Journal: JCI Insight

Article Title: CXCR3 regulates CD4 + T cell cardiotropism in pressure overload–induced cardiac dysfunction

doi: 10.1172/jci.insight.125527

Figure Lengend Snippet: (A and B) LV tissue sections from non-HF or nonischemic HF human subjects were stained for CXCR3 or isotype control by IHC (representative CXCR3+ leukocyte–shaped cells are indicated with red arrows) (A) and quantified in multiple fields of view using a 40× objective (B). (C and D) LV tissue sections were stained for DAPI (blue), CD3 (red), and CXCR3 (green) by immunofluorescence (C), and the number of cells showing colocalization were quantified in multiple fields of view per section using a 40× objective (D). Scale bar: 100 μm. n = 2 control, 3 HF. Error bars represent mean ± SEM (**P < 0.01; Mann-Whitney unpaired U test). (E and F) CD4+ T cells isolated from the LV tissue of mice 4 weeks after Sham or TAC surgery were analyzed (E) and quantified (F) for surface CXCR3 and LFA-1 expression within the CD4+ gate by flow cytometry. n = 3 Sham, 7 TAC. Error bars represent mean ± SEM (**P < 0.01; Mann-Whitney unpaired U test). (G–J) Chemokine and cytokine mRNA levels in the LV of WT mice at different time points after surgery were determined by qPCR for Cxcl9 (G), Cxcl10 (H), Ifng (I), and Tnfa (J). n = 4 Sham, 5 TAC 1 weeks (w); 5 Sham, 9 TAC 2w; 6 Sham, 8 TAC 4w. Error bars represent mean ± SEM (*P < 0.05, **P < 0.01; 1-way ANOVA with Bonferroni post hoc test).

Article Snippet: Immunofluorescence was performed using anti-CXCR3 (LSBio) (1:100 dilution) and anti-CD3 (Agilent Dako, clone F7.2.38) (1:50 dilution) overnight at 4°C, followed by incubation with Alexa Flour 488–conjugated anti-rabbit (Invitrogen, catalog A-11008) and Alexa Flour 568–conjugated anti-mouse secondary antibodies (Abcam, catalog ab175473).

Techniques: Staining, Immunofluorescence, MANN-WHITNEY, Isolation, Expressing, Flow Cytometry

Journal: JCI Insight

Article Title: CXCR3 regulates CD4 + T cell cardiotropism in pressure overload–induced cardiac dysfunction

doi: 10.1172/jci.insight.125527

Figure Lengend Snippet: (A and B) Representative flow cytometry plots (A) and quantification (B) of CD4+ T cells isolated from the mLNs of WT mice at the indicated times after Sham and TAC surgery, indicating surface CXCR3 and LFA-1 expression within the CD4+ gate. Relative values to Sham 1 week (w) are indicated. n = 3 Sham, 3 TAC 1w; 4 Sham, 4 TAC 2w; 3 Sham, 4 TAC 4w. Error bars represent mean ± SEM (*P < 0.05, **P < 0.01, ***P < 0.001; 1-way ANOVA with Bonferroni post hoc test). (C–F) Histogram representation (C and E) and quantification (D and F) of LFA-1 expression on CXCR3+CD4+ and CXCR3–CD4+ T cells in the mLNs of WT mice 4 weeks after TAC surgery (C and D) and in peripheral blood of nonischemic HF patients (E and F). n = 7 TAC mice, 20 nonischemic HF patients. Error bars represent mean ± SEM (*P < 0.05, **P < 0.01, ***P < 0.001; Mann-Whitney unpaired U test).

Article Snippet: Immunofluorescence was performed using anti-CXCR3 (LSBio) (1:100 dilution) and anti-CD3 (Agilent Dako, clone F7.2.38) (1:50 dilution) overnight at 4°C, followed by incubation with Alexa Flour 488–conjugated anti-rabbit (Invitrogen, catalog A-11008) and Alexa Flour 568–conjugated anti-mouse secondary antibodies (Abcam, catalog ab175473).

Techniques: Flow Cytometry, Isolation, Expressing, MANN-WHITNEY

Journal: JCI Insight

Article Title: CXCR3 regulates CD4 + T cell cardiotropism in pressure overload–induced cardiac dysfunction

doi: 10.1172/jci.insight.125527

Figure Lengend Snippet: CD4+ T cells were isolated from the mLN of WT and Cxcr3–/– mice, 4 weeks after Sham and TAC surgeries. (A and B) CD62LloCD44hi effector CD4+ T cells were identified by flow cytometry (A) and quantified (B). (C and D) CD4+ T cells recruited to the LV were identified by IHC (C) and quantified (D) per LV section. n = 4 Sham, 4 TAC WT; 3 Sham, 5 TAC Cxcr3–/–. Scale bars: 100 μm. Error bars represent mean ± SEM (*P < 0.05, **P < 0.01; 1-way ANOVA with Bonferroni post hoc test). (E–H) The indicated gating strategy shown for WT TAC (E) was used to quantify the total cell number per LV of CD4+ T cells (F), CD11b+ myeloid cells (G), and CD11b+MerTK+CCR2+ recruited macrophages (H) by flow cytometry in WT and Cxcr3–/– mice, 4 weeks after Sham and TAC surgeries. n = 3 Sham, 3 TAC WT; 6 TAC Cxcr3–/– mice. Error bars represent mean ± SEM (*P < 0.05, **P < 0.01; 1-way ANOVA with Bonferroni post hoc test). (I–K) mRNA levels in the LV of WT and Cxcr3–/– mice at 4 weeks after surgery was determined by qPCR for Cxcl9 (I), Cxcl10 (J), and Ifng (K). n = 9 Sham, 10 TAC WT; 3 Sham, 5 TAC Cxcr3–/–. Error bars represent mean ± SEM (*P < 0.05, **P < 0.01; 1-way ANOVA with Bonferroni post hoc test).

Article Snippet: Immunofluorescence was performed using anti-CXCR3 (LSBio) (1:100 dilution) and anti-CD3 (Agilent Dako, clone F7.2.38) (1:50 dilution) overnight at 4°C, followed by incubation with Alexa Flour 488–conjugated anti-rabbit (Invitrogen, catalog A-11008) and Alexa Flour 568–conjugated anti-mouse secondary antibodies (Abcam, catalog ab175473).

Techniques: Isolation, Flow Cytometry

Journal: JCI Insight

Article Title: CXCR3 regulates CD4 + T cell cardiotropism in pressure overload–induced cardiac dysfunction

doi: 10.1172/jci.insight.125527

Figure Lengend Snippet: LV tissue sections were isolated from WT and Cxcr3–/– mice, 4 weeks after Sham and TAC surgeries. IHC was used to determine perivascular fibrosis (A) (quantified in C) as well as interstitial fibrosis (B) (quantified in D) by Picrosirius red staining. Scale bars: 100 μm. (E and F) Mean cardiomyocyte area was quantified by wheat germ agglutinin (WGA) IHC of LV tissue sections (E) (as shown in F). n = 4 Sham, 5 TAC WT; 3 Sham, 5 TAC Cxcr3–/– mice. Scale bars: 50 μm. Error bars represent mean ± SEM (*P < 0.05, ***P < 0.001; 1-way ANOVA with Bonferroni post hoc test). (G) The relative LV mRNA expression of the α and β myosin heavy chain isoforms were determined by qPCR to assess pathological cardiomyocyte hypertrophy. n = 3 Sham, 3 TAC WT; 3 Sham, 5 TAC Cxcr3–/– mice. Error bars represent mean ± SEM (*P < 0.05; 1-way ANOVA with Bonferroni post hoc test).

Article Snippet: Immunofluorescence was performed using anti-CXCR3 (LSBio) (1:100 dilution) and anti-CD3 (Agilent Dako, clone F7.2.38) (1:50 dilution) overnight at 4°C, followed by incubation with Alexa Flour 488–conjugated anti-rabbit (Invitrogen, catalog A-11008) and Alexa Flour 568–conjugated anti-mouse secondary antibodies (Abcam, catalog ab175473).

Techniques: Isolation, Staining, Expressing

Journal: JCI Insight

Article Title: CXCR3 regulates CD4 + T cell cardiotropism in pressure overload–induced cardiac dysfunction

doi: 10.1172/jci.insight.125527

Figure Lengend Snippet: (A–C) Transthoracic short axis M mode images of the mid LV were acquired by echocardiography (A) of WT and Cxcr3–/– mice, 4 weeks after Sham and TAC surgeries to quantify fractional shortening (B) and ejection fraction (C). n = 5 Sham, 7 TAC WT; 5 Sham, 7 TAC Cxcr3–/– mice. Error bars represent mean ± SEM (***P < 0.001; 1-way ANOVA with Bonferroni post hoc test). (D–F) Intraventricular hemodynamic measurements were acquired by a pressure volume transducer catheter to quantify maximum LV pressure (D), as well as dP/dt max (E) and dP/dt min (F) as parameters of cardiac contractility and relaxation, respectively. n = 3 Sham, 3 TAC WT; 3 Sham, 4 TAC Cxcr3–/–. Error bars represent mean ± SEM (*P < 0.05; 1-way ANOVA with Bonferroni post hoc test).

Article Snippet: Immunofluorescence was performed using anti-CXCR3 (LSBio) (1:100 dilution) and anti-CD3 (Agilent Dako, clone F7.2.38) (1:50 dilution) overnight at 4°C, followed by incubation with Alexa Flour 488–conjugated anti-rabbit (Invitrogen, catalog A-11008) and Alexa Flour 568–conjugated anti-mouse secondary antibodies (Abcam, catalog ab175473).

Techniques:

Journal: JCI Insight

Article Title: CXCR3 regulates CD4 + T cell cardiotropism in pressure overload–induced cardiac dysfunction

doi: 10.1172/jci.insight.125527

Figure Lengend Snippet: Naive WT and Cxcr3–/– T cells were differentiated in vitro to Th1 cells and treated with either PMA (50 ng/ml), CXCL9 (100 ng/ml), or CXCL10 (100 ng/ml) for 5 minutes at 37°C to induce integrin LFA-1 activation, prior to perfusion over immobilized ICAM-1–coated coverslips at 1 dyne/cm2 in a parallel plate flow chamber. (A and B) Representative images (A) and quantification (B) of real-time videos of WT Th1 cell adhesion to ICAM-1 after treatment with anti–LFA-1 function-blocking antibody or IgG isotype control (20 μg/ml 30 minutes at 37°C). Scale bars: 100 μm. Statistical comparisons are indicated compared with untreated cells. n = 5 independent experiments, analysis of 6 different fields of view per experiment (*P < 0.05, ***P < 0.001; 1-way ANOVA with Bonferroni post hoc test). (C and D) Representative images (C) and quantification (D) of real-time videos of Cxcr3–/– Th1 cells. Scale bars: 100 μm. Statistical comparisons are indicated compared with untreated cells. n = 3 independent experiments, analysis of 6 different fields of view per experiment. Error bars represent mean ± SEM (***P < 0.001; 1-way ANOVA with Bonferroni post hoc test).

Article Snippet: Immunofluorescence was performed using anti-CXCR3 (LSBio) (1:100 dilution) and anti-CD3 (Agilent Dako, clone F7.2.38) (1:50 dilution) overnight at 4°C, followed by incubation with Alexa Flour 488–conjugated anti-rabbit (Invitrogen, catalog A-11008) and Alexa Flour 568–conjugated anti-mouse secondary antibodies (Abcam, catalog ab175473).

Techniques: In Vitro, Activation Assay, Blocking Assay

Journal: bioRxiv

Article Title: Mice carrying nonsense mutant p53 develop spontaneous tumors with frequent metastases

doi: 10.1101/2025.03.12.642796

Figure Lengend Snippet: (A) Representative histomicrographs of the thymus from the Trp53 R210X/R210X mouse X405 stained with HE (left) and CD3 antibody (IHC, right). Mats of CD3-positive, i.e. T-cell origin, pleomorphic neoplastic lymphocytes and dispersed, moderate numbers of tangible body macrophages (left panel, arrows) efface the normal thymic architecture. Scale bars = 50µm. (B) Representative flow cytometry to verify T-cell origin of the X405 Trp53 R210X/R210X T-lymphoma cell line established from a thymic lymphoma by staining with antibodies for CD3 (left panel), and CD4 and CD8 (right panel). (C) Immunofluorescence staining of X405 Trp53 R210X/R210X T-lymphoma cells, either untreated (NT, upper panels) or treated with 100µM G418 (lower panels) for 72 hours. p53 was detected using N1 (N-terminal epitope) and 280aa C-term (C-terminal epitope) antibodies. Rightmost panels show merged DAPI and 280aa C-term p53 staining, highlighting translational readthrough of the Trp53 -R210X nonsense mutation and production of full-length p53 protein upon treatment with G418. Scale bars = 100µm. (D) Western blot analysis showing dose-dependent induction of full-length p53 in X405 Trp53 R210X/R210X T-lymphoma cells following 72h treatment with G418. p53 was visualized using the anti-p53 antibody 1C12 that recognizes an N-terminal epitope and therefore detects both full-length and C-terminally truncated p53. Gapdh was used as a loading control. (E) qRT-PCR analysis showing dose-dependent induction of p53 mRNA levels in X405 Trp53 R210X/R210X T-lymphoma cells after 72h treatment with G418. mRNA levels of p53 target genes p21 , Puma and Zmat3 were significantly upregulated upon treatment with 200µM G418. Gene expression values were normalized to Gapdh expression and compared to the untreated (NT) negative control. (F) Representative flow cytometry for assessment of cell death by Annexin V staining of X405 Trp53 R210X/R210X T-lymphoma cells (left) and quantification of 3 flow cytometry replicates (right), showing significant increase in cell death following treatment with 200µM G418 for 72h. In E-F : statistical analysis was performed using one-way ANOVA followed by Dunnett’s (E) or Tukey’s (F) multiple comparisons tests. Values represent the mean ± SEM. Adjusted P-values: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Primary antibodies rabbit polyclonal p53 N-terminal antibody [N1] (GTX100629, Genetex, USA) and goat polyclonal Anti-TP53 (280aa C-Term) (ASJ-VN4O74-150, Nordic Biosite, Sweden) were diluted together at 1:100 in PBS, incubation 4°C ON.

Techniques: Staining, Flow Cytometry, Immunofluorescence, Mutagenesis, Western Blot, Control, Quantitative RT-PCR, Gene Expression, Expressing, Negative Control