t test, WT (n = 31 lysosomes), MTMR14 KO (n = 37 lysosomes). 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. See also .

Article Snippet: At 24 or 48 hours post-transfection, lysosomal integrity was assessed by monitoring Galectin-3 (Gal3) recruitment, a marker of lysosomal membrane damage.

Techniques: Control, Membrane, Expressing, Mutagenesis, Lactate Dehydrogenase Assay, Staining, Two Tailed Test

Journal: iScience

Article Title: An atom-edged magnetic nanomotor for cancer mechanotherapy

doi: 10.1016/j.isci.2026.114994

Figure Lengend Snippet: MagGO induces lysosomal disruption (A) CLSM images of MNPs and MagGO in U87 cells. Lysosomes were stained with LysoTracker red (red), and the MNPs and MagGO were labeled with FITC (green). Scale bars, 15 μm. (B and C) Intensity profiles (white dashed line) of signals from lysosome and MNPs/MagGO fluorescent channels in (A). (D) CLSM images of MNPs and MagGO in MDA-MB-231 cells. Lysosomes were stained with LysoTracker red (red), and the MNPs and MagGO were labeled with FITC (green). Scale bars, 15 μm. (E and F) Intensity profiles (white dashed line) of signals from lysosomes and MNPs/MagGO fluorescent channels in (D). (G) CLSM images of MNPs and MagGO in A549 cells. Lysosomes were stained with LysoTracker red (red), and the MNPs and MagGO were labeled with FITC (green). Scale bars, 15 μm. (H and I) Intensity profiles (white dashed line) of signals from lysosomes and MNPs/MagGO fluorescent channels in (G). (J) CLSM images of U87 cells transfected with the EGFP-Gal3 plasmid after MagGO treatment under 3D MF. The applied field strength is 75 mT. The duration of magnetic field application is 30 min. Scale bars, 15 μm. (K) Counts of Gal3 puncta per U87 cell ( n = 10). The data were presented as the mean ± SD. Data were analyzed by one-way ANOVA with Tukey’s post-hoc test. (L) Counts of Gal3 puncta per MDA-MB-231 cell ( n = 10). The data were presented as the mean ± SD. Data were analyzed by one-way ANOVA with Tukey’s post-hoc test. (M) Counts of Gal3 puncta per A549 cell ( n = 10). The data were presented as the mean ± SD. Data were analyzed by one-way ANOVA with Tukey’s post-hoc test. (N) Bio-TEM of lysosomal membrane morphology after mechanoporation for MagGO and MagGO+3D MF. The applied field strength is 75 mT. The duration of magnetic field application is 30 min. Scale bars, 1 μm. The dark blue arrow indicates the site of LMP, while the length of the blue arrow represents the size of the lysosomal membrane “wound”.

Article Snippet: The next day, cells were transfected with EGFP-Gal3 (provided by Dr. Bin Liu at Novo Nordisk company in Denmark) using Lipofectamine 3000 (Invitrogen) according to the manufacturer’s protocol.

Techniques: Disruption, Staining, Labeling, Transfection, Plasmid Preparation, Membrane

Journal: Journal of Extracellular Vesicles

Article Title: Development of a Live‐Cell Imaging Assay to Elucidate Spatiotemporal Dynamics of Extracellular Vesicle Fusion with Target Cells

doi: 10.1002/jev2.70228

Figure Lengend Snippet: EV‐FUSIM facilitates real‐time visualization of VSV‐G‐mediated EV‐fusion. (A) Live HeLa STAb‐GFP cells were subjected to time‐lapse imaging immediately after addition of medium only or medium containing 10 8 transfection ctl or VSV‐G EVs, taking Z‐stacks at a 15 min time interval. Images are maximum intensity projections acquired at 1 h post‐EV‐addition, showing fluorescence channels for mScar3 (top) and GFP (middle) in the same fields‐of‐view. Gamma was adjusted to 2 (mScar3) or 1.2 (GFP) for visualization purposes only. Scale bars represent 20 µm. White insets correspond to magnifications (bottom), which show mScar3, GFP and merged channels. Scale bar represents 5 µm. Images are representative of n = 3 independent experiments. (B–E) After segmentation of cells in 3D based on the GFP channel, cell‐associated fluorescent spots in the red and green channels were counted for each field‐of‐view. In tandem, the total cell volume per field‐of‐view was counted, which was used to correct spot counts for differences to the average cell volume per field‐of‐view. Graphs show the corrected mean spots per timepoint (B, D) or mean maximum spot detection over the course of the experiment (C, E) per field‐of‐view for both mScar3 and GFP channels ± SEM, calculated from n = 3 independent experiments with 2–6 fields‐of‐view per condition each. * p ≤0.05, ** p ≤0.01 and *** p ≤0.001 as determined by one‐way ANOVA with Tukey's multiple comparisons test. (F) Live HeLa STAb‐GFP were subjected to time‐lapse imaging 30 min after addition of medium containing 10 8 VSV‐G EVs, taking Z‐stacks at a 1 min time interval. Images are representative of n = 2 independent experiments. Overview image is a maximum intensity projection of the start of the experiment, showing a merge of fluorescence channels for mScar3 and GFP. Scale bar represents 20 µm. Insets shown in white correspond to magnified images of single EV‐containing endosomes on the right, showing fluorescence channels for mScar3, GFP and a merge of both at the indicated timepoints post‐EV‐addition. Scale bars represent 1 µm. (G) Live HeLa mAG‐Gal3 cells were subjected to time‐lapse imaging immediately after addition of medium only, medium containing 10 8 VSV‐G EVs or medium containing 1 mM LLOME, taking Z‐stacks at a 15 min time interval. Images are maximum intensity projections acquired at 1 h post‐EV‐addition, showing the fluorescence channel for mAG. Gamma was adjusted to 1.2 (mAG) for visualization purposes only. Scale bar represents 20 µm. (H, I) After segmentation of cells in 3D based on the mAG channel, cell‐associated fluorescent spots in the green channel were counted for each field‐of‐view. In tandem, the total cell volume per field‐of‐view was counted, which was used to correct spot counts for differences to the average cell volume per field‐of‐view. Graph shows the corrected mean spots per timepoint (H) or mean maximum spot detection over the course of the experiment (I) per field‐of‐view for the mAG channel ± SEM, calculated from n = 3 independent experiments with 5–6 fields‐of‐view per condition each. **** p≤0.0001 as determined by one‐way ANOVA with Tukey's multiple comparisons test.

Article Snippet: For the generation of endolysosomal rupture reporter cells (HeLa Gal3), a pHAGE2 mAG‐Gal3 plasmid (gift from Prof. Niels Geijsen—Addgene #62734) (D'Astolfo et al., ) for expression of mAzamiGreen‐tagged Galectin‐3, and a previously described pHR NLS‐BFP plasmid (Boersma et al., ) for tagging of the nucleus with Blue Fluorescent Protein, were used.

Techniques: Imaging, Transfection, Fluorescence

Journal: Neuroprotection

Article Title: Environmental enrichment modulates chronic poststroke inflammation and links white matter TREM2‐positive microglia in recovery in mice

doi: 10.1002/nep3.70028

Figure Lengend Snippet: Infarct size correlations with peri‐infarct immunofluorescence analysis of ionized calcium binding adaptor molecule 1 (Iba1), galectin‐3 (Gal3), and purinergic receptor P2Y12 (P2RY12). (A) Representative immunostaining of 4′,6‐diamidino‐2‐phenylindole (DAPI), Iba1, P2RY12, and Gal3 in a standard environment (SE) mouse at peri‐infarct area (at ×20 magnification). (B) Representative immunostaining of DAPI, Iba1, P2RY12, and Gal3 in an enriched environment (EE) mouse at peri‐infarct area (at ×20 magnification). (C) Quantification of indirect infarct area measurements. (D) Correlation of infarct area with Neuroscore per group. (E) Iba1 coverage quantification measured as the percentage of image covered by Iba1 area (%area). (F) Correlation of infarct area with Iba1 coverage. (G) Gal3 coverage quantification measured as the percentage of image covered by Iba1 + Gal3 + area (%area). (H) Correlation of infarct area with Gal3 coverage. (I) P2RY12 coverage quantification measured as the percentage of image covered by Iba1 + P2RY12 + area (%area). (J) Correlation of infarct area with P2RY12 coverage. Peri‐infarct area is shown as dashed red lines in A and B. In (C, E, G, and I), values are expressed as individual experimental replicates with mean ± SEM. In (D, F, H, and J), values are expressed as individual experimental replicates with simple linear regression lines; Pearson correlation's r value with p ‐value are also shown. In (C, E, G, and I), unpaired t ‐test was performed. n = 4/6 mice in SE and n = 7 mice in EE (2 mice in SE were not behaviorally characterized). P ‐values and r values are expressed with 3 decimals. P ‐values were not corrected for multiple comparisons.

Article Snippet: They were then incubated at 4°C overnight with one of the following antibodies: Iba1 (1:500, rabbit, Cat# 016‐26721; Wako), Gal3 (1:750, goat, Cat# AF1197; R&D Systems) P2RY12 (1:200, rat, Cat# S16007D; Biolegend).

Techniques: Immunofluorescence, Binding Assay, Immunostaining

Journal: Neuroprotection

Article Title: Environmental enrichment modulates chronic poststroke inflammation and links white matter TREM2‐positive microglia in recovery in mice

doi: 10.1002/nep3.70028

Figure Lengend Snippet: Infarct size correlations with white matter immunofluorescence analysis of ionized calcium binding adaptor molecule 1 (Iba1), galectin‐3 (Gal3), and purinergic receptor P2Y12 (P2RY12). (A) Representative immunostaining of 4′,6‐diamidino‐2‐phenylindole (DAPI), Iba1, P2RY12, and Gal3 (at ×20 magnification) in a standard environment (SE) mouse at white matter area (corpus callosum + external capsule). (B) Representative immunostaining of DAPI, Iba1, P2RY12, and Gal3 in an enriched environment (EE) mouse at white matter area (at ×20 magnification). (C) Iba1 coverage quantification measured as the percentage of image covered by Iba1 area (%area). (D) Gal3 coverage quantification measured as the percentage of image covered by Iba1 + Gal3 + area (%area). (E) P2RY12 coverage quantification measured as the percentage of image covered by Iba1 + P2RY12 + area (%area). (F) Correlation of infarct area with Iba1 coverage. (G) Correlation of infarct area with Gal3 coverage. (H) Correlation of infarct area with P2RY12 coverage. White matter area is shown as dashed red lines in (A, B). In (C–E) values are expressed as individual experimental replicates with mean ± SEM. In (F–H) values are expressed as individual experimental replicates with simple linear regression lines; Pearson correlation's r value with p ‐value are also shown. In (C–E) unpaired t ‐test was performed n = 6 mice in SE and n = 7 mice in EE. p ‐Values and r values are expressed with 3 decimals. p ‐Values were not corrected for multiple comparisons.

Article Snippet: They were then incubated at 4°C overnight with one of the following antibodies: Iba1 (1:500, rabbit, Cat# 016‐26721; Wako), Gal3 (1:750, goat, Cat# AF1197; R&D Systems) P2RY12 (1:200, rat, Cat# S16007D; Biolegend).

Techniques: Immunofluorescence, Binding Assay, Immunostaining

Journal: Neuroprotection

Article Title: Environmental enrichment modulates chronic poststroke inflammation and links white matter TREM2‐positive microglia in recovery in mice

doi: 10.1002/nep3.70028

Figure Lengend Snippet: Quantification of peri‐infarct myelin debris, white matter myelin loss, and their correlations with microglial markers. (A) Representative myelin staining in a standard environment mouse (at ×20 magnification). (B) Enlarged views of infarct contralateral cortical (green square) and peri‐infarct (red square) myelin in a standard environment (SE) mouse. (C) Representative myelin staining in an enriched environment mouse (at ×20 magnification). (D) Enlarged views of infarct contralateral cortical (green square) and peri‐infarct (red square) myelin in an enriched environment (EE) mouse. (E) Myelin debris coverage quantification measured as the percentage of peri‐infarct image covered by Black Gold Myelin dark debris area (%area). (F) Correlation of infarct area with myelin debris coverage. (G) Myelin loss quantification measured as the percentage of myelin lost at corpus callosum in ipsilateral versus contralateral infarct. (H) Correlation of infarct area with myelin loss. (I) Correlations of myelin debris with ionized calcium binding adaptor molecule 1 (Iba1), galectin‐3 (Gal3), purinergic receptor P2Y12 (P2RY12), cluster of differentiation 68 (CD68), and triggering receptor expressed on myeloid cells 2 (TREM2) coverages at peri‐infarct. (J) Correlations of myelin loss with Iba1, Gal3, P2RY12, CD68, and TREM2 coverages at white matter. In (E, G) values are expressed as individual experimental replicates with mean ± SEM. In (F, H, I, J), values are expressed as individual experimental replicates with simple linear regression lines; Pearson correlation's r value with p ‐value are also shown. In (E, G) unpaired t‐test was performed. n = 6 mice in SE and n = 7 mice in EE. p ‐Values and r values are expressed with 3 decimals. p ‐Values were not corrected for multiple comparisons.

Article Snippet: They were then incubated at 4°C overnight with one of the following antibodies: Iba1 (1:500, rabbit, Cat# 016‐26721; Wako), Gal3 (1:750, goat, Cat# AF1197; R&D Systems) P2RY12 (1:200, rat, Cat# S16007D; Biolegend).

Techniques: Staining, Binding Assay