Journal: Biosensors & bioelectronics

Article Title: Microarray method to monitor 40 intestinal bacterial species in the study of azo dye reduction

doi: 10.1016/j.bios.2004.04.011

Figure Lengend Snippet: Bacteria and the probe numbers in the microarray

Article Snippet: Anaerobic bacteria were cultured at 35 °C in either prereduced anaerobically sterilized (PRAS) Brain Heart Infusion (BIH) broth supplemented with vitamin K and hemin (Remel, Lenexa, KS, USA), inoculated under an oxygen-free cannula using 85% nitrogen, 10% hydrogen and 5% carbon dioxide, or on PRAS brucella blood agar plates supplemented with vitamin K and hemin (Remel). table ft1 table-wrap mode="anchored" t5 caption a7 Number Bacterial species and strain Probe number 1 B. thetaiotaomicron ATCC 29148 1, 2, 3 2 B. vulgatus ATCC 8482 4, 5, 6 3 B. fragilis ATCC 23745 7, 8, 9 4 B. distasonis ATCC 8503 10, 11, 12 5 C. clostridioforme ATCC 29084 13, 14, 15 6 C. leptum ATCC 29065 16, 17, 18 7 F. prausnitzii ATCC 27768 19, 20, 21 8 P. productus ATCC 27340 22, 23, 24 9 R. obeum ATCC 29174 25, 26, 27 10 R. bromii ATCC 27255 28, 29, 30 11 R. callidus ATCC 27760 31, 32, 33 12 R. albus ATCC 27210 34, 35, 36 13 B. longum ATCC 15707 37, 38, 39 14 B. adolescentis ATCC 15703 40, 41, 42 15 B. infantis ATCC 15697 43, 44, 45 16 E. biforme ATCC 27806 46, 47, 48 17 E. aerofaciens ATCC 25986 49, 50, 51 18 L. acidophilus ATCC 4356 52, 53, 54 19 E. coli ATCC 25922 55, 56, 57 20 E. faecium ATCC 19434 58, 59, 60 21 B. uniformis ATCC 8492 61, 62, 63 22 B. ovatus ATCC 8483 64, 65, 66 23 B. caccae ATCC 43185 67, 68, 69 24 C. perfringens ATCC 13124 70, 71, 72 25 C. butyricum ATCC 19398 73, 74, 75 26 C. ramosum ATCC 25582 76, 77, 78 27 C. difficile ATCC 9689 79, 80, 81 28 C. indolis ATCC 25771 82, 83, 84 29 F. russii ATCC 25533 85, 86, 87 30 F. nucleatum ATCC 25586 88, 89, 90 31 B. catenulatum ATCC 27539 91, 92, 93 32 B. angulatum ATCC 27535 94, 95, 96 33 E. rectale ATCC 33656 97, 98, 99 34 E. eligens ATCC 27750 100, 101, 102 35 E. limosum ATCC 8486 103, 104, 105 36 E. lentum ATCC 25553 106, 107, 108 37 L. fermentum ATCC 9338 109, 110, 111 38 E. faecalis ATCC 27274 112, 113, 114 39 P. magnus ATCC 14955 115, 116, 117 40 R. gnavus ATCC 291492 118, 119, 120 Open in a separate window Bacteria and the probe numbers in the microarray

Techniques: Bacteria

Journal: Biosensors & bioelectronics

Article Title: Microarray method to monitor 40 intestinal bacterial species in the study of azo dye reduction

doi: 10.1016/j.bios.2004.04.011

Figure Lengend Snippet: Microarray test results read from

Article Snippet: Anaerobic bacteria were cultured at 35 °C in either prereduced anaerobically sterilized (PRAS) Brain Heart Infusion (BIH) broth supplemented with vitamin K and hemin (Remel, Lenexa, KS, USA), inoculated under an oxygen-free cannula using 85% nitrogen, 10% hydrogen and 5% carbon dioxide, or on PRAS brucella blood agar plates supplemented with vitamin K and hemin (Remel). table ft1 table-wrap mode="anchored" t5 caption a7 Number Bacterial species and strain Probe number 1 B. thetaiotaomicron ATCC 29148 1, 2, 3 2 B. vulgatus ATCC 8482 4, 5, 6 3 B. fragilis ATCC 23745 7, 8, 9 4 B. distasonis ATCC 8503 10, 11, 12 5 C. clostridioforme ATCC 29084 13, 14, 15 6 C. leptum ATCC 29065 16, 17, 18 7 F. prausnitzii ATCC 27768 19, 20, 21 8 P. productus ATCC 27340 22, 23, 24 9 R. obeum ATCC 29174 25, 26, 27 10 R. bromii ATCC 27255 28, 29, 30 11 R. callidus ATCC 27760 31, 32, 33 12 R. albus ATCC 27210 34, 35, 36 13 B. longum ATCC 15707 37, 38, 39 14 B. adolescentis ATCC 15703 40, 41, 42 15 B. infantis ATCC 15697 43, 44, 45 16 E. biforme ATCC 27806 46, 47, 48 17 E. aerofaciens ATCC 25986 49, 50, 51 18 L. acidophilus ATCC 4356 52, 53, 54 19 E. coli ATCC 25922 55, 56, 57 20 E. faecium ATCC 19434 58, 59, 60 21 B. uniformis ATCC 8492 61, 62, 63 22 B. ovatus ATCC 8483 64, 65, 66 23 B. caccae ATCC 43185 67, 68, 69 24 C. perfringens ATCC 13124 70, 71, 72 25 C. butyricum ATCC 19398 73, 74, 75 26 C. ramosum ATCC 25582 76, 77, 78 27 C. difficile ATCC 9689 79, 80, 81 28 C. indolis ATCC 25771 82, 83, 84 29 F. russii ATCC 25533 85, 86, 87 30 F. nucleatum ATCC 25586 88, 89, 90 31 B. catenulatum ATCC 27539 91, 92, 93 32 B. angulatum ATCC 27535 94, 95, 96 33 E. rectale ATCC 33656 97, 98, 99 34 E. eligens ATCC 27750 100, 101, 102 35 E. limosum ATCC 8486 103, 104, 105 36 E. lentum ATCC 25553 106, 107, 108 37 L. fermentum ATCC 9338 109, 110, 111 38 E. faecalis ATCC 27274 112, 113, 114 39 P. magnus ATCC 14955 115, 116, 117 40 R. gnavus ATCC 291492 118, 119, 120 Open in a separate window Bacteria and the probe numbers in the microarray

Techniques: Microarray

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Lamin B1 Is a Novel Therapeutic Target of Betulinic Acid in Pancreatic Cancer

doi: 10.1158/1078-0432.CCR-12-3630

Figure Lengend Snippet: A. Suppression of lamin B1 expression at the levels of mRNA (qRT-PCR) and protein (Western blot) analysis. B. Dose-dependent antitumor effects of BA in xenograft models of human pancreatic cancer. Shown were gross tumors in mice (6 mice in each group). This was one representative experiment of three with similar results. *P<0.05 as compared with the control mice. The antitumor effect of BA in the primary pancreatic tumor xenograft model. C. Immunohistochemical analysis of the expression levels of lamin B1 and Sp1 in xenograft tumors of PANC-1 and AsPC-1 cells. Representative pictures showed that Sp1 (staining the nuclear) and lamin B1 (staining the nuclear envelope) levels were significantly decreased after BA treatment. D. Schematic structure of the lamin B1 promoter reporter with 8 Sp1 binding sites (CCGCCC or GGGCGG) were identified. BA treatment significantly suppressed the promoter activities (P=0.002) in PANC-1 cells.

Article Snippet: Standard western blot was performed using 35 μg whole-cell protein lysates with primary antibodies against lamin B1 (sc-6216, Santa Cruz Biotechnology) or Sp1 (sc-59, Santa Cruz Biotechnology), and proper secondary antibodies (anti-rabbit IgG and anti-goat IgG, Santa Cruz Biotechnology).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Immunohistochemical staining, Staining, Binding Assay

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Lamin B1 Is a Novel Therapeutic Target of Betulinic Acid in Pancreatic Cancer

doi: 10.1158/1078-0432.CCR-12-3630

Figure Lengend Snippet: A. Representative pictures from tissue microarray slides demonstrated the lamin B1 staining in tumor (T, in Blue Square) and paracancerous normal pancreatic tissues (N, in Red square). B. Analyses of lamin B1 expression at levels of mRNA (qRT-PCR) and protein (Western blot) in 5 pancreatic cancer samples (T) and their paired normal pancreatic tissues (N). Actin was used as a control. C. Representative images of IHC showed lamin B1 protein expression at different levels. D. Lamin B1 expression was negative or weak in tumor adjacent normal pancreatic ductal cells, whereas the expression was strong in pancreatic cancer tissues. Kaplan–Meier curve depicting survival of pancreatic cancer patients with high or low levels of lamin B1. The median survival duration of each group was 9.75 or 12.4 months, respectively.

Article Snippet: Standard western blot was performed using 35 μg whole-cell protein lysates with primary antibodies against lamin B1 (sc-6216, Santa Cruz Biotechnology) or Sp1 (sc-59, Santa Cruz Biotechnology), and proper secondary antibodies (anti-rabbit IgG and anti-goat IgG, Santa Cruz Biotechnology).

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

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Lamin B1 Is a Novel Therapeutic Target of Betulinic Acid in Pancreatic Cancer

doi: 10.1158/1078-0432.CCR-12-3630

Figure Lengend Snippet: A. Lamin B1 expression correlated with tumor differentiation. Representative images showed different levels of lamin B1 expression in well (a) and poorly (b) differentiated pancreatic ductal cells (left panel). An increased lamin B1 expression correlated with decreased levels of tumor differentiation and increased grade (between grade 1 and grade 3) of tumors (*P=0.008) (right panel). B. Lamin B1 expression was positively correlated with the presence of distant metastasis (*P<0.001 in a comparison between cases with distant metastasis and cases without distant metastasis) (right panel) and representative images of tumors from patients with or without distant metastasis are shown (left panel).

Article Snippet: Standard western blot was performed using 35 μg whole-cell protein lysates with primary antibodies against lamin B1 (sc-6216, Santa Cruz Biotechnology) or Sp1 (sc-59, Santa Cruz Biotechnology), and proper secondary antibodies (anti-rabbit IgG and anti-goat IgG, Santa Cruz Biotechnology).

Techniques: Expressing

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Lamin B1 Is a Novel Therapeutic Target of Betulinic Acid in Pancreatic Cancer

doi: 10.1158/1078-0432.CCR-12-3630

Figure Lengend Snippet: A. AsPC-1 and PANC-1 cells were transfected with siCtrl or siLMNB1. Lamin B1 silence efficiency were determined 48 hours after transfection and cell proliferation was analysed (data were mean±SEM calculated from three independent experiments). B. For cell scratch wound assay, the cultures were wounded by scratching and maintained at 37°C for additional 20 hours. Cell cultures were photographed and cell migration was assessed by measuring gap sizes (inserted number represented percentage area of mean±SD). C. For cell invasion assay, the transfected cells were maintained at 37 °C for additional 24 hours. Representative tumor cells invaded through Matrigel were photographed, whereas the numbers of invasive cells that penetrated through Matrigel-coated filter were counted in 15 random fields identified within the lower surface of the filters and expressed as percentage of control (inserted numbers). Data represented mean±SD of triplicates. (*P<0.05 in a comparison between the siLMNB1–transfected cells and the siCtrl cells.)

Article Snippet: Standard western blot was performed using 35 μg whole-cell protein lysates with primary antibodies against lamin B1 (sc-6216, Santa Cruz Biotechnology) or Sp1 (sc-59, Santa Cruz Biotechnology), and proper secondary antibodies (anti-rabbit IgG and anti-goat IgG, Santa Cruz Biotechnology).

Techniques: Transfection, Scratch Wound Assay Assay, Migration, Invasion Assay

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Lamin B1 Is a Novel Therapeutic Target of Betulinic Acid in Pancreatic Cancer

doi: 10.1158/1078-0432.CCR-12-3630

Figure Lengend Snippet: A. AsPC-1 and PANC-1 cells with lamin B1 knockdwon were injected subcutaneously (1×106 per mouse) into the right scapular region of nude mice. The tumor-bearing mice were sacrificed when they became moribund or on day 28. Shown were gross tumors in the mice. The tumors removed from the mice were weighed (*P<0.05 in a comparison between siLMNB1-transfected tumors and the parental or siCtrl ones): AsPC-1 (left panel) and PANC-1 (right panel). B. Knockdown of lamin B1 expression was observed in the xenograft tissues as determined by immunohistochemistry: AsPC-1 (left panel) and PANC-1 (right panel).

Article Snippet: Standard western blot was performed using 35 μg whole-cell protein lysates with primary antibodies against lamin B1 (sc-6216, Santa Cruz Biotechnology) or Sp1 (sc-59, Santa Cruz Biotechnology), and proper secondary antibodies (anti-rabbit IgG and anti-goat IgG, Santa Cruz Biotechnology).

Techniques: Injection, Transfection, Expressing, Immunohistochemistry

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Lamin B1 Is a Novel Therapeutic Target of Betulinic Acid in Pancreatic Cancer

doi: 10.1158/1078-0432.CCR-12-3630

Figure Lengend Snippet: A. Immunohistochemistry showed overexpression of Sp1 in human pancreatic cancer and xenograft tumors when compared with normal pancreatic ductal cells. However, no significant correlation between lamin B1 and Sp1 expressions was detected in pancreatic cancer samples. B. Lamin B1 and Sp1 expression in pancreatic cancer cell lines and their relationship. Western blot analysis of the lamin B1 and Sp1 protein expression in pancreatic cell lines was performed and GAPDH was used as a control. Correlation analysis was done between lamin B1 and Sp1 expressions in pancreatic cancer cell lines (Pearson's correlation test). C. Lamin B1 expression after knockdown of Sp1 and treatment with MIT. AsPC-1 and PANC-1 were transfected with siRNA targeting Sp1. The Sp1 knockdown efficiency was validated using western blot, whereas lamin B1 expression was not downregulated neither in AsPC-1 nor in PANC-1 cells. However, treatment of PANC-1 and AsPC-1 cells with indicated dose of MIT for 48 hours suppressed the expression of both Sp1 and lamin B1. D. Sp1 was knocked down in AsPC-1 and PANC-1 cells and proliferation assays were performed at indicated time points (data were mean ± SEM calculated from three independent experiments).

Article Snippet: Standard western blot was performed using 35 μg whole-cell protein lysates with primary antibodies against lamin B1 (sc-6216, Santa Cruz Biotechnology) or Sp1 (sc-59, Santa Cruz Biotechnology), and proper secondary antibodies (anti-rabbit IgG and anti-goat IgG, Santa Cruz Biotechnology).

Techniques: Immunohistochemistry, Over Expression, Expressing, Western Blot, Transfection

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: 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