Journal: PLoS ONE

Article Title: Molecular Characterization of Striated Muscle-Specific Gab1 Isoform as a Critical Signal Transducer for Neuregulin-1/ErbB Signaling in Cardiomyocytes

doi: 10.1371/journal.pone.0166710

Figure Lengend Snippet: A) Tyrosine phosphorylation of the high-MW Gab1 was analyzed by immunoprecipitation of the heart lysates. Mouse heart lysates were prepared at 5 min after injection with the cytokines and growth factors listed at top. Heart lysates were subjected to immunoprecipitation with anti-high-MW Gab1 serum, followed by western blot analysis using the antibodies indicated at the left. Arrowheads denote the high-MW Gab1. B) Activation level of ErbB4 was assessed by phospho-specific antibody. C) Activation levels of ERK1/2 and AKT were assessed by phospho-specific antibodies. Heart lysates were subjected to western blot analysis with the indicated antibodies. Representative blots of 3 experiments are shown. D) Phosphorylation of ERK1/2 was quantified against total ERK1/2 (n = 3). E) Phosphorylation of AKT on Ser-473 was quantified against total AKT (n = 3). Values are shown as means±SEM for 3 separate experiments. One-way ANOVA followed by Tukey’s test was used to analyze differences. * P <0.05, ** P <0.01, and *** P <0.001 for the indicated groups.

Article Snippet: Other antibodies were purchased as follows: anti-phospho-Gab1 (Tyr627) (1:1000, #3231), anti-Gab1 (1:1000, #3232), anti-phospho-AKT (Ser473) (1:1000, #9271), anti-AKT (1:1000, #9272), anti-phospho-p44/p42 (pERK1/2) (1:1000, #9101), anti-p44/p42 (ERK1/2) (1:1000, #9102), anti-pErbB4 (Tyr1284) (1:1000, #4757) antibodies for immunoblotting, and horseradish peroxidase-coupled horse anti-mouse (1:3000, #7076) and goat anti-rabbit IgG (1:3000, #7074) from Cell Signaling Technology; antibodies recognizing phospho-tyrosine (PY99) (1:1000, sc-7020), ErbB4 (1:400, sc-283), and SHP2 (1:400, sc-280) were from Santa Cruz Biotechnology Inc; anti-Gab1 (1:1000, 06–579) antibody for immunoblotting of immunoprecipitated proteins, and anti-p85 (1:1000, 06–195) antibody were from Millipore; antibodies against β-tubulin (mouse monoclonal) (1:3000, T5201), and anti-α-actinin (1:300, A7732) were from Sigma-Aldrich.

Techniques: Phospho-proteomics, Immunoprecipitation, Injection, Western Blot, Activation Assay

Journal: Journal for Immunotherapy of Cancer

Article Title: Optimal dosing regimen of CD73 blockade improves tumor response to radiotherapy through iCOS downregulation

doi: 10.1136/jitc-2023-006846

Figure Lengend Snippet: Tuning of CD73 blockade is required to improve the response of low CD73-expressing MC38 tumors to IR. (A) MC38 tumor cells were injected subcutaneously in C57BL/6 mice and when tumors reached 80–100 mm 3 , tumor were irradiated at 12 Gy, and mice were treated with anti-CD73 commencing 1 day before IR then 2, 6 and 9 day post-IR. (B) Tumor growth was monitored in treated mice. (C) The Kaplan-Meier survival curves for the treated mice are shown. Data were obtained from two independent experiments and are represented as the mean±SEM. n=11–12, *p<0.05 ****p<0.0001 (two-way ANOVA). (D, right panel) Representative histograms of CD73 expression in MC38 and TS/A cells 24-hour post-IR at 6 and 12 Gy compared with non-irradiated (NIR) cells. (D, left panel) cultured cells were analyzed by flow cytometry for their CD73 expression, which is represented as the mean fluorescence intensity (∆MFI=MFI of the isotype control - MFI of stained cells). Data were obtained from two independent experiments and are represented as the mean±SEM. n=6, **p<0.01, ***p<0.001, ****p<0.0001 (two-way ANOVA). (E) TS/A tumor cells were injected subcutaneously in BALB/c mice and when tumors reached 60–70 mm 3 , tumor were irradiated at 12 Gy, and mice were treated with anti-CD73 commencing 1 day before IR then 2, 6 and 9 days post-IR. (F) Tumor growth was monitored in treated mice. (G) The Kaplan-Meier survival curves for the treated mice are shown. (I) Tumor growth is shown for individual mice in each treatment group. Data were obtained from two independent experiments and are represented as the mean±SEM. n=12–13, **p<0.01 (two-way ANOVA). ANOVA, analysis of variance; IgG, immunoglobulin G; IR, irradiation.

Article Snippet: Anti-iCOS mAb (clone 7E.17G9) and the Rat IgG2b isotype control were purchased from Bio X Cell and i.p injected at 100 ug/mouse starting 2 days post-IR and then 6 and 9 days post-IR for a total of three injections.

Techniques: Expressing, Injection, Irradiation, Cell Culture, Flow Cytometry, Fluorescence, Control, Staining

Journal: Journal for Immunotherapy of Cancer

Article Title: Optimal dosing regimen of CD73 blockade improves tumor response to radiotherapy through iCOS downregulation

doi: 10.1136/jitc-2023-006846

Figure Lengend Snippet: CD73 expression level controls the MC38 tumor response to CD73 blockade treatment. (A) Representative histograms of transfected and non-transfected MC38 cell with CD73 gene analyzed by flow cytometry. (B) Cultured MC38 control (Ctrl) and CD73 high MC38 cells were analyzed by flow cytometry 24-hour post-IR at 12 Gy for their CD73 membrane expression, which is represented as the mean fluorescence intensity (∆MFI=MFI of the isotype control - MFI of stained cells). Data were obtained from two independent experiments and are represented as the mean±SEM. n=6, ***p<0.001, ****p<0.0001 (one-way ANOVA). (C) CD73 high MC38 tumor cells were injected subcutaneously in C57BL/6 mice and when tumors reached 80–100 mm 3 , tumor were irradiated at 12 Gy, and mice were treated with anti-CD73 starting 1 day before IR then 2, 6 and 9 days post-IR. (D, F) Tumor growth was monitored in treated mice. (E, G) The Kaplan-Meier survival curves for the treated mice are shown. Data were obtained from two independent experiments and are represented as the mean±SEM. n=13–14, *p<0.05, ***p<0.001 (two-way ANOVA). ANOVA, analysis of variance; IgG, immunoglobulin G; IR, irradiation; NIR, non-irradiated; ssc-a, side scatter-a.

Article Snippet: Anti-iCOS mAb (clone 7E.17G9) and the Rat IgG2b isotype control were purchased from Bio X Cell and i.p injected at 100 ug/mouse starting 2 days post-IR and then 6 and 9 days post-IR for a total of three injections.

Techniques: Expressing, Transfection, Flow Cytometry, Cell Culture, Control, Membrane, Fluorescence, Staining, Injection, Irradiation

Journal: Journal for Immunotherapy of Cancer

Article Title: Optimal dosing regimen of CD73 blockade improves tumor response to radiotherapy through iCOS downregulation

doi: 10.1136/jitc-2023-006846

Figure Lengend Snippet: CD73 blockade treatment regimen affects the expression level of iCOS in tumor infiltrating CD4 + T lymphocytes. C57BL/6 mice with the subcutaneous MC38 tumors and BALB/c mice with subcutaneous TS/A tumors were irradiated and treated with either one dose or four doses of anti-CD73 starting 1 day before IR, then 2, 6 and 9 days post-IR. At day 10 post-IR, tumors were harvested and analyzed for immune infiltrating tumor cells by flow cytometry. CD4 + T lymphocytes infiltrating MC38 tumor were analyzed by flow cytometry for iCOS (A, left panel) membrane expression, which is represented as the mean fluorescence intensity (∆MFI=MFI of the isotype control - MFI of stained cells). (A, right panel) Representative histograms of iCOS expression, in CD4 + T lymphocytes infiltrating MC38 tumors. (B, left panel) The percentages of iCOS + CD4 + T lymphocytes infiltrating MC38 tumor are presented for each treatment group. (B, right panel) Representative histograms of iCOS + CD4 + T lymphocytes infiltrating MC38 tumor are presented for each treatment group. Data were obtained from two independent experiments and are represented as the mean±SEM. n=8–10, *p<0.05 (two-way ANOVA). CD4 + T lymphocytes infiltrating MC38 CD73 high tumors were analyzed by flow cytometry for iCOS (C, left panel) membrane expression, which is represented as the mean fluorescence intensity (∆MFI=MFI of the isotype control - MFI of stained cells). (C, right panel) Representative histograms of iCOS expression, in CD4 + T lymphocytes infiltrating MC38 CD73 high tumors. (D) The percentages of iCOS + CD4 + T lymphocytes infiltrating MC38 CD73 high tumors are presented for each treatment group. CD4 + T lymphocytes infiltrating TS/A tumor were analyzed by flow cytometry for their iCOS (E, left panel) membrane expression, which is represented as the mean fluorescence intensity (∆MFI=MFI of the isotype control - MFI of stained cells). (E, right panel) Representative histograms of iCOS expression in CD4 + T lymphocytes infiltrating TS/A tumor. (F, left panel) The percentages of iCOS + CD4 + T lymphocytes infiltrating TS/A tumor are presented for each treatment group. (F, right panel) Representative histograms of iCOS + CD4 + T lymphocytes infiltrating MC38 tumor are presented for each treatment group. Data were obtained from two independent experiments and are represented as the mean±SEM. n=4–8, *p<0.05 (two-way ANOVA). ANOVA, analysis of variance; IgG, immunoglobulin G; IR, irradiation.

Article Snippet: Anti-iCOS mAb (clone 7E.17G9) and the Rat IgG2b isotype control were purchased from Bio X Cell and i.p injected at 100 ug/mouse starting 2 days post-IR and then 6 and 9 days post-IR for a total of three injections.

Techniques: Expressing, Irradiation, Flow Cytometry, Membrane, Fluorescence, Control, Staining

Journal: Journal for Immunotherapy of Cancer

Article Title: Optimal dosing regimen of CD73 blockade improves tumor response to radiotherapy through iCOS downregulation

doi: 10.1136/jitc-2023-006846

Figure Lengend Snippet: iCOS signaling is involved in CD73 blockade-mediated antitumor effect in MC38 tumor model. (A) MC38 tumor cells were injected subcutaneously in C57BL/6 mice and when tumors reached 80–100 mm 3 , tumor were irradiated at 12 Gy, and mice were treated with anti-CD73 (starting 1 day before IR then 2, 6 and 9 days post-IR) and anti-iCOS (starting 2 days post IR, then 6 and 9 days post-IR). (B and D) Tumor growth was monitored in treated mice. (C and E) The Kaplan-Meier survival curves for the treated mice are shown. (F) Tumor growth is shown for individual mice in each treatment group. Data were obtained from two independent experiments and are represented as the mean±SEM. n=6–7, *p<0.05, ****p<0.001, ***p<0.0001 (two-way ANOVA). ANOVA, analysis of variance; IgG, immunoglobulin G; IR, irradiation.

Article Snippet: Anti-iCOS mAb (clone 7E.17G9) and the Rat IgG2b isotype control were purchased from Bio X Cell and i.p injected at 100 ug/mouse starting 2 days post-IR and then 6 and 9 days post-IR for a total of three injections.

Techniques: Injection, Irradiation

Journal: Journal for Immunotherapy of Cancer

Article Title: Optimal dosing regimen of CD73 blockade improves tumor response to radiotherapy through iCOS downregulation

doi: 10.1136/jitc-2023-006846

Figure Lengend Snippet: One dose of aCD73 improves the antitumor effect of aPD-L1 and IR treatment in MC38 tumor model. (A) MC38 tumor cells were injected subcutaneously in C57BL/6 mice and when tumors reached 80–100 mm 3 , tumor were irradiated at 12 Gy, and mice were treated with one dose of anti-CD73 (starting 1 day before IR) and anti-PD-L1 (starting the same day as IR, then 3, 6 and 9 days post-IR). (B) Tumor growth was monitored in treated mice. (C) The Kaplan-Meier survival curves for the treated mice are shown. (D) Tumor growth is shown for individual mice in each treatment group. Data were obtained from two independent experiments and are represented as the mean±SEM. n=11–14, *p<0.05, **p<0.01, ****p<0.0001 (two-way ANOVA). ANOVA, analysis of variance; IgG, immunoglobulin G; IR, irradiation.

Article Snippet: Anti-iCOS mAb (clone 7E.17G9) and the Rat IgG2b isotype control were purchased from Bio X Cell and i.p injected at 100 ug/mouse starting 2 days post-IR and then 6 and 9 days post-IR for a total of three injections.

Techniques: Injection, Irradiation

Journal: bioRxiv

Article Title: Macrophages regulate meiotic initiation and germ cell clearance in the developing ovary

doi: 10.64898/2026.02.28.708733

Figure Lengend Snippet: (a) Representative whole-mount images of E13.5 ovaries from C57BL/6J embryos exposed at E6.5 to either control rat IgG2a antibody or anti-CSF1R blocking antibody to deplete YS-derived macrophages. For F4/80, PECAM1, and FOXL2 staining, n =5 control and n =6 anti-CSF1R-treated independent gonads; for CD11b and CD45 staining, n =3 control and n =3 anti-CSF1R-treated independent gonads. Insets show higher-magnification views of boxed regions; dashed lines mark the gonad-mesonephros boundary. (b) qRT-PCR analysis of macrophage-associated genes ( Adgre1, Cx3cr1, Csf1r, Mrc1 ) and endothelial marker Cdh5 , showing fold change in gene expression in E13.5 anti-CSF1R-treated ovaries versus controls. (c) Representative E14.5 sections stained for SYCP3 and TRA98 showing increased meiotic germ cells in anti-CSF1R-treated ovaries ( n =4) compared with control ovaries ( n =4). (d) qRT-PCR analysis of germ cell and meiotic genes ( Kit, Pou5f1, Ddx4, Stra8, Sycp1, Sycp3, Syce1, Smc1b ), showing fold change in gene expression in E14.5 anti-CSF1R-treated ovaries versus controls. (e) Representative E18.5 sections stained for F4/80 and CD45, and for SYCP3 and FOXL2, showing recovery of F4/80⁺ macrophages and advanced meiotic progression in anti-CSF1R-treated ovaries compared with control ovaries. For F4/80 and CD45 staining, n =4 control and n =4 anti-CSF1R-treated independent gonads; for SYCP3 and FOXL2 staining, n =3 control and n =3 anti-CSF1R-treated independent gonads. Arrows indicate SYCP3⁺ dictyate-stage oocytes. (f) Percentage of SYCP3⁺ germ cells at the dictyate stage among total SYCP3⁺ cells at E18.5. Data are shown as mean +/− SD. * P <0.05, ** P <0.01, *** P <0.001 (two-tailed Student’s t -tests). Scale bars, 100 μm (overview) and 50 μm (higher magnification/insets).

Article Snippet: To transiently deplete yolk-sac-derived and fetal CSF1R + macrophages, pregnant C57BL/6J females were injected intraperitoneally with 3 mg anti-CSF1R monoclonal antibody (mAb; clone AFS98, Bio X Cell #BP0213) or rat IgG2a isotype control (Bio X Cell #BP0089).

Techniques: Control, Blocking Assay, Derivative Assay, Staining, Quantitative RT-PCR, Marker, Gene Expression, Two Tailed Test

Journal: bioRxiv

Article Title: Macrophages regulate meiotic initiation and germ cell clearance in the developing ovary

doi: 10.64898/2026.02.28.708733

Figure Lengend Snippet: (a) Representative images of E18.5, P3, and P10 ovaries from C57BL/6J embryos exposed at E6.5 and E14.5 to either control rat IgG2a antibody or anti-CSF1R blocking antibody to deplete fetal ovarian macrophages. For E18.5, n =4 control and n =4 anti-CSF1R-treated independent gonads; for P3, n =6 control and n =6 anti-CSF1R-treated independent gonads; for P10, n =4 control and n =4 anti-CSF1R-treated independent gonads. Insets show higher-magnification views of boxed regions; red and green dashed outlines demarcate the ovary. (b) Representative sections stained for DDX4, showing developmental germ cell attrition at E18.5, P3, and P10 in control and anti-CSF1R-treated ovaries. For each time point, n =4 control and n =4 anti-CSF1R-treated independent gonads. (c) Quantification of CD45⁺IBA1 + macrophages per 0.1 mm² ovarian area at E18.5, P3, and P10. (d) Quantification of CD45⁺IBA1 − monocyte-like cells per 0.1 mm² ovarian area at the indicated stages. (e) Quantification of DDX4⁺ germ cell number per 0.1 mm² ovarian area at E18.5, P3, and P10, showing reduced physiological germ cell loss after macrophage depletion. Data are shown as mean +/− SD. * P <0.05, ** P <0.01, *** P <0.001 (two-tailed Student’s t -tests). Scale bars, 100 μm (overview) and 50 μm (higher magnification/insets).

Article Snippet: To transiently deplete yolk-sac-derived and fetal CSF1R + macrophages, pregnant C57BL/6J females were injected intraperitoneally with 3 mg anti-CSF1R monoclonal antibody (mAb; clone AFS98, Bio X Cell #BP0213) or rat IgG2a isotype control (Bio X Cell #BP0089).

Techniques: Control, Blocking Assay, Staining, Two Tailed Test

Journal: PLOS Pathogens

Article Title: B cells promote granulomatous inflammation during chronic Mycobacterium tuberculosis infection in mice

doi: 10.1371/journal.ppat.1011187

Figure Lengend Snippet: Mice were treated with the anti-CD20 mAb 5D2 beginning 2 days prior to a low-dose (100 CFU) aerogenic challenge with M . tuberculosis Erdman, as described in "Materials and Methods". B cell depletion was maintained throughout the duration of the experiment. The control mouse group (WT) received non-specific rat IgG. The lung tissues were examined at 5 months post-infection. The levels of granulomatous inflammation response were analyzed histologically by light microscopy on H&E-stained lung sections (A) and enumeration of total number of lung cells (B). The level of lung CD4 + T cell response was assessed by in vivo BrdU labeling to examine the proliferation capacity of this T cell subset (C), as well as by enumeration of IFN-γ-producing CD4 + T cells (D). Ex vivo evaluation of lung cells for the level of IL-10 production (E) was conducted as described in . Four to 5 mice per group were evaluated per group. Data depicted in B, C, D, and E are presented as means ± SEM. The data shown are representative of two experiments. The results demonstrated that the inflammation, Th1 response, and IL-10 phenotypes observed in the μMT mice are recapitulated in mice depleted for B cells.

Article Snippet: At day 90 post infection, 1 mg of anti-mouse IL-10 receptor (IL-10R; clone 1B1.3A; BioXcell) or isotype control Rat IgG1 antibody (BioXcell) was administered intraperitoneally (i.p.) per mouse.

Techniques: Control, Infection, Light Microscopy, Staining, In Vivo, Labeling, Ex Vivo

Journal: PLOS Pathogens

Article Title: B cells promote granulomatous inflammation during chronic Mycobacterium tuberculosis infection in mice

doi: 10.1371/journal.ppat.1011187

Figure Lengend Snippet: C57BL/6 mice were depleted for B cells via administration of 5D2 beginning 2 days prior to infection with a low dose (100 CFU) of M . tuberculosis Erdman delivered by aerosol. B cell depletion was maintained for the duration of the experiment. At 3 months after the infection, IL-10R blockade was initiated using the anti-mouse IL-10R antibody clone 1B1.3A. The control group received non-specific rat IgG. The IL-10R blockade was continued for two months. At five months post-infection (2 months after initiation of IL-10R blockade), mice were sacrificed and analyzed for the levels of inflammation in the lungs, as assessed by histological examination (A) and enumeration of total lung cells (B), CD4 + T cells proliferation via BrdU labeling (C), and Th1 response (D). Data shown are representation of two experiments. Three to four mice were analyzed per group. Data depicted in (B), (C), and (D) denote mean ± SEM.

Article Snippet: At day 90 post infection, 1 mg of anti-mouse IL-10 receptor (IL-10R; clone 1B1.3A; BioXcell) or isotype control Rat IgG1 antibody (BioXcell) was administered intraperitoneally (i.p.) per mouse.

Techniques: Infection, Aerosol, Control, Labeling

Journal: Infection and Immunity

Article Title: Role of T Cells and Gamma Interferon during Induction of Hypersensitivity to Lipopolysaccharide by Toxic Shock Syndrome Toxin 1 in Mice

doi: 10.1128/iai.69.3.1256-1264.2001

Figure Lengend Snippet: FIG. 4. Effects of Cs (A) or anti-IFN-g MAb (B) on LPS-induced serum TNF-a activity in BALB/c-AnNCr mice primed with TSST-1. Groups of four mice were pretreated with Cs (40 mg/kg, i.p.) or anti-IFN-g MAb (750 mg/mouse, i.p.) at 4 or 2 h, respectively, prior to injection of TSST-1. Mice were then injected i.p. with 200 mg of TSST-1 (dotted bars) per kg or PBS (clear bars). Control mice received equal volumes of PBS instead of Cs, rat IgG2a instead of anti-IFN-g MAb, or PBS instead of TSST-1. All mice were injected with LPS (400 mg/kg, i.p.) 12 h later. TNF-a activity in serum was measured 2 h postinjection of LPS. Solutions of Cs or MAb were treated with 2 mg of polymyxin B per ml to remove contaminating LPS. The percent reductions in circulating TNF-a activity caused by Cs or anti-IFN-g MAb are shown.

Article Snippet: Neutralizing rat monoclonal antibody (MAb) to mouse IFN-g, rat immunoglobulin G2a (IgG2a) isotype control MAb, and murine recombinant TNF-a were obtained from R&D Systems (Minneapolis, Minn.).

Techniques: Activity Assay, Injection, Control