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

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: Cell reports

Article Title: Distinct RORγt-dependent Th17 immune responses are required for autoimmune pathogenesis and protection against bacterial infection

doi: 10.1016/j.celrep.2024.114951

Figure Lengend Snippet: (A–E) RNA-seq analysis of WT and RORγt K256R/K256R CD4 + T cells polarized under Th17 conditions. (A) The number of differentially expressed genes (DEGs, black) including upregulated (red) and downregulated (blue) genes with a cutoff at p < 0.05 and fold change (FC) >1.9. (B) Volcano plot shows DEGs between indicated Th17 cells (log 10 p on y axis and log 2 FC on x axis). Top downregulated (left) and upregulated (right) candidates in RORγt K256R/K256R cells are indicated. Red font: the top three downregulated candidates. (C) A Venn diagram illustrates the overlapping 32 genes between 223 pathogenic Th17-specific genes and 746 downregulated genes in RORγt K256R/K256R Th17 cells (GEO: GSE39820). , , , (D) Heatmap of the 32 overlapping genes described in (C). (E) qPCR analysis of relative mRNA levels of Lgals3 in Th0 and Th17 cells ( n = 4). (F and G) qPCR analysis of Lgals3 mRNA levels (F) and flow cytometric analysis of Lgals3 protein (G, left panels) and percentage (G, right panel) of Lgals3 + cells among indicated CD4 + T cells polarized under Th17 conditions ( n = 6). (H and I) Representative flow cytometric analysis (H) and percentage (I) of Lgals3 + cells among CD4 + T cells from the spleens of indicated untreated mice (left two panels) or from the CNS of indicated EAE-induced mice (right two panels) as described in ( n = 7). (J) Representative flow cytometric analysis (left three panels) and percentage of Lgals3 + cells (right panel) among CD4 + T cells in the colons of C. rodentium -infected indicated mice described in . Lgals3 −/− group is a negative staining control. (K) Mean EAE clinical scores at different days of Rag1 −/− mice adoptively transferred with sorted 1 × 10 5 indicated CD4 + GFP + T cells retrovirally expressing GFP alone (EV) or with Ccr6 and polarized under Th17 conditions ( n = 8, two independent experiments). (L) Number of CD4 + T cells recovered from the CNS of EAE-induced mice described in (K). (M) Mean EAE clinical scores of Rag1 −/− -recipient mice adoptively transferred with sorted 1 × 10 5 Tg TCR2D GFP + CD4 + T cells retrovirally expressing GFP together with scrambled short hairpin RNA (shRNA) or shRNA targeting Lgals3 (shLgals3) and polarized under Th17 conditions ( n = 6, two independent experiments). (N) Number of CD4 + T cells recovered from the CNS of EAE-induced mice described in (M). Data are presented as mean ± SEM. Statistical significance is indicated as **p < 0.05; ***p < 0.001; ns, not significant (two-tailed unpaired Student’s t test). Also see .

Article Snippet: Lgals3 antibodies (M3/38) were obtained from Miltenyi Biotec for flow cytometric analysis or Santa Cruz Biotechnology for the Western blotting.

Techniques: RNA Sequencing, Infection, Negative Staining, Control, Expressing, shRNA, Two Tailed Test

Journal: Cell reports

Article Title: Distinct RORγt-dependent Th17 immune responses are required for autoimmune pathogenesis and protection against bacterial infection

doi: 10.1016/j.celrep.2024.114951

Figure Lengend Snippet: (A) Representative flow cytometric analysis of IL-17A expression in WT and Lgals3 −/− CD4 + T cells polarized under Th17 conditions in vitro for 3 days ( n = 4). (B) Mean clinical score of indicated mice on different days after EAE induction ( n = 6, two independent experiments). (C) The number of CD4 + T cells infiltrated into the CNS of mice described in (B). (D) Mean clinical score of Rag1 −/− recipients adoptively transferred with 3 × 10 6 naive CD4 + T cells from indicated mice, followed by induction of EAE with MOG 35–55 ( n = 7–8, two independent experiments). (E) The number of CD4 + T cells infiltrated into the CNS of Rag1 −/− mice described in (D). (F) Body weight of indicated mice on different days after oral infection with 2 × 10 9 C. rodentium ( n = 8–9, two independent experiments). (G–I) Bacterial load (G), colon length (H), and the number of CD4 + T cells in the colons (I) of mice described in (F) at day 21 post infection. (J) Representative flow cytometric analysis (left panels) and percentage (right panel) of IL-17A + cells among CD4 + T cells recovered from the colons of indicated mice shown in (F). Data are presented as mean ± SEM. Statistical significance is indicated as *p < 0.01; **p < 0.05; ***p < 0.001; ns, not significant (two-tailed unpaired Student’s t test). Also see .

Article Snippet: Lgals3 antibodies (M3/38) were obtained from Miltenyi Biotec for flow cytometric analysis or Santa Cruz Biotechnology for the Western blotting.

Techniques: Expressing, In Vitro, Infection, Two Tailed Test

Journal: Cell reports

Article Title: Distinct RORγt-dependent Th17 immune responses are required for autoimmune pathogenesis and protection against bacterial infection

doi: 10.1016/j.celrep.2024.114951

Figure Lengend Snippet: (A) Scheme of potential Runx1 DNA-binding sites. Region 1 (Rgn1) and Rgn2 are two conserved Runx1-binding sites identified from ChIP-seq for Runx1 (GEO: GSE158093). E, exon. (B) ChIP signals with anti-FLAG antibody in in vitro differentiated WT Th17 cells retrovirally transduced with GFP alone (EV) or with FLAG-tagged Runx1 ( n = 4). (C) Schematic representation of indicated luciferase reporter constructs. P, Lgals3 promoter; I, Lgals3 intron; TK, minimal thymidine kinase gene promoter; Δ, deletion; Luc, luciferase. (D and E) Relative luciferase activity from indicated reporter shown in (C) transfected into 293T cells together with expression plasmid for Runx1 or control empty plasmid (Ctrl). Basic is a promoterless reporter ( n = 4). (F and G) Flow cytometric analysis of Lgal3 expression (left panels) and percentage of Lgals3 + cells among Cas9-expressing CD4 + T cells retrovirally transduced with nontargeting (NonT) sgRNA controls or sgRNA targeting to delete Rgn1 (F) or Rgn2 (G) and polarized under Th17 conditions ( n = 4). Gray: controls unstained (Unst) with the Lgals3 antibody. (H) Number of CD4 + T cells infiltrating the CNS of Rag1 −/− adoptively transferred with Tg Tcr2D Th17 cells retrovirally transduced with NonT, sgRgn1, or sgRgn2 followed by induction of EAE with MOG 35–55 ( n = 5). (I) The endpoint clinical score of mice as described in (H). (J) Flow cytometric analysis of Lgals3 in indicated CD4 + T cells expressing GFP alone or together with Runx1. Data are presented as mean ± SEM. Statistical significance is indicated as *p < 0.01; **p < 0.05; ***p < 0.001; ns, not significant (two-tailed unpaired Student’s t test). Also see .

Article Snippet: Lgals3 antibodies (M3/38) were obtained from Miltenyi Biotec for flow cytometric analysis or Santa Cruz Biotechnology for the Western blotting.

Techniques: Binding Assay, ChIP-sequencing, In Vitro, Transduction, Luciferase, Construct, Activity Assay, Transfection, Expressing, Plasmid Preparation, Control, Two Tailed Test

Journal: Cell reports

Article Title: Distinct RORγt-dependent Th17 immune responses are required for autoimmune pathogenesis and protection against bacterial infection

doi: 10.1016/j.celrep.2024.114951

Figure Lengend Snippet: (A) Representative flow cytometric analysis (left panels) and percentage (right panel) of Ccr6 + cells among indicated CD4 + T cells polarized under Th17 conditions for 3 days ( n = 6). (B) The number (top panels) and percentage (bottom panels) of indicated types of cells in the CNS of indicated mice immunized with MOG 35–55 . (C) Representative flow cytometric analysis (top panels), percentage (two bottom-left panels), and number (two bottom-right panels) of monocytes/macrophages (Mono/Mac) and neutrophils in the CNS of Rag1 −/− mice adoptively transferred with indicated CD4 + GFP + T cells retrovirally expressing GFP alone (EV) or with Lgals3 and polarized under Th17 conditions, followed by EAE induction as described in . (D) Representative flow cytometric analysis (left panels) and percentage (right panel) of BMDMs migrated to the bottom wells containing indicated CD4 + GFP + T cells retrovirally expressing GFP alone (EV) or with Lgals3 and differentiated under Th17 cells conditions in a Transwell migration assay ( n = 4 independent experiments). (E) Immunoblot (IB) analysis of FLAG-Lgals3 immunoprecipitated (IP) from the supernatants of CD4 + T cells transduced with empty retrovirus (EV) or virus expressing 3xFLAG-Lgals3 and polarized under Th17 condition for 3 days ( n = 4). Bottom two lanes are the immunoblot analysis of Lgals3 in the whole-cell lysates (WCLs) with anti-FLAG or anti-Lgals3 antibody. (F) Representative flow cytometric analysis (left panels) and percentage (right panel) of Ccr6 + cells among in vitro differentiated RORγt K256R/K256R Th17 cells co-cultured without (None) or with BMDMs for 18 h ( n = 4). (G) Representative flow cytometric analysis (left panels) and percentage (right panel) of Ccr6 + cells among CD4 + T cells recovered from the CNS of Rag1 −/− mice adoptively transferred with indicated CD4 + GFP + T cells retrovirally expressing GFP alone (EV) or with Lgals3 and polarized under Th17 conditions followed by EAE induction as described in ( n = 7–8). Analysis was conducted on day 10 post immunization. (H) Percentage of Ccr6 + cells among CD4 + T cells infiltrated into the CNS of WT or Lgals3 −/− mice immunized with MOG 35–55 as described in ( n = 6). (I) Percentage of Ccr6 + cells among in vitro differentiated Th17 cells co-cultured without (None) or with BMDMs for 18 h in the absence or presence of indicated neutralizing antibodies, analyzed by flow cytometry ( n = 4). (J) Percentage of Ccr6 + cells among in vitro differentiated RORγt K256R/K256R Th17 cells treated with vehicle or recombinant IL-1β ( n = 5). (K) Representative flow cytometric analysis (left panels) and percentage (right panel) of IL-1β + cells among Mono/Mac cells in the spleens (left two panels) or the CNS (middle two panels) of indicated mice immunized with MOG 35–55 (n = 8–9). (L) Number of IL-1β + cells among lymphocytes recovered from CNS of indicated EAE-induced mice. Data are presented as mean ± SEM. Statistical significance is indicated as *p < 0.01; **p < 0.05; ***p < 0.001; ns, not significant (two-tailed unpaired Student’s t test). Also see .

Article Snippet: Lgals3 antibodies (M3/38) were obtained from Miltenyi Biotec for flow cytometric analysis or Santa Cruz Biotechnology for the Western blotting.

Techniques: Expressing, Transwell Migration Assay, Western Blot, Immunoprecipitation, Transduction, Virus, In Vitro, Cell Culture, Flow Cytometry, Recombinant, Two Tailed Test

Journal: Cell reports

Article Title: Distinct RORγt-dependent Th17 immune responses are required for autoimmune pathogenesis and protection against bacterial infection

doi: 10.1016/j.celrep.2024.114951

Figure Lengend Snippet:

Article Snippet: Lgals3 antibodies (M3/38) were obtained from Miltenyi Biotec for flow cytometric analysis or Santa Cruz Biotechnology for the Western blotting.

Techniques: Virus, Recombinant, Modification, Protease Inhibitor, Lysis, Radio Immunoprecipitation, Isolation, cDNA Synthesis, SYBR Green Assay, Emulsion, Reporter Assay, Microarray, CRISPR, Luciferase, Control, Software

Journal: Cell Death and Differentiation

Article Title: Galectin-3 promotes Aβ oligomerization and Aβ toxicity in a mouse model of Alzheimer’s disease

doi: 10.1038/s41418-019-0348-z

Figure Lengend Snippet: Aβ oligomerization is reduced in Gal-3 KO mice injected with Aβ. a WT and Gal-3 KO mice (3 months old) were injected with 1% NH 4 OH or Aβ (5 μg) in the CA1 area. Animals were sacrificed 48 h after injection and dissected hippocampal tissues were subjected to Western blot analysis of Aβ oligomerization and Gal-3 expression. b Quantified results of HMW and LMW Aβ oligomerization [ n = 4 per group; F (3,12) = 149.28, P < 0.001 for HMW; q = 26.22, P < 0.001 for the WT+NH 4 OH group versus the WT+Aβ group and q = 13.78, P < 0.001 for the WT+Aβ group versus the Gal-3 KO+Aβ group; F (3,12) = 48.02 and P < 0.001 for LMW; q = 14.31, P < 0.001 for the WT+NH 4 OH group versus the WT+Aβ group and q = 6.74, P < 0.001 for the WT+Aβ group versus the Gal-3 KO+Aβ group]. c Quantified results for Gal-3 expression in the same mouse groups [ F (3,12) = 83.43, P < 0.001; q = 15.63, P < 0.001 for the WT+NH 4 OH group versus the WT+Aβ group]. d Mice (3 months old) received the following intra-hippocampal injections: NH 4 OH (1%) injection and Flag-vector plasmid (0.4 μg) transfection; Aβ (5 μg) injection and Flag-vector plasmid transfection; NH 4 OH injection and Flag-Gal-3 plasmid transfection; and Aβ injection and Flag-Gal-3 plasmid transfection. The two injections were given 2 h apart and animals were sacrificed 48 h after the injection of NH 4 OH or Aβ. Dissected hippocampal tissues were analyzed for Aβ oligomerization. Tissue lysates were also subjected to immunoprecipitation and immunoblotting with anti-Flag antibody to confirm transfection and expression of the plasmid. e Quantified results for HMW and LMW Aβ oligomerization [ n = 4 per group; F (3,12) = 52.71, P < 0.001 for HMW; q = 9.09, P < 0.001 for the NH 4 OH+Flag-vector group versus the Aβ+Flag-vector group and q = 5.8, P < 0.01 for the Aβ+Flag-vector group versus the Aβ+Flag-Gal-3 group; F (3,12) = 72.63, P < 0.001 for LMW; q = 5.65, P < 0.01 for the NH 4 OH+Flag-vector group versus the Aβ+Flag-vector group and q = 12.38, P < 0.001 for the Aβ+Flag-vector group versus the Aβ+Flag-Gal-3 group]. Data are expressed as mean ± SEM. ** P < 0.01, # P < 0.001

Article Snippet: Immunoblotting was carried out using the following antibodies: rat anti-Gal-3 (1:5000, R&D systems), goat anti-Gal-1 (1:5000, R&D systems), rabbit anit-PIAS1 (1:3000, Epitomics, Burlingame, CA), mouse anti-CD10/NEP (1:500, Santa Cruz Biotechnology, Dallas, TX), mouse anti-prealbumin/transthyretin (1:500, Santa Cruz Biotechnology), rabbit anti-insulin degrading enzyme (1:3000, Abcam, Cambridge, UK), rabbit anti-FAK (1:2000, Abcam), rabbit anti-pFAK (phospho-Y397, 1:2000, Abcam), rabbit anti-CREB (1:2000, Cell Signaling, Danvers, MA), rabbit anti-pCREB (phospho-S133, 1:2000, Cell Signaling), rat anti-TREM2 (1:3000, Millipore, Bedford, MA), mouse anti-TLR4 (1:500, Santa Cruz Biotechnology), rabbit anti-CD40 (1:2000, Abcam), mouse anti-actin (1:150000, Millipore), and mouse anti-Flag M2 (1:10000, Sigma-Aldrich) antibodies.

Techniques: Injection, Western Blot, Expressing, Plasmid Preparation, Transfection, Immunoprecipitation

Journal: Cell Death and Differentiation

Article Title: Galectin-3 promotes Aβ oligomerization and Aβ toxicity in a mouse model of Alzheimer’s disease

doi: 10.1038/s41418-019-0348-z

Figure Lengend Snippet: Aβ oligomerization and Gal-3 expression are age-dependently increased in APP/PS1 mice. a Wild-type (3 months old) and APP/PS1 mice of different ages (3, 5, 8, and 11 months) were sacrificed and dissected hippocampal tissues were subjected to Western blot analysis for endogenous Aβ oligomers. b Quantified results for HMW and LMW Aβ oligomerization [ n = 4 per group; F (4,15) = 39.3, P < 0.001 for HMW and F (4,15) = 74.07, P < 0.001 for LMW]. Statistical significances for various comparisons are shown in the figure. c Western blot analysis showing the expression levels of Gal-3 and PIAS1 in hippocampal samples from the same batch of WT mice and APP/PS1 mice of different ages ( n = 4 per group). d Quantified results for Gal-3 expression in WT and APP/PS1 mice [ F (4,15) = 14.59, P < 0.001]. e Quantified results for PIAS1 expression in WT and APP/PS1 mice [ F (4,15) = 5.86, P < 0.01] The letter “m” indicates month. Statistical significances of various comparisons are shown in the figure. Data are expressed as mean ± SEM. * P < 0.05, ** P < 0.01, # P < 0.001

Article Snippet: Immunoblotting was carried out using the following antibodies: rat anti-Gal-3 (1:5000, R&D systems), goat anti-Gal-1 (1:5000, R&D systems), rabbit anit-PIAS1 (1:3000, Epitomics, Burlingame, CA), mouse anti-CD10/NEP (1:500, Santa Cruz Biotechnology, Dallas, TX), mouse anti-prealbumin/transthyretin (1:500, Santa Cruz Biotechnology), rabbit anti-insulin degrading enzyme (1:3000, Abcam, Cambridge, UK), rabbit anti-FAK (1:2000, Abcam), rabbit anti-pFAK (phospho-Y397, 1:2000, Abcam), rabbit anti-CREB (1:2000, Cell Signaling, Danvers, MA), rabbit anti-pCREB (phospho-S133, 1:2000, Cell Signaling), rat anti-TREM2 (1:3000, Millipore, Bedford, MA), mouse anti-TLR4 (1:500, Santa Cruz Biotechnology), rabbit anti-CD40 (1:2000, Abcam), mouse anti-actin (1:150000, Millipore), and mouse anti-Flag M2 (1:10000, Sigma-Aldrich) antibodies.

Techniques: Expressing, Western Blot

Journal: Cell Death and Differentiation

Article Title: Galectin-3 promotes Aβ oligomerization and Aβ toxicity in a mouse model of Alzheimer’s disease

doi: 10.1038/s41418-019-0348-z

Figure Lengend Snippet: Endogenous Aβ oligomerization, Gal-3 expression, Iba1 and GFAP distribution, and amyloid plaque are reduced in APP/PS1;Gal-3 +/− mice. a Endogenous Aβ oligomerization was examined by Western blot analysis of samples obtained from 3-month-old WT;WT, APP/PS1;WT, WT;Gal-3 −/− and APP/PS1;Gal-3 +/− mice. b Quantified results for HMW and LMW Aβ oligomerization [ n = 4 per group; F (3,12) = 110.36; P < 0.001 for HMW; q = 19.91, P < 0.001 for the WT;WT group versus the APP/PS1;WT group and q = 4.13, P = 0.01 for the APP/PS1;WT group versus the APP/PS1;Gal-3 +/− group; F (3,12) = 77.72, P < 0.001 for LMW; q = 15.87, P < 0.001 for the WT;WT group versus the APP/PS1;WT group and q = 3.32, P < 0.05 for the APP/PS1;WT group versus the APP/PS1;Gal-3 +/− group]. c Immunohistochemical analysis of Gal-3 and Iba1 and merged images for 7-month-old mice of the four genotypes. Scale bar, 25 μm. d Immunohistochemical analysis of Gal-3 and GFAP and merged image in 7-month-old mice of the four genotypes. Scale bar, 25 μm. e Immunohistochemical analysis showing the distribution of Gal-3 and ProteoStat in 11-month-old mice of the four genotypes. Scale bar, 200 μm. Data are expressed as mean ± SEM. * P < 0.05, ** P < 0.01, # P < 0.001

Article Snippet: Immunoblotting was carried out using the following antibodies: rat anti-Gal-3 (1:5000, R&D systems), goat anti-Gal-1 (1:5000, R&D systems), rabbit anit-PIAS1 (1:3000, Epitomics, Burlingame, CA), mouse anti-CD10/NEP (1:500, Santa Cruz Biotechnology, Dallas, TX), mouse anti-prealbumin/transthyretin (1:500, Santa Cruz Biotechnology), rabbit anti-insulin degrading enzyme (1:3000, Abcam, Cambridge, UK), rabbit anti-FAK (1:2000, Abcam), rabbit anti-pFAK (phospho-Y397, 1:2000, Abcam), rabbit anti-CREB (1:2000, Cell Signaling, Danvers, MA), rabbit anti-pCREB (phospho-S133, 1:2000, Cell Signaling), rat anti-TREM2 (1:3000, Millipore, Bedford, MA), mouse anti-TLR4 (1:500, Santa Cruz Biotechnology), rabbit anti-CD40 (1:2000, Abcam), mouse anti-actin (1:150000, Millipore), and mouse anti-Flag M2 (1:10000, Sigma-Aldrich) antibodies.

Techniques: Expressing, Western Blot, Immunohistochemical staining

Journal: Cell Death and Differentiation

Article Title: Galectin-3 promotes Aβ oligomerization and Aβ toxicity in a mouse model of Alzheimer’s disease

doi: 10.1038/s41418-019-0348-z

Figure Lengend Snippet: Endogenous Gal-3 expression is decreased but memory performance is improved in APP/PS1;Gal-3 +/− mice. a Endogenous expression levels of Gal-3 and NEP were examined by Western blot analysis in 3-month-old WT;WT, APP/PS1;WT, WT;Gal-3 −/− and APP/PS1;Gal-3 +/− mice. b Quantified results for Gal-3 and NEP expression [ n = 4 per group; for Gal-3, F (3,12) = 53.47, P < 0.001; q = 10.43, P < 0.001 for the WT;WT group versus the APP/PS1;WT group; q = 6.46, P < 0.001 for the APP/PS1;WT group versus the APP/PS1;Gal-3 +/− group and q = 3.98, P < 0.05 for the WT;WT group versus the APP/PS1;Gal-3 +/− group; for NEP, F (3,12) = 27.76, P < 0.001; q = 12.04, P < 0.001 for the WT;WT group versus the APP/PS1;WT group; q = 9.72, P < 0.001 for the WT;WT group versus the WT;Gal-3 −/− group and q = 6.29, P < 0.01 for the WT;WT group versus the APP/PS1;Gal-3 +/− group]. c Acquisition performance obtained by assessing the water maze learning of 8-month-old mice of the four genotypes [ n = 7 per group; F (3,24) = 28.72, P < 0.001; q = 9.33, P < 0.001 for the APP/PS1;WT group versus the WT;WT group and q = 3.59, P < 0.05 for the APP/PS1;Gal-3 +/− group versus the APP/PS1;WT group]. d Retention performance of the same mice described in ( c ) [ n = 7 per group; F (3,24) = 4.76, P < 0.01; q = 3.94, P < 0.05 for the APP/PS1;WT group versus the WT;WT group; q = 3.25, P < 0.05 for the APP/PS1;Gal-3 +/− group versus the APP/PS1;WT group for the target quadrant]. Representative swim patterns from each group are also shown (upper panel). Data are expressed as mean ± SEM. * P < 0.05, ** P < 0.01, # P < 0.001

Article Snippet: Immunoblotting was carried out using the following antibodies: rat anti-Gal-3 (1:5000, R&D systems), goat anti-Gal-1 (1:5000, R&D systems), rabbit anit-PIAS1 (1:3000, Epitomics, Burlingame, CA), mouse anti-CD10/NEP (1:500, Santa Cruz Biotechnology, Dallas, TX), mouse anti-prealbumin/transthyretin (1:500, Santa Cruz Biotechnology), rabbit anti-insulin degrading enzyme (1:3000, Abcam, Cambridge, UK), rabbit anti-FAK (1:2000, Abcam), rabbit anti-pFAK (phospho-Y397, 1:2000, Abcam), rabbit anti-CREB (1:2000, Cell Signaling, Danvers, MA), rabbit anti-pCREB (phospho-S133, 1:2000, Cell Signaling), rat anti-TREM2 (1:3000, Millipore, Bedford, MA), mouse anti-TLR4 (1:500, Santa Cruz Biotechnology), rabbit anti-CD40 (1:2000, Abcam), mouse anti-actin (1:150000, Millipore), and mouse anti-Flag M2 (1:10000, Sigma-Aldrich) antibodies.

Techniques: Expressing, Western Blot

Journal: Cell Death and Differentiation

Article Title: Galectin-3 promotes Aβ oligomerization and Aβ toxicity in a mouse model of Alzheimer’s disease

doi: 10.1038/s41418-019-0348-z

Figure Lengend Snippet: Galectin-3 associates with Aβ and interacts with Aβ. a Immunohistochemical analysis showing the distributions of endogenous Gal-3 and Aβ in hippocampal samples of WT (left panel) and APP/PS1 (right panel) mice at 11 months of age. Scale bar, 200 μm. b Co-IP experiment showing preferential association of Gal-3 with endogenous Aβ monomers in 8-month-old APP/PS1 mice. Experiments were performed in duplicate. c Different amounts of recombinant human Gal-3 protein (0.25, 0.5, 1, and 2 μg) were added to a solution containing Aβ42 peptide (15 μg), the mixture was subjected to a thioflavin-T assay, and fluorescence was measured hourly at different time points. The fluorescence plateaued at the 7-h time point. Experiments were performed in triplicate. The abbreviation “rh” indicates “recombinant human”. Data are expressed as mean ± SEM

Article Snippet: Immunoblotting was carried out using the following antibodies: rat anti-Gal-3 (1:5000, R&D systems), goat anti-Gal-1 (1:5000, R&D systems), rabbit anit-PIAS1 (1:3000, Epitomics, Burlingame, CA), mouse anti-CD10/NEP (1:500, Santa Cruz Biotechnology, Dallas, TX), mouse anti-prealbumin/transthyretin (1:500, Santa Cruz Biotechnology), rabbit anti-insulin degrading enzyme (1:3000, Abcam, Cambridge, UK), rabbit anti-FAK (1:2000, Abcam), rabbit anti-pFAK (phospho-Y397, 1:2000, Abcam), rabbit anti-CREB (1:2000, Cell Signaling, Danvers, MA), rabbit anti-pCREB (phospho-S133, 1:2000, Cell Signaling), rat anti-TREM2 (1:3000, Millipore, Bedford, MA), mouse anti-TLR4 (1:500, Santa Cruz Biotechnology), rabbit anti-CD40 (1:2000, Abcam), mouse anti-actin (1:150000, Millipore), and mouse anti-Flag M2 (1:10000, Sigma-Aldrich) antibodies.

Techniques: Immunohistochemical staining, Co-Immunoprecipitation Assay, Recombinant, ThT Assay, Fluorescence

Journal: Cell Death and Differentiation

Article Title: Galectin-3 promotes Aβ oligomerization and Aβ toxicity in a mouse model of Alzheimer’s disease

doi: 10.1038/s41418-019-0348-z

Figure Lengend Snippet: Neprilysin (NEP) expression is increased and integrin signaling is enhanced in Gal-3 KO mice. a Dissected hippocampi of naïve WT and Gal-3 KO mice (3-month-old) were subjected to various Western blot assays. Representative gel patterns obtained for NEP, IDE, TTR, and Gal-3 are shown. b Quantified results for NEP expression [ t (1,8) = 8.61, P < 0.001; left panel], IDE expression [ t (1,8) = 0.72, P > 0.05; middle panel] and TTR expression [ t (1,8) = 1.32, P > 0.05; right panel]. c Representative gel patterns obtained for pFAK, FAK, pCREB, and CREB, and their quantified results [ t (1,8) = 11.14, P < 0.001 for pFAK/FAK; t (1,8) = 0.57, P > 0.05 for FAK/Actin; t (1,8) = 6.72, P < 0.001 for pCREB/CREB and t (1,8) = 0.67, P > 0.05 for CREB/Actin]. d ChIP assay examining the binding of endogenous CREB to the NEP promoter in WT and Gal-3 KO mice, and the quantified results [ t (1,8) = 9.06, P < 0.001]. Data are expressed as mean ± SEM. # P < 0.001

Article Snippet: Immunoblotting was carried out using the following antibodies: rat anti-Gal-3 (1:5000, R&D systems), goat anti-Gal-1 (1:5000, R&D systems), rabbit anit-PIAS1 (1:3000, Epitomics, Burlingame, CA), mouse anti-CD10/NEP (1:500, Santa Cruz Biotechnology, Dallas, TX), mouse anti-prealbumin/transthyretin (1:500, Santa Cruz Biotechnology), rabbit anti-insulin degrading enzyme (1:3000, Abcam, Cambridge, UK), rabbit anti-FAK (1:2000, Abcam), rabbit anti-pFAK (phospho-Y397, 1:2000, Abcam), rabbit anti-CREB (1:2000, Cell Signaling, Danvers, MA), rabbit anti-pCREB (phospho-S133, 1:2000, Cell Signaling), rat anti-TREM2 (1:3000, Millipore, Bedford, MA), mouse anti-TLR4 (1:500, Santa Cruz Biotechnology), rabbit anti-CD40 (1:2000, Abcam), mouse anti-actin (1:150000, Millipore), and mouse anti-Flag M2 (1:10000, Sigma-Aldrich) antibodies.

Techniques: Expressing, Western Blot, Binding Assay

Journal: Cell Death and Differentiation

Article Title: Galectin-3 promotes Aβ oligomerization and Aβ toxicity in a mouse model of Alzheimer’s disease

doi: 10.1038/s41418-019-0348-z

Figure Lengend Snippet: Aβ oligomerization and Gal-3 expression are increased in AD patients. The frontal lobe lysates of normal subjects and AD patients were subjected to Western blot analysis for Aβ oligomerization and the expression of Gal-3 and Gal-1 ( n = 4 per group). b – d Quantified results for ( b ) HMW [ t (1,6) = 9.72, P < 0.001] and LMW [ t (1,6) = 7.21, P < 0.001] Aβ oligomerization, c Gal-3 expression [ t (1,6) = 4.92, P < 0.01] and d Gal-1 expression [ t (1,6) = 0.43, P > 0.05]. e Immunohistochemical analysis showing the distributions of Gal-3 and ProteoStat staining in normal subjects and AD patients ( n = 3 per group). Scale bar, 25 μm. DAPI was used to stain nuclei. Data are expressed as mean ± SEM. ** P < 0.01, # P < 0.001

Article Snippet: Immunoblotting was carried out using the following antibodies: rat anti-Gal-3 (1:5000, R&D systems), goat anti-Gal-1 (1:5000, R&D systems), rabbit anit-PIAS1 (1:3000, Epitomics, Burlingame, CA), mouse anti-CD10/NEP (1:500, Santa Cruz Biotechnology, Dallas, TX), mouse anti-prealbumin/transthyretin (1:500, Santa Cruz Biotechnology), rabbit anti-insulin degrading enzyme (1:3000, Abcam, Cambridge, UK), rabbit anti-FAK (1:2000, Abcam), rabbit anti-pFAK (phospho-Y397, 1:2000, Abcam), rabbit anti-CREB (1:2000, Cell Signaling, Danvers, MA), rabbit anti-pCREB (phospho-S133, 1:2000, Cell Signaling), rat anti-TREM2 (1:3000, Millipore, Bedford, MA), mouse anti-TLR4 (1:500, Santa Cruz Biotechnology), rabbit anti-CD40 (1:2000, Abcam), mouse anti-actin (1:150000, Millipore), and mouse anti-Flag M2 (1:10000, Sigma-Aldrich) antibodies.

Techniques: Expressing, Western Blot, Immunohistochemical staining, Staining

Journal: Cancers

Article Title: FOXD1 and Gal-3 Form a Positive Regulatory Loop to Regulate Lung Cancer Aggressiveness

doi: 10.3390/cancers11121897

Figure Lengend Snippet: High FOXD1 and Gal-3 expression are associated with the poor prognosis in lung cancer. ( A ) The ranking of candidate molecules and the corresponding z-score and p -value using the Ingenuity Pathway Analyses (IPA) database from a microarray experiment performed with CL1-0 FOXD1 compared to CL1-0 vector and CL1-5 scramble compared to CL1-5 siRNA targeting FOXD1 (si- FOXD1 ) using a 5-fold change cut-off. ( B ) Statistical p -values and fold change of the expression of Gal-3 ( LGALS3 ) and its relative molecules are shown. Prediction legend: upregulation (red round icon), downregulation (green round icon), predicted activation (brown round icon), predicted inhibition (blue round icon), leads to activation (brown line), leads to inhibition (blue line), findings inconsistent with stage of downstream molecule (yellow line), effect not predicted (gray line). ( C ) RT-qPCR analysis of Gal-3 and FOXD1 mRNA expression in various lung cancer cell lines. GAPDH was used as an internal control for mRNA loading. ( D ) Western blot analysis of Gal-3 and FOXD1 protein expression in various lung cancer cell lines. GAPDH was used as an internal control for protein loading. ( E ) Lung cancer patients with high gene level transcription of Gal-3 or FOXD1 expression level show a correlation with high risk and ( F ) poor disease-free survival. The data were retrieved and analyzed from the TCGA-LUAD samples ( n = 475) and lung meta-base ( n = 1044) of the SurvExpress database ( http://bioinformatica.mty.itesm.mx:8080/Biomatec/SurvivaX.jsp ).

Article Snippet: The slide was de-paraffinized with xylene, rehydrated with alcohol and antigen retrieval, and then blocked with 5% normal goat serum for one hour at room temperature and then incubated with the FOXD1 antibody (Aviva Systems Biology, OAAB10686, USA) or Gal-3 antibody (R&D, MAB11541, USA) at 4 °C overnight.

Techniques: Expressing, Microarray, Plasmid Preparation, Activation Assay, Inhibition, Quantitative RT-PCR, Western Blot

Journal: Cancers

Article Title: FOXD1 and Gal-3 Form a Positive Regulatory Loop to Regulate Lung Cancer Aggressiveness

doi: 10.3390/cancers11121897

Figure Lengend Snippet: FOXD1 is a transcription factor of Gal-3. ( A ) CL1-0 cells were co-transfected with plasmids of the Gal-3 promoter reporter (pGL3- Gal-3 2000 bp) or Gal-3 promoter mutation reporter (pGL3- Gal-3 2000 bp-mut) and FOXD1 ( pFOXD1 ). The cell lysates were collected, and the promoter activity of Gal-3 was analyzed by luciferase assay. ( B ) ChIP-qPCR assay using IgG as a control or FOXD1 antibody in CL1-0 and CL1-5 showed the binding of FOXD1 on the Gal-3 promoter. ( C ) CL1-0 cells transfected with the empty vector or plasmid of FOXD1 ( pFOXD1 ) for 48 h and ( D ) CL1-5 cells transfected with scramble or siRNA targeting FOXD1 (si- FOXD1 ) for 72 h were collected. The mRNA and protein levels of FOXD1 and Gal-3 were analyzed by qPCR (left) and immunoblotting (right), respectively. ( E ) CCK-8 assay in different groups. FOXD1-stimulated CL1-0 proliferation ability was reduced by knocking down Gal-3 . ( F ) The colony-formation ability of FOXD1-stimulated CL1-0 cells was suppressed by the depletion of Gal-3. ( G ) The numbers of migrating or invading cells were increased in the FOXD1 group compared with the FOXD1-stimulated CL1-0-si Gal-3 group and control group.

Article Snippet: The slide was de-paraffinized with xylene, rehydrated with alcohol and antigen retrieval, and then blocked with 5% normal goat serum for one hour at room temperature and then incubated with the FOXD1 antibody (Aviva Systems Biology, OAAB10686, USA) or Gal-3 antibody (R&D, MAB11541, USA) at 4 °C overnight.

Techniques: Transfection, Mutagenesis, Activity Assay, Luciferase, Binding Assay, Plasmid Preparation, Western Blot, CCK-8 Assay

Journal: Cancers

Article Title: FOXD1 and Gal-3 Form a Positive Regulatory Loop to Regulate Lung Cancer Aggressiveness

doi: 10.3390/cancers11121897

Figure Lengend Snippet: Proto-oncogene 1 (ETS-1) is involved in Gal-3-mediated FOXD1 expression. ( A ) The ranking of candidate transcription factors and its corresponding z-score and p -value using the IPA database from a microarray experiment performed with CL1-0 Gal-3 compared with the CL1-0 vector and CL1-5 scramble compared with CL1-5 si- Gal-3 using a 5-fold change cut-off. ( B ) Statistical p -values and fold change of the expressions of ETS-1 ( ets-1 ) and its relative molecules are shown. Prediction legend: upregulation (red round icon), downregulation (green round icon), predicted activation (brown round icon), predicted inhibition (blue round icon), leads to activation (brown line), leads to inhibition (blue line), findings inconsistent with stage of downstream molecule (yellow line), effect not predicted (gray line). ( C ) CL1-0 and CL1-5 cells were transfected with specific cDNA for 48 h or siRNA for 72 h; RT-qPCR analysis of ets-1, which targets downstream mRNA expression in CL1-0 cells. ( D ) Western blot analysis of Gal-3 and ETS-1 from CL1-0 with expression of the vector control (VC) or exogenous Gal-3 gene and CL1-5 cells with scramble and siRNA targeting Gal-3 ( si-Gal-3 ). ( E , F ) CL1-0 and CL1-5 cells were transfected with the empty vector or plasmid of Gal-3 ( p Ets- 1 ) for 48 h or scramble and siRNA targeting ETS-1 ( si- ets-1 ) for 72 h, respectively, as indicated. The mRNA and protein levels of ETS-1, FOXD1, and Gal-3 were analyzed by qPCR and immunoblotting, respectively. Cells were transfected with scramble and siRNA targeting Ets- 1 for 72 h, or empty vector or plasmid of Ets- 1 for 48 h, respectively, as indicated, and immunoprecipitation was analyzed by immunoblotting. Input, the whole cell lysates. ( G ) CL1-0 cells were co-transfected with plasmids of Gal-3 promoter reporter (pGL3- Gal-3 2000 bp), empty vector or plasmid of FOXD1 ( p FOXD1 ) or scramble and siRNA targeting ETS-1 ( si- ets-1 ). Cell lysates were collected, and the promoter activity of Gal-3 was analyzed by luciferase assay. ( H ) CL1-5 cells were transfected with scramble and siRNA targeting ETS-1 ( si- ets-1 ). ChIP-qPCR assay using IgG as a control or FOXD1 antibody in CL1-5 showed the binding of FOXD1 on the Gal-3 promoter.

Article Snippet: The slide was de-paraffinized with xylene, rehydrated with alcohol and antigen retrieval, and then blocked with 5% normal goat serum for one hour at room temperature and then incubated with the FOXD1 antibody (Aviva Systems Biology, OAAB10686, USA) or Gal-3 antibody (R&D, MAB11541, USA) at 4 °C overnight.

Techniques: Expressing, Microarray, Plasmid Preparation, Activation Assay, Inhibition, Transfection, Quantitative RT-PCR, Western Blot, Immunoprecipitation, Activity Assay, Luciferase, Binding Assay

Journal: Cancers

Article Title: FOXD1 and Gal-3 Form a Positive Regulatory Loop to Regulate Lung Cancer Aggressiveness

doi: 10.3390/cancers11121897

Figure Lengend Snippet: Gal-3 regulates FOXD1 expression through integrin-β1 (ITGβ1) signaling. ( A , B ) CL1-0 and CL1-5 cells were transfected with the empty vector or plasmid of Gal-3 ( pGal-3 ) for 48 h or scramble and siRNA targeting Gal-3 ( si-Gal-3 ) for 72 h, respectively, as indicated. The mRNA and protein levels of FOXD1 and Gal-3 were analyzed by qPCR and immunoblotting. CL1-5 cells were transfected with scramble and siRNA targeting Gal-3 for 72 h or empty vector or plasmid of Gal-3 for 48 h, respectively, as indicated, and immunoprecipitation was analyzed by immunoblotting. Input, the whole cell lysates. ( C ) CL1-0 cells were transfected with scramble, Gal-3 , ITGβ1 or si- Gal-3, and si- ITG β 1 for 72 h, as indicated. Cells lysates were collected and analyzed by immunoblotting. ( D ) CL1-0 cells were transfected with Gal-3 or ITGβ1 expression vector for 48 h, and immunoprecipitation was analyzed by immunoblotting.

Article Snippet: The slide was de-paraffinized with xylene, rehydrated with alcohol and antigen retrieval, and then blocked with 5% normal goat serum for one hour at room temperature and then incubated with the FOXD1 antibody (Aviva Systems Biology, OAAB10686, USA) or Gal-3 antibody (R&D, MAB11541, USA) at 4 °C overnight.

Techniques: Expressing, Transfection, Plasmid Preparation, Western Blot, Immunoprecipitation

Journal: Cancers

Article Title: FOXD1 and Gal-3 Form a Positive Regulatory Loop to Regulate Lung Cancer Aggressiveness

doi: 10.3390/cancers11121897

Figure Lengend Snippet: FOXD1 and Gal-3 form a positive regulatory loop to promote lung cancer cell growth and motility. CL1-5 cells were transfected with scramble, si-FOXD1 , or si-Gal-3 or si-FOXD1 with si-Gal-3 for 72 h. Cells lysates were collected and analyzed by immunoblotting. ( A ) The proliferation ability was analyzed by CCK-8. ( B ) Representative images of the colony-forming assay. ( C ) Representative images of the migration and invasion assay. CL1-0 cells were transfected with the empty vector, FOXD1 , Gal-3 , or both FOXD1 and Gal-3 expression vector for 48 h. ( D ) Proliferation ability was analyzed by CCK-8 assay. ( E ) Representative images of colony-forming assay. ( F ) Representative images of migration and invasion assay.

Article Snippet: The slide was de-paraffinized with xylene, rehydrated with alcohol and antigen retrieval, and then blocked with 5% normal goat serum for one hour at room temperature and then incubated with the FOXD1 antibody (Aviva Systems Biology, OAAB10686, USA) or Gal-3 antibody (R&D, MAB11541, USA) at 4 °C overnight.

Techniques: Transfection, Western Blot, CCK-8 Assay, Migration, Invasion Assay, Plasmid Preparation, Expressing

Journal: Cancers

Article Title: FOXD1 and Gal-3 Form a Positive Regulatory Loop to Regulate Lung Cancer Aggressiveness

doi: 10.3390/cancers11121897

Figure Lengend Snippet: FOXD1 and Gal-3 are positively correlated in human lung cancer tissues. ( A ) Immunohistochemistry images show that FOXD1 was increased in human lung cancer tissue microarray. ( B ) Immunohistochemistry images show that Gal-3 was highly expressed in human lung cancer tissue microarray.

Article Snippet: The slide was de-paraffinized with xylene, rehydrated with alcohol and antigen retrieval, and then blocked with 5% normal goat serum for one hour at room temperature and then incubated with the FOXD1 antibody (Aviva Systems Biology, OAAB10686, USA) or Gal-3 antibody (R&D, MAB11541, USA) at 4 °C overnight.

Techniques: Immunohistochemistry, Microarray

Journal: Cancers

Article Title: FOXD1 and Gal-3 Form a Positive Regulatory Loop to Regulate Lung Cancer Aggressiveness

doi: 10.3390/cancers11121897

Figure Lengend Snippet: Schematic diagram illustrates cooperation between FOXD1 and Gal-3 in promoting lung cancer proliferation and metastasis.

Article Snippet: The slide was de-paraffinized with xylene, rehydrated with alcohol and antigen retrieval, and then blocked with 5% normal goat serum for one hour at room temperature and then incubated with the FOXD1 antibody (Aviva Systems Biology, OAAB10686, USA) or Gal-3 antibody (R&D, MAB11541, USA) at 4 °C overnight.

Techniques:

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: Molecular Therapy Oncolytics

Article Title: Galectin-3 inhibition boosts the therapeutic efficacy of Semliki Forest virus in pediatric osteosarcoma

doi: 10.1016/j.omto.2022.07.004

Figure Lengend Snippet: Expression of Gal3 and related genes in human patients and mouse models of osteosarcoma (A) mRNA expression of Gal3 ( LGALS3 ), Gal3bp ( LGALS3BP ), IL-6 , and C1GALT1 in tumor versus paired healthy samples from osteosarcoma patients (n = 6). The level of expression was determined using microarray analysis with the robust multiarray analysis (RMA) algorithm. Correlation of IL-6 versus C1GALT1 mRNA expression in tumor samples, ∗∗p < 0.01 by Pearson’s r. (B and C) mRNA (B) and protein (C) expression of Gal3 and Gal3bp in murine osteosarcoma cell lines (K7M2, MOS-J, and POS-1) and the murine melanoma cell line B16OVA determined by qRT-PCR (n = 3) and western blotting. (D) mRNA expression of Gal3, Gal3bp, and IL-6 in tibias and lungs representing healthy versus tumor tissue from orthotopic K7M2 tumor-bearing mice determined by qRT-PCR (n = 3). The data in (B) and (D) were calculated as 2E(−ΔCt) normalized to GAPDH × 10,000 and are presented as the mean ± SD. ∗p < 0.05; ∗∗p < 0.01; ns, not significant. Student’s t test.

Article Snippet: Samples were subjected to SDS-Tris-Gly gel electrophoresis under denaturing conditions and then transferred to nitrocellulose membranes, which were incubated with antibodies specific for the following molecules ( ): nsp2 subunit of SFV replicase, HA tag (BioLegend), Gal3 (R&D Systems, Minneapolis, MN), Gal3-BP (ProteinTech, Rosemont, IL), and α-tubulin (Cell Signaling Technologies, Danvers, MA).

Techniques: Expressing, Microarray, Quantitative RT-PCR, Western Blot

Journal: Molecular Therapy Oncolytics

Article Title: Galectin-3 inhibition boosts the therapeutic efficacy of Semliki Forest virus in pediatric osteosarcoma

doi: 10.1016/j.omto.2022.07.004

Figure Lengend Snippet: Characterization of SFV vectors expressing Gal3 inhibitors (A) Diagrams of SFV vectors expressing Gal3 inhibitors: SFV-Gal3-C, SFV-Gal3-N, SFV-C12, and SFV-Gal3-N-C12. Constructs contained an SFV replicase sequence (composed of four nonstructural subunits [nsps]) followed by the viral subgenomic promoter (sgPr), a translation enhancer (b1) linked to the 2A FMDV protease fused in-frame to each Gal3 inhibitor sequences, and an HA tag. (B and C) Gal3 inhibitor expression in BHK-21 cells 24 h after infection with SFV vectors at an MOI of 20, as determined by western blot analysis of cell extracts (CEs) and supernatants (SNs) using an anti-HA antibody (B) and by immunofluorescence staining (C) using anti-nsp2 and anti-HA antibodies. Cell nuclei were stained with DAPI (magnification, 200×; scale bar, 100 μm). (D) Luciferase activity was determined in orthotopic K7M2 tumor-bearing mice at the indicated times after intratumoral injection of 1 × 10 8 VPs SFV-Luc; signal is measured in photons/s. Data represent the mean ± SD (n = 3). Images of luciferase expression in mice are shown. (E and F) Inhibition of Gal3 binding to activated T cells. CD8 + and CD4 + T cells activated with anti-CD3 and anti-CD28 antibodies and incubated with IL-10 were treated with the indicated recombinant Gal3 inhibitors at 50 μM (CD8 + ) or 25 μM (CD4 + ), with an anti-Gal3 antibody (a-Gal3) at 20 μg/mL (CD8 + ) or 10 μg/mL (CD4 + ) in the presence of 5 μg/mL recombinant Gal3 (+) for 30 min (CD8 + ) or 48 h (CD4 + ). Cells incubated without inhibitors and Gal3 indicated by (−). The binding of Gal3 was determined by flow-cytometric measurement of the mean fluorescence intensity (MFI) of Gal3 on total CD8 + and CD4 + T cells (E) or in CD8 + PD1 + and CD4 + PD1 + T cells (F). Data are presented as the mean ± SD (n = 3). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001; one-way ANOVA. N, Gal3-N; C, Gal3C; N-C12, Gal3-N-C12.

Article Snippet: Samples were subjected to SDS-Tris-Gly gel electrophoresis under denaturing conditions and then transferred to nitrocellulose membranes, which were incubated with antibodies specific for the following molecules ( ): nsp2 subunit of SFV replicase, HA tag (BioLegend), Gal3 (R&D Systems, Minneapolis, MN), Gal3-BP (ProteinTech, Rosemont, IL), and α-tubulin (Cell Signaling Technologies, Danvers, MA).

Techniques: Expressing, Construct, Sequencing, Infection, Western Blot, Immunofluorescence, Staining, Luciferase, Activity Assay, Injection, Inhibition, Binding Assay, Incubation, Recombinant, Fluorescence

Journal: Molecular Therapy Oncolytics

Article Title: Galectin-3 inhibition boosts the therapeutic efficacy of Semliki Forest virus in pediatric osteosarcoma

doi: 10.1016/j.omto.2022.07.004

Figure Lengend Snippet: Evaluation of the antitumor effect of SFV vectors expressing Gal3 inhibitors in osteosarcoma (A) Treatment schedule for orthotopic osteosarcoma mouse models. Tumor cells were injected intratibially on day 0. The tumors were treated with 1 × 10 8 VPs SFV on day 7, and tumor size and survival were monitored. (B) K7M2 tumor growth in mice treated with the indicated vectors (n = 10) or PBS (n = 9). A representative experiment is shown of two experiments with similar results. Data are shown as the mean ± SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001; extra sum-of-squares F test. (C) Individual tumor growth of the mice presented in (B). Discontinuous red line indicates time when control mice developed tumors >400 mm 2 . (D) Kaplan-Meier survival plot of the mice described in (A). The graph corresponds to pooled data from two experiments using SFV-Gal3-C (n = 10), SFV-Gal3-N (n = 19), SFV-Gal3-N-C12 (n = 19), SFV-C12 (n = 9), SFV-Luc (n = 10), and PBS (n = 17). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001; log-rank test. (E) Kaplan-Meier survival curves of cured K7M2 tumor-bearing mice rechallenged with K7M2 cells (n = 5). p > 0.05 (not significant); log-rank test. (F) Tumor growth evaluation of MOS-J tumor-bearing mice treated as described in (A) with the indicated vectors (n = 9–10). Data are shown as the mean ± SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001; extra sum-of-squares F test. (G) Kaplan-Meier survival plot of the MOS-J tumor-bearing mice described in (A). ∗p < 0.05, log-rank test. (H) Kaplan-Meier survival plot of cured MOS-J tumor-bearing mice rechallenged with MOS-J cells (n = 3). ∗p < 0.05, log-rank test.

Article Snippet: Samples were subjected to SDS-Tris-Gly gel electrophoresis under denaturing conditions and then transferred to nitrocellulose membranes, which were incubated with antibodies specific for the following molecules ( ): nsp2 subunit of SFV replicase, HA tag (BioLegend), Gal3 (R&D Systems, Minneapolis, MN), Gal3-BP (ProteinTech, Rosemont, IL), and α-tubulin (Cell Signaling Technologies, Danvers, MA).

Techniques: Expressing, Injection, Control

Journal: Molecular Therapy Oncolytics

Article Title: Galectin-3 inhibition boosts the therapeutic efficacy of Semliki Forest virus in pediatric osteosarcoma

doi: 10.1016/j.omto.2022.07.004

Figure Lengend Snippet: Assessment of the antimetastatic effect of SFV vectors in an orthotopic K7M2 osteosarcoma mouse model (A) Summary table for survival, presence of metastases, and presence of bone tumors from two pooled experiments evaluating K7M2 tumor-bearing mice treated with the indicated SFV vectors or PBS. (B) Analysis of lung metastases. K7M2 tumor-bearing mice were analyzed on day 15 after treatment with PBS, SFV-Luc, or SFV-Gal3-N-C12, and healthy mice without tumors were used as controls. MicroCT analysis (upper images) and H&E staining (lower images) of lung tissue samples from one representative mouse in each group (magnification, 20×; scale bar, 4 mm). Quantification of the volume of the healthy lung parenchyma in all mice in the different treatment groups. Data are presented as the mean ± SD (n = 3, each group). p > 0.05 (not significant); one-way ANOVA. (C and D) Analysis of gene expression by RNA-seq. Mice bearing K7M2 tumors were treated with SFV-Gal3-N-C12 (NC12, n = 4), SFV-Luc (LUC, n = 3), or PBS (n = 5) as described in Figure 3 A. On day 14 the mice were sacrificed, and total RNA was extracted from the tumors for sequencing. (C) Upon gene set enrichment analysis, an enriched gene set of prometastatic genes involved in osteosarcoma pathology was downregulated in the SFV-Gal3-N-C12 (NC12) group compared with the SFV-Luc (LUC) group at nominal p < 0.01 and false discovery rate (FDR) < 0.05. Normalized enrichment score (NES), −1.82; ∗∗p adjusted < 0.01. (D) A heatmap and hierarchical clustering representing the differential expression of the most significant prometastatic genes between the treatment groups.

Article Snippet: Samples were subjected to SDS-Tris-Gly gel electrophoresis under denaturing conditions and then transferred to nitrocellulose membranes, which were incubated with antibodies specific for the following molecules ( ): nsp2 subunit of SFV replicase, HA tag (BioLegend), Gal3 (R&D Systems, Minneapolis, MN), Gal3-BP (ProteinTech, Rosemont, IL), and α-tubulin (Cell Signaling Technologies, Danvers, MA).

Techniques: Staining, Gene Expression, RNA Sequencing, Sequencing, Quantitative Proteomics

Journal: Molecular Therapy Oncolytics

Article Title: Galectin-3 inhibition boosts the therapeutic efficacy of Semliki Forest virus in pediatric osteosarcoma

doi: 10.1016/j.omto.2022.07.004

Figure Lengend Snippet: Analysis of immune cell populations in primary K7M2 tumors after treatment with SFV vectors by immunohistochemistry and RNA-seq (A) Immunohistochemistry (IHC) analysis of CD3 + T cells in primary osteosarcoma tumors from mice sacrificed at 14–17 days after intratumoral treatment with the indicated vectors or PBS. Representative IHC images are shown. Quantification of CD3 + T cells presented as the percentage of cells stained positive for CD3 in IHC images (magnification, 400×; scale bar, 200 μm). CD3 + T cells were counted in five different fields in each sample, and the mean was used to perform statistical analysis. Data are shown as the mean ± SD (n = 3). p > 0.05 (not significant); one-way ANOVA. (B) Relative abundances (in percentages) of 29 different immune cell populations determined by analysis of RNA-seq data for primary tumors from K7M2 tumor-bearing mice obtained as described in Figure 4 C with the online tool ImmuCellAI-mouse. The abundance of each population was normalized by considering 1 to be the total (100%) population abundance. (C) Normalized abundances of natural killer (NK) cells, type 1 dendritic cells (cDC1s), plasmacytoid dendritic cells (pDCs), M1 macrophages, and M2 macrophages. Data are shown as the mean ± SD (n = 3–5). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001; one-way ANOVA. (D) Heatmap representing the differential expression and hierarchical clustering of the most significant immunomodulatory genes between treatment groups. NC12, SFV-Gal3-N-C12; LUC, SFV-Luc.

Article Snippet: Samples were subjected to SDS-Tris-Gly gel electrophoresis under denaturing conditions and then transferred to nitrocellulose membranes, which were incubated with antibodies specific for the following molecules ( ): nsp2 subunit of SFV replicase, HA tag (BioLegend), Gal3 (R&D Systems, Minneapolis, MN), Gal3-BP (ProteinTech, Rosemont, IL), and α-tubulin (Cell Signaling Technologies, Danvers, MA).

Techniques: Immunohistochemistry, RNA Sequencing, Staining, Quantitative Proteomics

Journal: Molecular Therapy Oncolytics

Article Title: Galectin-3 inhibition boosts the therapeutic efficacy of Semliki Forest virus in pediatric osteosarcoma

doi: 10.1016/j.omto.2022.07.004

Figure Lengend Snippet: Characterization of the tumor microenvironment of K7M2 tumors after treatment with SFV vectors (A and B) Flow-cytometric analyses of different immune cell populations in primary K7M2 tumors (tibias) (A) and lung metastases (B) on day 3 after treatment with PBS, SFV-Gal3-N-C12 (N-C12), or SFV-Luc (Luc). Data are shown as the number of cells/mg tissue and as the mean ± SD (n = 5). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001; one-way ANOVA. (C) Ratios of CD4 + /CD8 + T cells, CD8 + T cells/M2 macrophages, and CD8 + /CD4 + Foxp3 + T cells in the tumor samples analyzed in (A). Data are shown as the mean ± SD (n = 5). ∗p < 0.05; one-way ANOVA. (D) Gp70 tetramer (Tet + ) staining (%) of the CD8 + T cell population (%) and surface expression of Gal3 in the CD8 + Tet + T cell population (MFI) in K7M2 tumors on day 14 after treatment with the indicated vectors. Data are shown as the mean ± SD (n = 4). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001; one-way ANOVA. (E) IFN-γ production in TILs isolated from K7M2 primary tumors on day 14 after treatment with the indicated SFV vectors. IFN-γ levels were measured by ELISA, and IFN-γ spot numbers and IFN-γ mean spot sizes were measured by ELISPOT. Data are shown as the mean ± SD (n = 3). ∗p < 0.05, ∗∗p < 0.01; one-way ANOVA. +, splenocytes plus mitogen; −, only splenocytes; K7M2, only K7M2 cells.

Article Snippet: Samples were subjected to SDS-Tris-Gly gel electrophoresis under denaturing conditions and then transferred to nitrocellulose membranes, which were incubated with antibodies specific for the following molecules ( ): nsp2 subunit of SFV replicase, HA tag (BioLegend), Gal3 (R&D Systems, Minneapolis, MN), Gal3-BP (ProteinTech, Rosemont, IL), and α-tubulin (Cell Signaling Technologies, Danvers, MA).

Techniques: Staining, Expressing, Isolation, Enzyme-linked Immunosorbent Assay, Enzyme-linked Immunospot

Journal: Molecular Therapy Oncolytics

Article Title: Galectin-3 inhibition boosts the therapeutic efficacy of Semliki Forest virus in pediatric osteosarcoma

doi: 10.1016/j.omto.2022.07.004

Figure Lengend Snippet: Analysis of exhaustion markers expressed by tumor-infiltrating lymphocytes in K7M2 tumors after treatment with SFV vectors (A) Expression of PD1, LAG3, or TIM3 (MFI) in CD8 + T cells from K7M2 tumors on day 3 and day 7 after treatment with PBS, SFV-Luc (LUC), or SFV-Gal3-N-C12 (N-C12). Data are shown as the mean ± SD (n = 5, each group). ∗p < 0.05, ∗∗p < 0.01; one-way ANOVA. (B) Pie charts showing the percentage of CD8 + T cells coexpressing the activation/exhaustion markers PD1, LAG3, and TIM3. (C) Same analysis as in (A) performed with tumor-infiltrating CD4 + T cells. Data are shown as the mean ± SD (n = 5, each group). p > 0.05 (not significant); one-way ANOVA. (D) Pie charts showing the percentage of CD4 + T cells coexpressing the activation/exhaustion markers PD1, LAG3, and TIM3.

Article Snippet: Samples were subjected to SDS-Tris-Gly gel electrophoresis under denaturing conditions and then transferred to nitrocellulose membranes, which were incubated with antibodies specific for the following molecules ( ): nsp2 subunit of SFV replicase, HA tag (BioLegend), Gal3 (R&D Systems, Minneapolis, MN), Gal3-BP (ProteinTech, Rosemont, IL), and α-tubulin (Cell Signaling Technologies, Danvers, MA).

Techniques: Expressing, Activation Assay

Journal: Molecular Therapy Oncolytics

Article Title: Galectin-3 inhibition boosts the therapeutic efficacy of Semliki Forest virus in pediatric osteosarcoma

doi: 10.1016/j.omto.2022.07.004

Figure Lengend Snippet: Modulation of immune cell populations involved in pulmonary osteosarcoma metastases Flow-cytometric analyses of CD4 + or CD8 + T cells expressing PD1, Foxp3, CD25, and/or Gal3 in the primary tumors (tibias) (A) and pulmonary metastases (B) of mice bearing K7M2 tumors on day 14 after treatment with PBS, SFV-Gal3-N-C12 (N-C12), or SFV-Luc (Luc). Data are shown as the mean percentage ± SD of the total CD4 + or CD8 + T cells (n = 5). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001; one-way ANOVA.

Article Snippet: Samples were subjected to SDS-Tris-Gly gel electrophoresis under denaturing conditions and then transferred to nitrocellulose membranes, which were incubated with antibodies specific for the following molecules ( ): nsp2 subunit of SFV replicase, HA tag (BioLegend), Gal3 (R&D Systems, Minneapolis, MN), Gal3-BP (ProteinTech, Rosemont, IL), and α-tubulin (Cell Signaling Technologies, Danvers, MA).

Techniques: Expressing