Journal: Pediatric research

Article Title: Galectin-3 Expression and Effect of Supplementation in Neonatal Mice with Disseminated Candida albicans Infection

doi: 10.1038/s41390-019-0279-x

Figure Lengend Snippet: Tissue fungal burden and galectin-3 concentrations in adult mice with disseminated candidiasis. Mice (n=7) were infected via tail-vein injection with C. albicans and euthanized at 48 h after injection. Panel A: Tissue fungal burden. Panel B: Mean gal3 concentration in tissue homogenates compared to animals receiving saline (n=10). Error bars represent standard deviation. No differences in tissue gal3 concentration were detected by ANOVA with inter-group comparisons by the Student-Newman-Keuls Method.

Article Snippet: In a subsequent experiment, pups were delivered and randomized to receive 5 μg carrier-free recombinant mouse gal3 (R&D Systems, Minneapolis, MN) or saline in 20 μl i.p. injection prior to infection with C. albicans as described above.

Techniques: Infection, Injection, Concentration Assay, Saline, Standard Deviation

Journal: Pediatric research

Article Title: Galectin-3 Expression and Effect of Supplementation in Neonatal Mice with Disseminated Candida albicans Infection

doi: 10.1038/s41390-019-0279-x

Figure Lengend Snippet: Tissue fungal burden and galectin-3 concentrations in neonatal mice with disseminated candidiasis. Two-day-old mouse pups (n=14) were infected via intraperitoneal injection with C. albicans and euthanized at 24 h after injection. Panel A: Tissue fungal burden. Panel B: Mean gal3 concentration in tissue homogenates compared to pups receiving saline (n=14). Error bars represent standard deviation. Mean gal3 concentration was reduced in the spleen of infected pups relative to controls based on ANOVA with inter-group comparisons by the Student-Newman-Keuls Method (p=0.04).

Article Snippet: In a subsequent experiment, pups were delivered and randomized to receive 5 μg carrier-free recombinant mouse gal3 (R&D Systems, Minneapolis, MN) or saline in 20 μl i.p. injection prior to infection with C. albicans as described above.

Techniques: Infection, Injection, Concentration Assay, Saline, Standard Deviation

Journal: Pediatric research

Article Title: Galectin-3 Expression and Effect of Supplementation in Neonatal Mice with Disseminated Candida albicans Infection

doi: 10.1038/s41390-019-0279-x

Figure Lengend Snippet: Survival curve of neonatal mice with disseminated candidiasis after pretreatment with recombinant galectin-3. Two-day-old mouse pups were given intraperitoneal injections of either saline or recombinant gal3, 2 h prior to infection with C. albicans . Uninfected pups receiving gal3 only were included as a control. Pretreatment with gal3 reduced mortality in infected compared to saline treated pups based on log-rank test (p=0.02).

Article Snippet: In a subsequent experiment, pups were delivered and randomized to receive 5 μg carrier-free recombinant mouse gal3 (R&D Systems, Minneapolis, MN) or saline in 20 μl i.p. injection prior to infection with C. albicans as described above.

Techniques: Recombinant, Saline, Infection, Control

Journal: Pediatric research

Article Title: Galectin-3 Expression and Effect of Supplementation in Neonatal Mice with Disseminated Candida albicans Infection

doi: 10.1038/s41390-019-0279-x

Figure Lengend Snippet: Tissue fungal burden in neonatal mice with disseminated candidiasis pretreated with galectin-3. Two-day-old mouse pups were given intraperitoneal injections of either saline or recombinant gal3, 2 h prior to infection with C. albicans (n=10 pups per group). Tissues were collected at the time of death or at 72 h in surviving animals. Fungal burden is depicted with the bars representing median values. P values were derived based on analysis using a negative binomial model to account for the variability in these data which are not normally distributed.

Article Snippet: In a subsequent experiment, pups were delivered and randomized to receive 5 μg carrier-free recombinant mouse gal3 (R&D Systems, Minneapolis, MN) or saline in 20 μl i.p. injection prior to infection with C. albicans as described above.

Techniques: Saline, Recombinant, Infection, Derivative Assay

Journal: Pediatric research

Article Title: Galectin-3 Expression and Effect of Supplementation in Neonatal Mice with Disseminated Candida albicans Infection

doi: 10.1038/s41390-019-0279-x

Figure Lengend Snippet: Tissue fungal burden in neonatal mice with disseminated candidiasis at early time points following infection and pretreatment with galectin-3. Two-day-old mouse pups were given intraperitoneal injections of either saline or recombinant gal3, 2 h prior to infection with C. albicans (n=5 pups per group). Pups were euthanized and tissues were collected at 24 and 36 h following infection. Fungal burden is depicted with the bars representing median values. P values were derived based on analysis using a negative binomial model to account for the variability in these data which are not normally distributed. NS – not significant.

Article Snippet: In a subsequent experiment, pups were delivered and randomized to receive 5 μg carrier-free recombinant mouse gal3 (R&D Systems, Minneapolis, MN) or saline in 20 μl i.p. injection prior to infection with C. albicans as described above.

Techniques: Infection, Saline, Recombinant, Derivative Assay

Journal: Nature Communications

Article Title: Galectin-3 is required for the microglia-mediated brain inflammation in a model of Huntington’s disease

doi: 10.1038/s41467-019-11441-0

Figure Lengend Snippet: Up-regulation of Gal3 in the plasma and brains of HD patients. a The levels of plasma Gal3 of non-HD ( n = 16), pre-symptomatic HD (Pre-HD, n = 4), and HD individuals ( n = 26) were measured by ELISA. b – f Correlations between the plasma Gal3 levels of HD patients ( n = 26) and their disease burden b , MMSE c , UHDRS-motor d , UHDRS-Independence e , or UHDRS-functional capacity f scores were analyzed by Pearson correlation coefficients. The levels of transcripts of LGALS3 in the g caudate nucleus and h cerebellum of HD patients or non-HD subjects were measured by RT-qPCR ( n = 5 in each group). Each dot represents the plasma Gal3 level or the relative LGALS3 level of a subject. Data in ( a ) were analyzed by one-way ANOVA followed by Holm–Sidak’s multiple comparisons test, * P < 0.05. Data in g and h are presented as the means ± SEM and were analyzed by the unpaired Student’s t -test. * P < 0.05. n.s., not significant. Source data is available as a Source Data File

Article Snippet: Primary antibodies included a marker of microglia, IbaI (1:500 for mouse and human brain sections, 1:1000 for cell samples, 019-19741, Wako Laboratory Chemicals), an antibody for brain tissue staining of Gal3 (1:300 for brain sections, AF1197, R&D System), antibodies for the cell staining of Gal3 (0.5 μg/ml, mouse anti-Gal3 B2C10; 1.0 μg/ml goat anti-Gal3 , ), and antibodies for NFκB-p65 (1:100 for tissue sections, 1:200 for cell samples, MAB3026, Millipore), LAMP1 and LAMP2 (1:500, ab24170 and 1:200, ab25339, Abcam), LC3 (1:2000, GTX127375 GeneTex), SDHB (1:500, GTX113833 GeneTex), EM48 (1:500, MAB5374, Millipore), NeuN (1:1000, ABN78 and 1:1000, MAB377, Millipore), S100 (1:1000, Z0311, Dako), CD68 (1:100, ab31630, Abcam), NLRP3 (1:200, AG-20B-0014, AdipoGen), and DARPP32 (1:50, #2302, Cell Signaling).

Techniques: Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Functional Assay, Quantitative RT-PCR

Journal: Nature Communications

Article Title: Galectin-3 is required for the microglia-mediated brain inflammation in a model of Huntington’s disease

doi: 10.1038/s41467-019-11441-0

Figure Lengend Snippet: Up-regulation of Gal3 in HD microglia. a An ELISA of Gal3 was performed on plasma collected from 7-week-old, 10.5-week-old, and 12-week-old R6/2 mice and their controls ( n = 7–11 in each group). Each dot represents the value of an individual animal. b qRT-PCR analysis of Lgals3 was performed on cDNA prepared from striatal tissues collected from 5-week-old, 7-week-old, and 12-week-old R6/2 mice and their littermate controls ( n = 6 in each group). c The protein levels of Gal3 in the striatum of R6/2 and their littermate controls (12 weeks old) were analyzed by western blot analysis ( n = 4 in each group). d , e Brain sections from WT and R6/2 mice (12 weeks old, n = 4 in each group) were stained with anti-Gal3 antibody (green) and a microglial marker (IbaI, red). Nuclei were stained with Hoechst (blue). Arrows mark the IbaI-positive cells (i.e., microglia). f , g Primary cultures of microglia prepared from R6/2 and littermates as described in the “Methods” section. The cells were collected 2 days after plating and were stained with anti-Gal3 antibody (red) and anti-IbaI antibody (gray). Three independent experiments were performed. h Representative histogram plot of WT and R6/2 microglia stained with Gal3 and assessed by flow cytometry. i The levels of Lgal3 transcripts in primary microglia were quantified by RT-qPCR. Three independent experiments were performed. j Brain sections from WT and Hdh 150Q mice (21 months; n = 3 in each group) were stained with anti-Gal3 (green) and anti-IbaI antibodies (red). Nuclei were stained with Hoechst (blue). Arrows mark the IbaI-positive cells (i.e., microglia). k Striatal tissues of Hdh 150Q (15 months old; n = 3 in each group) were carefully removed for RNA preparation and were subjected to RT-qPCR as detailed in the “Methods” section. Data in a and b were analyzed by two-way ANOVA followed by Sidak’s multiple comparisons test. * P < 0.05. Data in c – k are presented as the means ± SEM and were analyzed by the unpaired Student’s t -test. * P < 0.05, *** P < 0.001, **** P < 0.0001. n.s., not significant. Scale bar: 10 µm. Source data is available as a Source Data File

Article Snippet: Primary antibodies included a marker of microglia, IbaI (1:500 for mouse and human brain sections, 1:1000 for cell samples, 019-19741, Wako Laboratory Chemicals), an antibody for brain tissue staining of Gal3 (1:300 for brain sections, AF1197, R&D System), antibodies for the cell staining of Gal3 (0.5 μg/ml, mouse anti-Gal3 B2C10; 1.0 μg/ml goat anti-Gal3 , ), and antibodies for NFκB-p65 (1:100 for tissue sections, 1:200 for cell samples, MAB3026, Millipore), LAMP1 and LAMP2 (1:500, ab24170 and 1:200, ab25339, Abcam), LC3 (1:2000, GTX127375 GeneTex), SDHB (1:500, GTX113833 GeneTex), EM48 (1:500, MAB5374, Millipore), NeuN (1:1000, ABN78 and 1:1000, MAB377, Millipore), S100 (1:1000, Z0311, Dako), CD68 (1:100, ab31630, Abcam), NLRP3 (1:200, AG-20B-0014, AdipoGen), and DARPP32 (1:50, #2302, Cell Signaling).

Techniques: Enzyme-linked Immunosorbent Assay, Clinical Proteomics, Quantitative RT-PCR, Western Blot, Staining, Marker, Flow Cytometry

Journal: Nature Communications

Article Title: Galectin-3 is required for the microglia-mediated brain inflammation in a model of Huntington’s disease

doi: 10.1038/s41467-019-11441-0

Figure Lengend Snippet: Inhibition of the NFκB pathway reduces the abnormal up-regulation of Gal3. a , b Primary microglia harvested from R6/2 mice and their littermate controls (WT) were cultured for 24 h and then treated with Bay11-702 (3 µM) or vehicle (0.1% DMSO) as indicated for 24 h and were then fixed for immunofluorescence staining of Gal3 (green), IbaI (gray), and p65 (red). Nuclei were stained with Hoechst (blue). The localization of nuclei in the right-most panels is outlined by dotted lines. The color bars labeled p65 intensity represent the level of p65 intensity, from low to high fluorescence signals (blue → red, respectively). c An NFκB transcription factor assay was performed on the nuclear extracts prepared from the indicated primary microglia ( n = 4). d ELISA was performed on the supernatants collected from the indicated primary microglia to measure the levels of IL1β, IL6, TNFα, and IL10 released by the cells. One dot represents the mean value of each sample. e The levels of nitrite (NO) in the supernatants were measured using the Griess reagent ( n = 5). The results were analyzed by two-way ANOVA followed by Tukey’s post hoc test. Data are presented as the means ± SEM. *Specific comparison between WT and R6/2 cells of the same treatment; #Specific comparison between the Bay11-treated and DMSO-treated groups of the same genotype; * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Same P -value denotation for #. Scale bar: 10 µm. Source data is available as a Source Data File

Article Snippet: Primary antibodies included a marker of microglia, IbaI (1:500 for mouse and human brain sections, 1:1000 for cell samples, 019-19741, Wako Laboratory Chemicals), an antibody for brain tissue staining of Gal3 (1:300 for brain sections, AF1197, R&D System), antibodies for the cell staining of Gal3 (0.5 μg/ml, mouse anti-Gal3 B2C10; 1.0 μg/ml goat anti-Gal3 , ), and antibodies for NFκB-p65 (1:100 for tissue sections, 1:200 for cell samples, MAB3026, Millipore), LAMP1 and LAMP2 (1:500, ab24170 and 1:200, ab25339, Abcam), LC3 (1:2000, GTX127375 GeneTex), SDHB (1:500, GTX113833 GeneTex), EM48 (1:500, MAB5374, Millipore), NeuN (1:1000, ABN78 and 1:1000, MAB377, Millipore), S100 (1:1000, Z0311, Dako), CD68 (1:100, ab31630, Abcam), NLRP3 (1:200, AG-20B-0014, AdipoGen), and DARPP32 (1:50, #2302, Cell Signaling).

Techniques: Inhibition, Cell Culture, Immunofluorescence, Staining, Labeling, Fluorescence, Transcription Factor Assay, Enzyme-linked Immunosorbent Assay, Comparison

Journal: Nature Communications

Article Title: Galectin-3 is required for the microglia-mediated brain inflammation in a model of Huntington’s disease

doi: 10.1038/s41467-019-11441-0

Figure Lengend Snippet: Suppression of Gal3 reduces the activation of NFκB and inflammation. a , b Primary microglia were infected with the indicated lentiviruses expressing shRNA against Gal3 (i.e., sh Lgals3 ) to knockdown Gal3 or the control lentivirus (sh GFP ), as described in the “Methods” section. One week after infection, the levels of Gal3 (green) and nuclear p65 (red) were analyzed by immunofluorescence staining. Nuclei were stained with Hoechst. The localization of nuclei in the right-most panels is outlined by dotted lines. The color bars labeled p65 intensity represent the level of p65 intensity, from low to high fluorescence signals (blue → red, respectively). c An NFκB transcription factor assay was performed on the nuclear extracts prepared from the indicated primary microglia ( n = 4). d , e ELISA and NO assays were performed on the supernatants collected from the indicated primary microglia to measure the levels of IL1β, IL6, TNFα, IL10, and nitrite released by the cells. One dot represents the mean value of each sample. The results were analyzed by two-way ANOVA, followed by Tukey’s post hoc test. Data are presented as the means ± SEM. *Specific comparison between WT and R6/2 cells infected with the same lentivirus; #Specific comparison between the sh GFP -infected and sh Lgals3 -infected groups of the same genotype; * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Same P -value denotation for #. Scale bar: 10 µm. Source data is available as a Source Data File

Article Snippet: Primary antibodies included a marker of microglia, IbaI (1:500 for mouse and human brain sections, 1:1000 for cell samples, 019-19741, Wako Laboratory Chemicals), an antibody for brain tissue staining of Gal3 (1:300 for brain sections, AF1197, R&D System), antibodies for the cell staining of Gal3 (0.5 μg/ml, mouse anti-Gal3 B2C10; 1.0 μg/ml goat anti-Gal3 , ), and antibodies for NFκB-p65 (1:100 for tissue sections, 1:200 for cell samples, MAB3026, Millipore), LAMP1 and LAMP2 (1:500, ab24170 and 1:200, ab25339, Abcam), LC3 (1:2000, GTX127375 GeneTex), SDHB (1:500, GTX113833 GeneTex), EM48 (1:500, MAB5374, Millipore), NeuN (1:1000, ABN78 and 1:1000, MAB377, Millipore), S100 (1:1000, Z0311, Dako), CD68 (1:100, ab31630, Abcam), NLRP3 (1:200, AG-20B-0014, AdipoGen), and DARPP32 (1:50, #2302, Cell Signaling).

Techniques: Activation Assay, Infection, Expressing, shRNA, Knockdown, Control, Immunofluorescence, Staining, Labeling, Fluorescence, Transcription Factor Assay, Enzyme-linked Immunosorbent Assay, Comparison

Journal: Nature Communications

Article Title: Galectin-3 is required for the microglia-mediated brain inflammation in a model of Huntington’s disease

doi: 10.1038/s41467-019-11441-0

Figure Lengend Snippet: Inhibition of Gal3 by TD139 intracellularly suppresses microglial inflammation. a Primary microglia were cultured for 24 h and then treated with a cell-permeable Gal3 inhibitor, TD139 (1 and 10 µM), or vehicle (0.1% DMSO) for 48 h, and the supernatants were then collected for measurement of IL1β, IL6, TNFα, and IL10 levels using ELISA ( n = 6). b Primary microglia were treated with lactose (10 and 100 mM) to block the binding of extracellular Gal3. Sucrose served as the osmolarity control. The supernatant was collected and subjected to ELISA ( n = 6–7). Suc sucrose, Lac lactose. The results of a were analyzed by two-way ANOVA followed by Tukey’s post hoc test. *Specific comparison between WT and R6/2 cells of the same treatment; #Specific comparison between the DMSO-treated and TD139-treated groups of the same genotype; P < 0.01. Results in b were analyzed by one-way ANOVA followed by Tukey’s post hoc test. *Specific comparison between WT and R6/2 cells; #Specific comparison between R6/2 cells treated with Lac and Suc. Data are presented as the means ± SEM from the indicated sets of cells. ** P < 0.01, *** P < 0.001, **** P < 0.0001. Same P -value denotation for #. Source data is available as a Source Data File

Article Snippet: Primary antibodies included a marker of microglia, IbaI (1:500 for mouse and human brain sections, 1:1000 for cell samples, 019-19741, Wako Laboratory Chemicals), an antibody for brain tissue staining of Gal3 (1:300 for brain sections, AF1197, R&D System), antibodies for the cell staining of Gal3 (0.5 μg/ml, mouse anti-Gal3 B2C10; 1.0 μg/ml goat anti-Gal3 , ), and antibodies for NFκB-p65 (1:100 for tissue sections, 1:200 for cell samples, MAB3026, Millipore), LAMP1 and LAMP2 (1:500, ab24170 and 1:200, ab25339, Abcam), LC3 (1:2000, GTX127375 GeneTex), SDHB (1:500, GTX113833 GeneTex), EM48 (1:500, MAB5374, Millipore), NeuN (1:1000, ABN78 and 1:1000, MAB377, Millipore), S100 (1:1000, Z0311, Dako), CD68 (1:100, ab31630, Abcam), NLRP3 (1:200, AG-20B-0014, AdipoGen), and DARPP32 (1:50, #2302, Cell Signaling).

Techniques: Inhibition, Cell Culture, Enzyme-linked Immunosorbent Assay, Blocking Assay, Binding Assay, Control, Comparison

Journal: Nature Communications

Article Title: Galectin-3 is required for the microglia-mediated brain inflammation in a model of Huntington’s disease

doi: 10.1038/s41467-019-11441-0

Figure Lengend Snippet: Knockdown of Gal3 improves the clearance of damaged lysosomes. a Primary cultures of microglia were prepared as described in the “Methods” section. The cells were harvested 2 days after plating and stained with a lysosomal marker (LAMP1, green) and anti-Gal3 antibody (red). b The percentages of microglial cells exhibiting Gal3 puncta (6–10 pixels in width) were quantified from more than 100 cells in each group. Galectin-3 puncta is the marker of vesicle rupture. b , c The signals of LAMP1 and the percentage of colocalization with Gal3 puncta were quantified. Data are presented as the means ± SEM and were analyzed by the unpaired Student’s t -test. d Primary microglia were infected with the indicated lentiviruses expressing shRNA against Gal3 (i.e., sh Lgals3 ) to knockdown Gal3 or the control lentivirus (sh GFP ), as described in the “Methods” section. One week after infection, the levels of Gal3 (red) and LAMP1 (green) were analyzed by immunofluorescence staining. Nuclei were stained with Hoechst. e , f Primary microglia were treated with TD139 (10 µM) or vehicle (0.1% DMSO) for 48 h. After 48 h, cells of the indicated treatment were incubated with the Magic Red solution for 30 min and visualized by confocal microscopy e or monitored by a fluorescence microplate reader as described in the “Methods” section f . The results were analyzed by two-way ANOVA, followed by Tukey’s post hoc test. Data are presented as the means ± SEM. *Specific comparison between WT and R6/2 cells infected with the same lentivirus or treatment; #Specific comparison between the sh GFP -infected and sh Lgals3 -infected groups or DMSO-treated and TD139-treated groups of the same genotype; * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Same P -value denotation for #. Scale bar: 10 µm. Source data is available as a Source Data File

Article Snippet: Primary antibodies included a marker of microglia, IbaI (1:500 for mouse and human brain sections, 1:1000 for cell samples, 019-19741, Wako Laboratory Chemicals), an antibody for brain tissue staining of Gal3 (1:300 for brain sections, AF1197, R&D System), antibodies for the cell staining of Gal3 (0.5 μg/ml, mouse anti-Gal3 B2C10; 1.0 μg/ml goat anti-Gal3 , ), and antibodies for NFκB-p65 (1:100 for tissue sections, 1:200 for cell samples, MAB3026, Millipore), LAMP1 and LAMP2 (1:500, ab24170 and 1:200, ab25339, Abcam), LC3 (1:2000, GTX127375 GeneTex), SDHB (1:500, GTX113833 GeneTex), EM48 (1:500, MAB5374, Millipore), NeuN (1:1000, ABN78 and 1:1000, MAB377, Millipore), S100 (1:1000, Z0311, Dako), CD68 (1:100, ab31630, Abcam), NLRP3 (1:200, AG-20B-0014, AdipoGen), and DARPP32 (1:50, #2302, Cell Signaling).

Techniques: Knockdown, Staining, Marker, Infection, Expressing, shRNA, Control, Immunofluorescence, Incubation, Confocal Microscopy, Fluorescence, Comparison

Journal: Nature Communications

Article Title: Galectin-3 is required for the microglia-mediated brain inflammation in a model of Huntington’s disease

doi: 10.1038/s41467-019-11441-0

Figure Lengend Snippet: Knockdown of Gal3 in the brains of R6/2 mice reduces the activation of microglia. Mice of 6 weeks were intrastriatally injected with lentiviruses carrying the indicated shRNA and monitored for an additional 7 weeks. Brain tissues were carefully harvested and subjected to immunofluorescence assays using the indicated antibodies. Nuclei were stained with Hoechst (blue). a The expression levels of Gal3 (green), IbaI (red, a microglia marker), and CD68 (gray, a marker for activated microglia) in the striatum of the indicated mice were determined using immunofluorescence staining. b The expression levels of Gal3 (green), IbaI (red), and p65 (gray) in the striatum of the indicated mice were determined using immunofluorescence staining. The color bars labeled p65 intensity represent the level of p65 intensity, from low to high fluorescence signals (blue → red, respectively). Twelve image frames of each animal were analyzed (4 animals in each group). c Striatal homogenates were analyzed to determine the levels of IL1β and IL10 by ELISA. Each dot represents the mean value of each mouse (3–6 animals in each group). Data are presented as the means ± SEM. The results were analyzed by two-way ANOVA followed by Tukey’s post hoc test. *Specific comparison between WT and R6/2 mice infected with the same lentivirus; #Specific comparison between mice of the same genotype. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Same P -value denotation for #. Scale bar: 10 µm. Source data is available as a Source Data File

Article Snippet: Primary antibodies included a marker of microglia, IbaI (1:500 for mouse and human brain sections, 1:1000 for cell samples, 019-19741, Wako Laboratory Chemicals), an antibody for brain tissue staining of Gal3 (1:300 for brain sections, AF1197, R&D System), antibodies for the cell staining of Gal3 (0.5 μg/ml, mouse anti-Gal3 B2C10; 1.0 μg/ml goat anti-Gal3 , ), and antibodies for NFκB-p65 (1:100 for tissue sections, 1:200 for cell samples, MAB3026, Millipore), LAMP1 and LAMP2 (1:500, ab24170 and 1:200, ab25339, Abcam), LC3 (1:2000, GTX127375 GeneTex), SDHB (1:500, GTX113833 GeneTex), EM48 (1:500, MAB5374, Millipore), NeuN (1:1000, ABN78 and 1:1000, MAB377, Millipore), S100 (1:1000, Z0311, Dako), CD68 (1:100, ab31630, Abcam), NLRP3 (1:200, AG-20B-0014, AdipoGen), and DARPP32 (1:50, #2302, Cell Signaling).

Techniques: Knockdown, Activation Assay, Injection, shRNA, Immunofluorescence, Staining, Expressing, Marker, Labeling, Fluorescence, Enzyme-linked Immunosorbent Assay, Comparison, Infection

Journal: Nature Communications

Article Title: Galectin-3 is required for the microglia-mediated brain inflammation in a model of Huntington’s disease

doi: 10.1038/s41467-019-11441-0

Figure Lengend Snippet: Gal3 triggers IL1β production via an NLRP3 inflammasome-dependent pathway. a Mice of 6 weeks were intrastriatally injected with lentiviruses harboring sh Lgals3 to down-regulate Gal3 and then monitored for an additional 7 weeks. Brain tissues were carefully removed and subjected to immunofluorescence staining to determine the levels of Gal3 (green) and NLRP3 (gray). Four animals in each group were examined. Data are presented as the means ± SEM. *Specific comparison between WT and R6/2 mice infected with the same lentivirus; #Specific comparison between mice of the same genotype. b Primary microglia were treated with MCC950 (1 µM, an inhibitor of NLRP3) or PBS (0.1%) for 24 h. The levels of IL1β released by these cells were measured by ELISA. Each dot represents the mean value of one sample. Three independent experiments were conducted. Data are presented as the mean ± SEM. The results were analyzed by two-way ANOVA followed by Tukey’s post hoc test. *Specific comparison between WT and R6/2 cells; #Specific comparison between R6/2 cells treated with PBS and MCC950. * P < 0.05, ** P < 0.01, **** P < 0.0001. Same P -value denotation for #. Scale bar: 10 µm. Source data is available as a Source Data File

Article Snippet: Primary antibodies included a marker of microglia, IbaI (1:500 for mouse and human brain sections, 1:1000 for cell samples, 019-19741, Wako Laboratory Chemicals), an antibody for brain tissue staining of Gal3 (1:300 for brain sections, AF1197, R&D System), antibodies for the cell staining of Gal3 (0.5 μg/ml, mouse anti-Gal3 B2C10; 1.0 μg/ml goat anti-Gal3 , ), and antibodies for NFκB-p65 (1:100 for tissue sections, 1:200 for cell samples, MAB3026, Millipore), LAMP1 and LAMP2 (1:500, ab24170 and 1:200, ab25339, Abcam), LC3 (1:2000, GTX127375 GeneTex), SDHB (1:500, GTX113833 GeneTex), EM48 (1:500, MAB5374, Millipore), NeuN (1:1000, ABN78 and 1:1000, MAB377, Millipore), S100 (1:1000, Z0311, Dako), CD68 (1:100, ab31630, Abcam), NLRP3 (1:200, AG-20B-0014, AdipoGen), and DARPP32 (1:50, #2302, Cell Signaling).

Techniques: Injection, Immunofluorescence, Staining, Comparison, Infection, Enzyme-linked Immunosorbent Assay

Journal: Nature Communications

Article Title: Galectin-3 is required for the microglia-mediated brain inflammation in a model of Huntington’s disease

doi: 10.1038/s41467-019-11441-0

Figure Lengend Snippet: Knockdown of Gal3 ameliorates HD symptoms in R6/2 mice. Mice of 6 weeks were intrastriatally injected with the sh Lgals3 -expressing or shGFP -expressing lentivirus, and monitored for an additional 7 weeks ( n = 9–11 mice in each group). a The motor functions of R6/2 mice and their littermate controls were assessed by rotarod performance. b Kaplan–Meier plots of R6/2 and WT mice ( P = 0.0089). c Body weight of the indicated animal was measured from 7 to 13 weeks of age. Brain tissues were carefully removed and subjected to immunofluorescence staining to determine the amount of mHTT aggregates (green, EM48; d , e ) or DARPP32 (green, a marker for the medium spiny neurons (MSNs), g , h ), on the neurons (NeuN, red; a neuronal marker; d , f , g ). Nuclei were stained with Hoechst (blue). Four animals were analyzed in each group. The results were analyzed by two-way ANOVA followed by Tukey’s post hoc test. Data are presented as the means ± SEM. *Specific comparison between WT and R6/2 mice infected with the same lentivirus; #Specific comparison between mice of the same genotype. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Same P -value denotation for #. Scale bar: 10 µm. Source data is available as a Source Data File

Article Snippet: Primary antibodies included a marker of microglia, IbaI (1:500 for mouse and human brain sections, 1:1000 for cell samples, 019-19741, Wako Laboratory Chemicals), an antibody for brain tissue staining of Gal3 (1:300 for brain sections, AF1197, R&D System), antibodies for the cell staining of Gal3 (0.5 μg/ml, mouse anti-Gal3 B2C10; 1.0 μg/ml goat anti-Gal3 , ), and antibodies for NFκB-p65 (1:100 for tissue sections, 1:200 for cell samples, MAB3026, Millipore), LAMP1 and LAMP2 (1:500, ab24170 and 1:200, ab25339, Abcam), LC3 (1:2000, GTX127375 GeneTex), SDHB (1:500, GTX113833 GeneTex), EM48 (1:500, MAB5374, Millipore), NeuN (1:1000, ABN78 and 1:1000, MAB377, Millipore), S100 (1:1000, Z0311, Dako), CD68 (1:100, ab31630, Abcam), NLRP3 (1:200, AG-20B-0014, AdipoGen), and DARPP32 (1:50, #2302, Cell Signaling).

Techniques: Knockdown, Injection, Expressing, Immunofluorescence, Staining, Marker, Comparison, Infection

Journal: Nature Communications

Article Title: Galectin-3 is required for the microglia-mediated brain inflammation in a model of Huntington’s disease

doi: 10.1038/s41467-019-11441-0

Figure Lengend Snippet: Schematic representation of the detrimental role of Gal3 in HD microglia. Under normal conditions, microglia express low levels of Gal3 and pro-inflammatory cytokines. In Huntington’s disease, the presence of mHTT activates NFκB, which evokes the expression of Gal3, NLRP3, and pro-inflammatory cytokines (such as IL1β). Gal3 further activates NFκB through a positive feedback loop. On the other hand, the presence of mHTT triggers lysosomal damages as demonstrated by aggregation of Gal3 on the ruptured lysosomes. Furthermore, Gal3 inhibits the clearance of damaged lysosomes and promotes the assembly of NLRP3 inflammasomes, which results in the maturation of IL1β and subsequent inflammatory events

Article Snippet: Primary antibodies included a marker of microglia, IbaI (1:500 for mouse and human brain sections, 1:1000 for cell samples, 019-19741, Wako Laboratory Chemicals), an antibody for brain tissue staining of Gal3 (1:300 for brain sections, AF1197, R&D System), antibodies for the cell staining of Gal3 (0.5 μg/ml, mouse anti-Gal3 B2C10; 1.0 μg/ml goat anti-Gal3 , ), and antibodies for NFκB-p65 (1:100 for tissue sections, 1:200 for cell samples, MAB3026, Millipore), LAMP1 and LAMP2 (1:500, ab24170 and 1:200, ab25339, Abcam), LC3 (1:2000, GTX127375 GeneTex), SDHB (1:500, GTX113833 GeneTex), EM48 (1:500, MAB5374, Millipore), NeuN (1:1000, ABN78 and 1:1000, MAB377, Millipore), S100 (1:1000, Z0311, Dako), CD68 (1:100, ab31630, Abcam), NLRP3 (1:200, AG-20B-0014, AdipoGen), and DARPP32 (1:50, #2302, Cell Signaling).

Techniques: Expressing

Journal: Upsala Journal of Medical Sciences

Article Title: Surface glycans contribute to differences between seminal prostasomes from normozoospermic and oligozoospermic men

doi: 10.1080/03009734.2019.1592266

Figure Lengend Snippet: Surface glycosylation of seminal prostasomes: lectin- and immune-transmission electron microscopy. A: Lectin-TEM using SNA. Inserts show enlarged characteristic pattern of SNA-reactivity to each sample group. B: Lectin-TEM using ConA. Inserts show enlarged characteristic pattern of ConA-reactivity to vesicles in each sample group. In O, staining of some proteinaceous material was also observed (arrowheads). C: Immune-TEM using anti-galectin-3 antibodies. Inserts show enlarged characteristic pattern of anti-gal-3-reactivity to each sample group. Micrographs show most characteristic patterns obtained. Although differences in the reactivity of particular vesicles could be noticed, it does not affect the general reactivity of the sample (as in IEC when taking all vesicles into account). (N = seminal prostasomes from normozoospermic men; O = seminal prostasomes from oligozoospermic men).

Article Snippet: Monoclonal anti-CD63 antibody (clone TS63) was from Abcam (Cambridge, UK) and biotinylated goat anti-galectin-3 (gal-3) antibodies from R&D Systems (Minneapolis, MN, USA); 3,3′,5,5′-tetramethylbenzidine (TMB), bovine serum albumin (BSA), D-lactose, and methyl- alpha D-mannopyranoside were from Sigma (St. Louis, MO, USA).

Techniques: Transmission Assay, Electron Microscopy, Staining