human/mouse/rat galectin-3 antibody  (Bio-Techne corporation)

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