Journal: bioRxiv

Article Title: Human effector CD8 + T cells with an exhausted-like phenotype control tumor growth in vivo

doi: 10.1101/2023.10.11.561856

Figure Lengend Snippet: Splenocytes (A-D) and tumor-infiltrating lymphocytes (TIL) (E-H) were isolated from HIS mice 16-18 days after tumor implantation and analyzed by flow cytometry. A, frequency of splenic T cells in tumor-bearing or naïve HIS mice; parent population refers to frequency (%) of CD3 + T cells within human CD45 + cells and CD4 + and CD8 + T cells within CD3 + T cells. B , CD8 + T cell differentiation defined as T naïve (CD45RA + CD62L + ), T CM (CD45RA - CD62L + ), T EM (CD45RA - CD62L - ), T EMRA (CD45RA + CD62L - ) in tumor-bearing or naïve HIS mice. C-D , expression of indicated markers on CD8 + T cells from spleen of tumor-bearing or naïve HIS mice. E, frequency of CD4 + and CD8 + T cells within TILs, gated on total CD3 + T cells. F, CD8 + T cell differentiation within TILs. G-H, expression of indicated markers on CD8 + T cells within TILs. I, expression of CD137 on splenic CD8 + and CD4 + T cells of tumor-bearing or naïve HIS mice. J, expression of PD-1 and CD137 on splenic CD8 + T cells of tumor-bearing mice. K , expression of PD-1 on splenic CD8 + T cells based on CD137 in tumor-bearing HIS mice or bulk CD8 + T cells from naïve HIS mice. L, CD8 + T cell differentiation in spleen of tumor-bearing HIS mice. M-O , expression of indicated markers on splenocyte-derived CD8 + T cell populations based on expression of CD137 and PD-1. Data are pooled from at least 3 independent experiments, n=10-23 mice per group. For each experiment, a different HPC donor was used for HIS mouse reconstitution and generation of autologous tumor. Significance by paired t-test, one-way ANOVA or 2way ANOVA, as appropriate. Data from individual experiments are indicated by different symbols.

Article Snippet: Antibodies for flow cytometry-based sorting of T cells: CD3-FITC (Biolegend, UCHT1, Cat. 300406), CD4-APC-Cy7 (Biolegend, RPA-T4, Cat. 300518), CD8-BV650 (Biolegend, SK1, Cat. 344730), CD45-Pacific Blue (Biolegend, HI30, Cat. 304029), CD137-APC (Miltenyi, REA765, Cat. 130-110-901), PD-1-PE-Dazzle (Biolegend, EH12.2H7, Cat. 329940), Zombie AquaTM Fixable Viability Kit (Biolegend, Cat. 423101).

Techniques: Isolation, Tumor Implantation, Flow Cytometry, Cell Differentiation, Expressing, Derivative Assay

Journal: bioRxiv

Article Title: Human effector CD8 + T cells with an exhausted-like phenotype control tumor growth in vivo

doi: 10.1101/2023.10.11.561856

Figure Lengend Snippet: A , schematic of generation, expansion and characterization of T cells from HIS mice bearing autologous LCL tumors. B , fold expansion of FACS sorted splenic CD8 + T cells from tumor-bearing HIS mice (CD137 + , CD137 - and CD137 - PD1 - ) or naïve HIS mice (bulk). C , CD8 + T cell differentiation after ex vivo expansion defined as T naïve (CD45RA + CD62L + ), T CM (CD45RA - CD62L + ), T EM (CD45RA - CD62L - ), T EMRA (CD45RA + CD62L - ). D, IFN-ψ ELISpot of expanded T cells in 5:1 (E:T) co-culture with autologous LCL tumor cells for 24 hours. Spot count is normalized to the spots produced by expanded CD8 + bulk T cells from naïve HIS mice. E , TNF⍺ ELISA of supernatant of expanded T cells in co-culture (5:1, E:T) with autologous LCL tumor cells for 24 hours. TNF⍺ concentration is normalized to the TNF⍺ secretion from expanded CD8 + bulk T cells derived from naïve mice. Data are pooled from at least 3 independent experiments, n=6-19 mice per group. For each experiment, a different HPC donor was used for HIS mouse reconstitution and generation of autologous tumor. Significance by mixed-effects analysis (paired), RM one-way ANOVA or 2way ANOVA, as appropriate. Data from individual experiments are indicated by different symbols.

Article Snippet: Antibodies for flow cytometry-based sorting of T cells: CD3-FITC (Biolegend, UCHT1, Cat. 300406), CD4-APC-Cy7 (Biolegend, RPA-T4, Cat. 300518), CD8-BV650 (Biolegend, SK1, Cat. 344730), CD45-Pacific Blue (Biolegend, HI30, Cat. 304029), CD137-APC (Miltenyi, REA765, Cat. 130-110-901), PD-1-PE-Dazzle (Biolegend, EH12.2H7, Cat. 329940), Zombie AquaTM Fixable Viability Kit (Biolegend, Cat. 423101).

Techniques: Cell Differentiation, Ex Vivo, Enzyme-linked Immunospot, Co-Culture Assay, Produced, Enzyme-linked Immunosorbent Assay, Concentration Assay, Derivative Assay

Journal: bioRxiv

Article Title: Human effector CD8 + T cells with an exhausted-like phenotype control tumor growth in vivo

doi: 10.1101/2023.10.11.561856

Figure Lengend Snippet: Transcriptome analysis and pathway analysis of expanded CD8 + T cells from tumor-bearing HIS mice (CD137 + , CD137 - and CD137 - PD1 - ) or naïve HIS mice (bulk). A, Upset plot (intersect) showing number of differentially expressed genes between groups in bulk RNAseq. p(FDR) < 0.05, log2 FC > 1.5. B , PCA plot of RNAseq showing PC1 and PC2. C, Top 50 upregulated and D, downregulated genes of T cell subsets based on the DEG between CD137 + vs. CD137 - PD1 - CD8 + T cells. p(FDR) < 0.05, log2 FC > 1.5. E, Volcano plot showing DEG between CD137 + and CD137 - PD1 - CD8 + T cells with genes of interest highlighted in yellow. F , differential expression of genes of interest between groups. G , Overrepresentation analysis (ORA) of upregulated pathways in CD137 + CD8 + T cells. H , Gene set enrichment analysis (GSEA) of signatures described on the y-axis. Gene ratio (# genes related to GO term / total number of sig genes) is displayed on the x-axis. Signatures with an adjusted p-value <0.05 are highlighted with a red box. Shown are data from 5 individual experiments, each experiment with different human HPC donor for reconstitution of HIS mice and autologous tumor.

Article Snippet: Antibodies for flow cytometry-based sorting of T cells: CD3-FITC (Biolegend, UCHT1, Cat. 300406), CD4-APC-Cy7 (Biolegend, RPA-T4, Cat. 300518), CD8-BV650 (Biolegend, SK1, Cat. 344730), CD45-Pacific Blue (Biolegend, HI30, Cat. 304029), CD137-APC (Miltenyi, REA765, Cat. 130-110-901), PD-1-PE-Dazzle (Biolegend, EH12.2H7, Cat. 329940), Zombie AquaTM Fixable Viability Kit (Biolegend, Cat. 423101).

Techniques: Quantitative Proteomics

Journal: bioRxiv

Article Title: Human effector CD8 + T cells with an exhausted-like phenotype control tumor growth in vivo

doi: 10.1101/2023.10.11.561856

Figure Lengend Snippet: A , total number of individual clonotypes found per population. Left: data from individual experiments, right: pooled data for group analysis. Wilcoxon test. B, TCR (CDR3 of TRA , TRB , TRG and TRD ) sequence sample diversity estimation using Hill numbers method, with Q=1 describing the Shannon diversity. C, rare clonal proportion showing the occupied repertoire space by clonotypes with defined counts (1, 2-3, 4-10, etc.). Left: data from individual experiments, right: pooled data for group analysis. Wilcoxon test. D, relative abundance of clonotypes with defined frequencies (size). Left: data from individual experiments, right: pooled data for group analysis. Wilcoxon test. E, repertoire overlap analysis cross-comparing every population from every experiment (each with different donor). F , repertoire overlap comparing the repertoire of bulk CD8 + T cells from naïve HIS mice to the populations from tumor-bearing HIS mice from individual experiments (each with different donor; data from individual experiments are indicated by different symbols). Mixed effects analysis with Tukey’s multiple comparisons test. G , tracking of clonotypes over populations. The top 10 most abundant clonotypes of the TCR repertoire of CD137 + CD8 + T cells from one representative experiment are shown. H , proportion of the top 10 most abundant clonotypes (from repertoires of CD137 + CD8 + T cells) in the repertoire of all populations, correlated with the spot count of IFN-ψ ELISpot. Shown are data from 4 individual experiments, each experiment with different human HPC donor for reconstitution of HIS mice and autologous tumor.

Article Snippet: Antibodies for flow cytometry-based sorting of T cells: CD3-FITC (Biolegend, UCHT1, Cat. 300406), CD4-APC-Cy7 (Biolegend, RPA-T4, Cat. 300518), CD8-BV650 (Biolegend, SK1, Cat. 344730), CD45-Pacific Blue (Biolegend, HI30, Cat. 304029), CD137-APC (Miltenyi, REA765, Cat. 130-110-901), PD-1-PE-Dazzle (Biolegend, EH12.2H7, Cat. 329940), Zombie AquaTM Fixable Viability Kit (Biolegend, Cat. 423101).

Techniques: Sequencing, Enzyme-linked Immunospot

Journal: bioRxiv

Article Title: Human effector CD8 + T cells with an exhausted-like phenotype control tumor growth in vivo

doi: 10.1101/2023.10.11.561856

Figure Lengend Snippet: A , schematic of generation of tumor-reactive T cells and subsequent ACT. NSG mice were injected with 2 x 10 LCL s.c. in the flank and after three days, 10 x 10 ex vivo expanded T cells were adoptively transferred intravenously. Transferred T cells and LCL tumors were autologous to each other. B , tumor volume on the day of sacrifice in NSG recipient mice after ACT of the indicated cell populations. C, waterfall plot of tumor size in NSG recipient mice of ACT on the day of sacrifice relative to the tumor volume of control mice (no ACT). Bars depict individual mice. D , frequency of CD3 + T cells (% of human CD45 + cells) in TIL from NSG mice after ACT of the indicated cell populations, measured by flow cytometry. E , frequency of CD8 + T cells (% of total cells) in tumors of NSG mice after ACT of the indicated cell populations, measured by immunohistochemistry. F , differentiation of CD8 + T cells in TIL of NSG mice after ACT of the indicated cell populations defined as T naïve (CD45RA + CD62L + ), T CM (CD45RA - CD62L + ), T EM (CD45RA - CD62L - ) and T EMRA (CD45RA + CD62L - ). G, correlation between tumor volume and infiltration of CD8 + T cells (measured by IHC) in tumors of NSG mice after adoptive transfer of CD137 + CD8 + T cells. H , schematic of ACT. CD137 + CD8 + T cells, CD137 - CD8 + T cells and CD137 - PD-1 - CD8 + T cells were isolated from spleen of tumor-bearing HIS mice or bulk CD8 + T cells from spleen of naïve HIS mice and expanded ex vivo . Recipient HIS mice were injected with 2 x 10 LCL s.c. in the flank and after three days, ex vivo expanded T cells were adoptively transferred intravenously. Tumor-bearing HIS recipient mice received 2 x 10 T cells without prior conditioning/lymphodepletion. Donor and recipient HIS mice as well as LCL were autologous to each other. I, tumor volume on the day of sacrifice in HIS recipient mice after ACT of the indicated cell populations. J, waterfall plot of tumor size on the day of sacrifice of HIS mice receiving ACT relative to the tumor volume of control HIS mice (no ACT). Bars depict individual mice. B-G: Data are pooled from 2-3 independent experiments, n=6-13 per group. I, data are pooled from 2-3 independent experiments (n=6-13 per group); J, data are from 1-3 independent experiments (n=3-13 per group). For each experiment, a with different HPC donor was used for HIS mouse reconstitution and generation of autologous tumor. Significance by one-way ANOVA. Data from individual experiments are indicated by different symbols.

Article Snippet: Antibodies for flow cytometry-based sorting of T cells: CD3-FITC (Biolegend, UCHT1, Cat. 300406), CD4-APC-Cy7 (Biolegend, RPA-T4, Cat. 300518), CD8-BV650 (Biolegend, SK1, Cat. 344730), CD45-Pacific Blue (Biolegend, HI30, Cat. 304029), CD137-APC (Miltenyi, REA765, Cat. 130-110-901), PD-1-PE-Dazzle (Biolegend, EH12.2H7, Cat. 329940), Zombie AquaTM Fixable Viability Kit (Biolegend, Cat. 423101).

Techniques: Injection, Ex Vivo, Control, Flow Cytometry, Immunohistochemistry, Adoptive Transfer Assay, Isolation

Journal: iScience

Article Title: Ad26.COV2.S priming provided a solid immunological base for mRNA-based COVID-19 booster vaccination

doi: 10.1016/j.isci.2022.105753

Figure Lengend Snippet:

Article Snippet: Anti-human CD137 PE clone 4B4-1 , Miltenyi Biotec , Cat#130-119-885; RRID: AB_2783944.

Techniques: Staining, Virus, Clinical Proteomics, Recombinant, Enzyme-linked Immunosorbent Assay, Cell Analysis, Software, Imaging

Journal: Circulation Research

Article Title: High Levels of Costimulatory Receptors OX40 and 4-1BB Characterize CD4 + CD28 null T Cells in Patients With Acute Coronary Syndrome

doi: 10.1161/circresaha.111.261933

Figure Lengend Snippet: Figure 2. Expression of costimulatory receptors in CD4CD28null T cells. Peripheral blood mononuclear cells (PBMCs) from acute coronary syndrome (ACS, n25) and stable angina (SA, n21) patients were cultured alone (w/o) or activated with anti-CD3 antibodies (aCD3) and the expression of costimulatory receptors (OX40, 4–1BB, inducible costimulator [ICOS]) was analyzed on day 4. A, Illustra- tive histograms show the expression of OX40, 4-1BB, and ICOS (green histograms) and control staining with isotype-matched antibod- ies (red histograms) by activated CD4CD28null (28null) and classical CD4CD28 (28pos) T cells from an ACS patient. B, Scatter plots show the distribution and mean fluorescence intensity (MFI) of cells expressing costimulatory receptors in the ACS group. C, Scatter plots show the distribution and mean MFI of cells expressing costimulatory receptors in the SA group. Probability values (P) were cal- culated by 2-tailed t test. a.u. indicates arbitrary units.

Article Snippet: Stimulation of 4- 1BB and OX40 was done with 10 g/ml stimulating antibodies for 4-1BB (R&D Systems, AF838) or OX40 (AF3388) or with 5 g/ml recombinant ligands 4-1BBL (R&D Systems, 2295-4L) and OX40L (R&D Systems, 1054-OX).

Techniques: Expressing, Cell Culture, Control, Staining

Journal: Circulation Research

Article Title: High Levels of Costimulatory Receptors OX40 and 4-1BB Characterize CD4 + CD28 null T Cells in Patients With Acute Coronary Syndrome

doi: 10.1161/circresaha.111.261933

Figure Lengend Snippet: Figure 5. Analysis of CD4CD28null T cells, costimulatory receptors and ligands in atherosclerotic plaques. A, The frequency of CD4CD28null (CD28neg), CD4CD28 (CD28pos) T cells and the expression of OX40 and 4-1BB costimulatory receptors was quantified in cells isolated from human endarterectomy atherosclerotic plaques using flow cytometry. Representative plots show expression of OX40 and 4-1BB on plaque-derived T cells from atherosclerotic plaques B, Paraffin embedded tissue sections were immunohistochemically labeled

Article Snippet: Stimulation of 4- 1BB and OX40 was done with 10 g/ml stimulating antibodies for 4-1BB (R&D Systems, AF838) or OX40 (AF3388) or with 5 g/ml recombinant ligands 4-1BBL (R&D Systems, 2295-4L) and OX40L (R&D Systems, 1054-OX).

Techniques: Expressing, Isolation, Cytometry, Derivative Assay, Labeling

Journal: Circulation Research

Article Title: High Levels of Costimulatory Receptors OX40 and 4-1BB Characterize CD4 + CD28 null T Cells in Patients With Acute Coronary Syndrome

doi: 10.1161/circresaha.111.261933

Figure Lengend Snippet: Figure 6. Effects of OX40 and 4-1BB blockade on cytokine production by CD4CD28null T cells. Peripheral blood mononuclear cells (PBMCs) from acute coronary syndrome (ACS) patients (n9) were activated with anti-CD3 antibodies alone (aCD3) or in the presence of blocking antibodies against OX40 (aOX40) and 4-1BB (a4–1BB) or control antibodies (ctrl Ig) for 4 days. A, Repre- sentative contour plots and bar graphs display percentage of interferon (IFN)-

Article Snippet: Stimulation of 4- 1BB and OX40 was done with 10 g/ml stimulating antibodies for 4-1BB (R&D Systems, AF838) or OX40 (AF3388) or with 5 g/ml recombinant ligands 4-1BBL (R&D Systems, 2295-4L) and OX40L (R&D Systems, 1054-OX).

Techniques: Blocking Assay, Control

Journal: Circulation Research

Article Title: High Levels of Costimulatory Receptors OX40 and 4-1BB Characterize CD4 + CD28 null T Cells in Patients With Acute Coronary Syndrome

doi: 10.1161/circresaha.111.261933

Figure Lengend Snippet: Figure 7. Effects of OX40 and 4-1BB blockade on perforin and granzyme B in CD4CD28null T cells. Peripheral blood mononuclear cells (PBMCs) from acute coronary syndrome (ACS) patients (n9) were cultured alone (w/o) or acti- vated with anti-CD3 antibodies alone (aCD3) or in the presence of blocking antibodies against OX40 (aOX40) and 4-1BB (a4–1BB) for 4 days. A, Repre- sentative contour plots and bar graphs show surface expression of CD107a in CD4CD28null (28null) T cells. B, Repre- sentative contour plots and bar graphs display percentage of perforin

Article Snippet: Stimulation of 4- 1BB and OX40 was done with 10 g/ml stimulating antibodies for 4-1BB (R&D Systems, AF838) or OX40 (AF3388) or with 5 g/ml recombinant ligands 4-1BBL (R&D Systems, 2295-4L) and OX40L (R&D Systems, 1054-OX).

Techniques: Cell Culture, Blocking Assay, Expressing

Journal: Circulation Research

Article Title: High Levels of Costimulatory Receptors OX40 and 4-1BB Characterize CD4 + CD28 null T Cells in Patients With Acute Coronary Syndrome

doi: 10.1161/circresaha.111.261933

Figure Lengend Snippet: Figure 8. Schematic outline of the effects of costimulatory receptors OX40 and 4-1BB on the functions of CD4CD28null T cells in acute coro- nary syndrome (ACS). Classical CD4CD28 T cells constitutively express the costimulatory receptor CD28. Activation of CD4CD28 T cells induces transient expression of costimu- latory (OX40, 4–1BB) and coinhibitory (cytotoxic T lymphocyte associated antigen-4 [CTLA-4], programmed death [PD]-1) receptors. CD4CD28 T cells produce moderate levels of inflammatory cytokines (interferon [IFN]-, tumor necrosis factor [TNF]-) when activated and do not express cytotoxic molecules (perforin, granzyme B). CD4CD28null T cells from ACS patients lack CD28 and upregulate expression of OX40 and 4-1BB. These receptors drive production of high levels of IFN-, TNF-, and per- forin. We hypothesize that these media- tors could potentially enable CD4CD28null T cells to trigger plaque rupture via macrophage activation and endothelial and vascular smooth muscle cell lysis. VSMC indicates vascular smooth muscle cell.

Article Snippet: Stimulation of 4- 1BB and OX40 was done with 10 g/ml stimulating antibodies for 4-1BB (R&D Systems, AF838) or OX40 (AF3388) or with 5 g/ml recombinant ligands 4-1BBL (R&D Systems, 2295-4L) and OX40L (R&D Systems, 1054-OX).

Techniques: Activation Assay, Expressing, Lysis

Journal: Nutrition & Metabolism

Article Title: Evaluation of markers of beige adipocytes in white adipose tissue of the mouse

doi: 10.1186/s12986-016-0081-2

Figure Lengend Snippet: List or thermoregulatory or beige markers employed in this study and their function

Article Snippet: Primary antibodies were: anti-FGF21, AF3057, and anti-CD137, AF937 (R&D Systems, Minneapolis, MN), anti-P2RX5, sc-398682 (Santa Cruz Biotechnology, Dallas, TX), anti-beta-actin, sc-47778 (Santa Cruz, Dallas, TX), and anti-UCP1, ab10983, (abcam, Cambridge, MA).

Techniques: Marker

Journal: Nutrition & Metabolism

Article Title: Evaluation of markers of beige adipocytes in white adipose tissue of the mouse

doi: 10.1186/s12986-016-0081-2

Figure Lengend Snippet: List of primer/probe sets

Article Snippet: Primary antibodies were: anti-FGF21, AF3057, and anti-CD137, AF937 (R&D Systems, Minneapolis, MN), anti-P2RX5, sc-398682 (Santa Cruz Biotechnology, Dallas, TX), anti-beta-actin, sc-47778 (Santa Cruz, Dallas, TX), and anti-UCP1, ab10983, (abcam, Cambridge, MA).

Techniques:

Journal: Nutrition & Metabolism

Article Title: Evaluation of markers of beige adipocytes in white adipose tissue of the mouse

doi: 10.1186/s12986-016-0081-2

Figure Lengend Snippet: Markers of beige adipocytes in vitro . The transcripts for FGF21 a , P2RX5 b , PAT2 c , CITED1 d , CAR4 e , TBX1 f , CD137 g , and TMEM26 ( h ) were evaluated in white and beige adipocytes in culture treated with isoproterenol (+ Iso) or without (− Iso) for 6 hrs. before harvesting the cells. Transcripts were normalized to white – Iso. Values are means ± SEM ( n = 3). (*) different from white – Iso, (#) different from white + Iso, (+) different from beige + Iso, (@) different from beige – Iso, p ≤ 0.05, two way ANOVA followed by multiple comparisons using the Holm-Sidak method

Article Snippet: Primary antibodies were: anti-FGF21, AF3057, and anti-CD137, AF937 (R&D Systems, Minneapolis, MN), anti-P2RX5, sc-398682 (Santa Cruz Biotechnology, Dallas, TX), anti-beta-actin, sc-47778 (Santa Cruz, Dallas, TX), and anti-UCP1, ab10983, (abcam, Cambridge, MA).

Techniques: In Vitro

Journal: Nutrition & Metabolism

Article Title: Evaluation of markers of beige adipocytes in white adipose tissue of the mouse

doi: 10.1186/s12986-016-0081-2

Figure Lengend Snippet: Summary of beigeing criteria met by the thermoregulatory genes and putative beige markers when studied under in vitro and in vivo conditions

Article Snippet: Primary antibodies were: anti-FGF21, AF3057, and anti-CD137, AF937 (R&D Systems, Minneapolis, MN), anti-P2RX5, sc-398682 (Santa Cruz Biotechnology, Dallas, TX), anti-beta-actin, sc-47778 (Santa Cruz, Dallas, TX), and anti-UCP1, ab10983, (abcam, Cambridge, MA).

Techniques: In Vitro, In Vivo, Cell Culture

Journal: Nutrition & Metabolism

Article Title: Evaluation of markers of beige adipocytes in white adipose tissue of the mouse

doi: 10.1186/s12986-016-0081-2

Figure Lengend Snippet: Markers of beige adipocytes in inguinal adipose tissue. The transcripts for FGF21, P2RX5, PAT2, CITED1, CAR4, TBX1, CD137, and TMEM26 were evaluated in inguinal adipose tissue from mice maintained at room temperature (22 °C) or exposed to cold (5 °C) for one week in 3 separate experiments were the ages of the mice at the end of the experiment were 2 months and 10 days (Exp-1), 2 months and 23 days (Exp-2), and 4 months and 10 days (Exp-3). The levels of the transcripts at 5 °C were normalized to the transcript of the same gene at 22 °C. Values are means ± SEM ( n = 4–5). (*) different from 22 °C, p ≤ 0.05, two-tailed t-test

Article Snippet: Primary antibodies were: anti-FGF21, AF3057, and anti-CD137, AF937 (R&D Systems, Minneapolis, MN), anti-P2RX5, sc-398682 (Santa Cruz Biotechnology, Dallas, TX), anti-beta-actin, sc-47778 (Santa Cruz, Dallas, TX), and anti-UCP1, ab10983, (abcam, Cambridge, MA).

Techniques: Two Tailed Test

Journal: Nutrition & Metabolism

Article Title: Evaluation of markers of beige adipocytes in white adipose tissue of the mouse

doi: 10.1186/s12986-016-0081-2

Figure Lengend Snippet: Markers of beige adipocytes in the adipocyte and SVF derived from inguinal adipose tissue. The transcripts for FGF21 a , P2RX5 b , PAT2 c , CITED1 d , CAR4 e , TBX1 f , CD137 g , and TMEM26 ( h ) were evaluated in the adipocyte and SVF of 4 month old mice kept at room temperature (22 °C) or exposed to the cold (5 °C) for one week. Transcripts were normalized to 22 °C, SVF. Values are means ± SEM ( n = 3–5). (*) different from SVF 22 °C, (#) different from SVF 5 °C, (+) different from adipocytes 5 °C, (@) different from adipocytes 22 °C, p ≤ 0.05, two way ANOVA followed by multiple comparisons using the Holm-Sidak method

Article Snippet: Primary antibodies were: anti-FGF21, AF3057, and anti-CD137, AF937 (R&D Systems, Minneapolis, MN), anti-P2RX5, sc-398682 (Santa Cruz Biotechnology, Dallas, TX), anti-beta-actin, sc-47778 (Santa Cruz, Dallas, TX), and anti-UCP1, ab10983, (abcam, Cambridge, MA).

Techniques: Derivative Assay

Journal: Nutrition & Metabolism

Article Title: Evaluation of markers of beige adipocytes in white adipose tissue of the mouse

doi: 10.1186/s12986-016-0081-2

Figure Lengend Snippet: Beige markers in preadipocytes and SVF cells in vitro . The transcripts for CAR4, CITED1, PAT2, FGF21, P2RX5, CD137, TBX1, and TMEM26 were evaluated in SVF cells and beige adipocytes in culture. Transcripts were normalized to SVF cells. Values are means ± SEM ( n = 3). (*) different from SVF, p ≤ 0.05, two-tailed t-test

Article Snippet: Primary antibodies were: anti-FGF21, AF3057, and anti-CD137, AF937 (R&D Systems, Minneapolis, MN), anti-P2RX5, sc-398682 (Santa Cruz Biotechnology, Dallas, TX), anti-beta-actin, sc-47778 (Santa Cruz, Dallas, TX), and anti-UCP1, ab10983, (abcam, Cambridge, MA).

Techniques: In Vitro, Two Tailed Test

Journal: bioRxiv

Article Title: Basic-Leucine-Zipper Transcription Factors Regulate Selective Molecular Phenotypes in Regulatory T Cells During IL-2-Induced Activation

doi: 10.1101/2025.02.25.638325

Figure Lengend Snippet: (A) Percentage of live Treg cells that co-express ST2 and 4-1BB surface proteins compared to those that do not co-express them for each single gene knockout condition, where color filled circles represent means of technical triplicates and error bars represent standard deviations. (B) Percentage of live Treg cells that co-express ST2 and 4-1BB surface proteins compared to those that do not co-express them for each dual gene knockout condition, where color filled squares represent means of technical triplicates and error bars represent standard deviations. (C) Integrated Genomics Viewer (IGV) view of bulk ATAC-seq peaks comparing Fosl2 and Jund dual and Batf single gene knockout with the control at the Il1rl1 locus. (D) Integrated Genomics Viewer (IGV) view of bulk ATAC-seq peaks comparing Fosl2 and Jund dual and Batf single gene knockout with the control at the Sell locus.

Article Snippet: After two washes in PBS containing 2% FBS (#SH30071.03, Hyclone, GE Life Sciences), an antibody cocktail containing CD45-BV510 (#563891, BD Biosciences), CD4-BUV395 (#740208, BD Biosciences), CD25-PE (#553866, BD Biosciences), CD73-FITC (#567701, BD Biosciences), GITR-PE-Cy7 (#25-5874-82, Invitrogen), ST2-BV605 (#745257, BD Biosciences) and 4-1BB-AlexaFluor647 (#FAB937R, R&D Systems) were added to the cell suspension and incubated for 45 minutes on ice in the dark.

Techniques: Gene Knockout, Control

Journal: Scientific reports

Article Title: Fluid flow impacts endothelial-monocyte interactions in a model of vascular inflammatory fibrosis.

doi: 10.1038/s41598-025-85987-z

Figure Lengend Snippet: Fig. 3. Effect of fluid flow on surface and junctional adhesion molecule expression with or without TNF-α stimulation. (a) ECs cultured for 72 h, either statically or with 1.5 dynes/cm2 of fluidic shear stress. Junctions labeled with VE-cadherin (green) and PECAM-1 (red). Insets show an enlarged view of the VE-cadherin morphological differences between fluidic and static cultures. (b) Quantification of VE-cadherin junctional borders, demonstrating a significant decrease in width for fluidic cultures. Unpaired t-test was used to determine statistical significance, ****p < 0.0001. (c) ECs cultured with basal 10 ng/ml TNF-α stimulation for 24 h of static culture or fluidic shear stress of 1.5 or 10 dynes/cm2. ECs were preconditioned with 1.5 dynes/ cm2 of shear stress for 24 h before stimulation. Devices were stained with ICAM-1 (green) and Hoechst (blue). (d). ICAM-1 expression is reported as the percent of cells with high, low, or no ICAM-1 levels. A fluorescence intensity threshold was set across all images to determine expression levels. Results indicate that a greater percentage of ECs express high levels of ICAM-1 in the 1.5 dynes/cm2 condition with TNF-α stimulation compared to static culture (p < 0.001, one-way ANOVA with Tukey’s post-hoc test).

Article Snippet: Nuclei were then stained with Hoechst 33342 (Invitrogen, H3570) diluted 1:10,000 in PBS for 5 min. For VE-cadherin (R&D Systems, MAB8381) and PECAM-1 (Invitrogen, PA5-32,321), antibodies were diluted (1:100) and added after the fixation step.

Techniques: Expressing, Cell Culture, Shear, Labeling, Staining, Fluorescence