Journal: The American Journal of Pathology

Article Title: Identification and Characterization of Mesenchymal-Epithelial Progenitor-Like Cells in Normal and Injured Rat Liver

doi: 10.1016/j.ajpath.2014.08.029

Figure Lengend Snippet: Primary Antibodies Used in This Study

Article Snippet: Rabbit anti-GFP , Polyclonal , Novus Biologicals, LLC , NB-600-308 , 1:1000.

Techniques: Transduction

Journal: Journal of Leukocyte Biology

Article Title: Epigenetic regulation of IL-12-dependent T cell proliferation

doi: 10.1189/jlb.1a0814-375rr

Figure Lengend Snippet: Figure 1. Mll1+/2 mice have an altered response to PPD antigen challenge at 4 d post-PPD bead challenge. (A and B) H&E staining of lungs from WT and Mll1+/2 mice at 4 d postbead injection and a graph quantifying significant differences in granu- loma size. *P , 0.03, as determined by Student’s t test. (C and D) Quantification of IFN-g at protein and mRNA levels. (E) Analysis of infiltrating myeloid cell populations in the lung. Results are representative of 3 separate experiments. *P , 0.007, as determined by Student’s t test. Neutrophils [polymorphonuclear neutrophils (PMN)] were defined as autofluorescent- negative CD11b+CD11c2Ly6G+, DCs as autofluores- cent-negative CD11c+CD11b+Ly6C+MHC II+, and macrophages (MF) as autofluorescent-negative CD11b+F/480+MHC IImidhigh. (F) Analysis of total CD4+ T cells, as well as T cell subsets in the lung and lymph node. Effector CD4 cells were defined as CD44hiCD62Llo, and memory cells were defined as CD44hiCD62L+CCR7+. *P , 0.05, as determined by Student’s t test. (G) Staining of lung sections with antibodies to H3K4Me3 in WT and Mll1+/2 mice. The beads at the center of the granulomas are represented by the dashed circles. (H) Mll1 expression in T cell subsets isolated from the spleen by FACS at 14 d post- PPD immunization. Effector and memory cells were isolated, as described in F. Na¨ıve cells were defined as CD44loCD62L+. Expression of Mll1 in CD4+ effector cells isolated from the lungs of PPD-immunized and -challenged mice at 4 d postinjection. For all data, n = 3–5 animals/experiment. *P , 0.05, as de- termined by Student’s t test. Results are representative of 3 independent experiments.

Article Snippet: Flow cytometry for Mll1 was performed by use of Mll1 antibody NB-600-256 at 1:1000 dilution (Novus Biologicals, Littleton, CO, USA).

Techniques: Staining, Injection, Expressing, Isolation

Journal: Journal of Leukocyte Biology

Article Title: Epigenetic regulation of IL-12-dependent T cell proliferation

doi: 10.1189/jlb.1a0814-375rr

Figure Lengend Snippet: Figure 2. Mll1+/2 CD4+ T cells cause altered granuloma pathology in Rag22/2 mice. Quantifica- tion of IFN-g protein (A) and Mll1 mRNA expression (B) in the lungs of Rag22/2 mice receiving Mll1+/2

Article Snippet: Flow cytometry for Mll1 was performed by use of Mll1 antibody NB-600-256 at 1:1000 dilution (Novus Biologicals, Littleton, CO, USA).

Techniques: Expressing

Journal: Journal of Leukocyte Biology

Article Title: Epigenetic regulation of IL-12-dependent T cell proliferation

doi: 10.1189/jlb.1a0814-375rr

Figure Lengend Snippet: Figure 3. Mll1+/2 Th1 cells have reduced proliferative capacity and have reduced ex- pression of CCND3. (A) Assessment of pro- liferation of WT and Mll1+/2 T cells under Th0 and Th1 conditions by [3H]thymidine uptake. *P # 0.01, as determined by 1-way ANOVA. (B) Depiction of the ratio of T cells proliferating in the presence (Th1) vs. the absence (Th0) of IL-12. This ratio is shown for WT and Mll1+/2

Article Snippet: Flow cytometry for Mll1 was performed by use of Mll1 antibody NB-600-256 at 1:1000 dilution (Novus Biologicals, Littleton, CO, USA).

Techniques:

Journal: Journal of Leukocyte Biology

Article Title: Epigenetic regulation of IL-12-dependent T cell proliferation

doi: 10.1189/jlb.1a0814-375rr

Figure Lengend Snippet: Figure 4. STAT4 binds to the Mll1 promoter. (A) PCR data assessing the levels of Mll1 in T cells cultured under normal Th0 or Th1 conditions or Th1 conditions with the addition of 1 mM of the JAK inhibitor tofacitinab (CP690,550). *P # 0.05, as determined by 1-way ANOVA. (B) Assessment of Mll1+

Article Snippet: Flow cytometry for Mll1 was performed by use of Mll1 antibody NB-600-256 at 1:1000 dilution (Novus Biologicals, Littleton, CO, USA).

Techniques: Cell Culture

Journal: Journal of Leukocyte Biology

Article Title: Epigenetic regulation of IL-12-dependent T cell proliferation

doi: 10.1189/jlb.1a0814-375rr

Figure Lengend Snippet: Figure 5. MLL1 binds to genes critical to Th1 cell differentiation. ChIP assay to quantify H3K4Me3 modifications and MLL1 binding to the promoters of Tbx21, Ifng, IL12RB2, and Ccnd3 in murine Th0 and Th1 cells. TSS, Transcription start site. Negative numbers on the abscissa represent kilobases upstream of the TSS. Data are pooled from 3 independent experiments performed with 1.0 3 107 pooled cells from 3 to 5 animals for each replicate. *P # 0.05 by 1-way ANOVA.

Article Snippet: Flow cytometry for Mll1 was performed by use of Mll1 antibody NB-600-256 at 1:1000 dilution (Novus Biologicals, Littleton, CO, USA).

Techniques: Cell Differentiation, Binding Assay

Journal: Journal of Leukocyte Biology

Article Title: Epigenetic regulation of IL-12-dependent T cell proliferation

doi: 10.1189/jlb.1a0814-375rr

Figure Lengend Snippet: Figure 6. Mll1 is essential for the T cell recall response. (A) IFN-g production by Mll1+/2 CD4+ T cells in vitro after 5 d of activation and 3 d of rest, followed by an additional 48 h of stimulation with anti- CD3/anti-CD28. *P # 0.03 by Student’s t test. (B) Proliferation of Mll1+/2 T cells in the same conditions as in (A). *P # 0.03 by Student’s t test. (C) IFN-g production from CD4+ T cells isolated from the lungs of PPD-sensitized and -challenged mice that were then cocultured with BMDCs pulsed with PPD for 48 h. **P # 0.001 by Student’s t test. (D) Assessment of pro- liferation and IFN-g production during the tetanus toxoid recall response when cocultures of T cells and monocyte-derived DCs from the same donor were incubated with the Mll1/menin inhibitor MI-2-2 for 5 d in the presence of tetanus toxoid. All comparisons in (D) are made by use of the DMSO control. *P , 0.05 by Student’s t test compared with the 15 mM dose of inhibitor. Culture supernatants were analyzed by bioplex (A and C) or standard sandwich ELISA (D). Proliferation was measured by pulsing with [3H]thymidine on day 4 of culture and measuring uptake over an 18 h period. (A–C) Data are pooled from 3 independent experiments with the pooled cells of 2–3 animals for each replicate.

Article Snippet: Flow cytometry for Mll1 was performed by use of Mll1 antibody NB-600-256 at 1:1000 dilution (Novus Biologicals, Littleton, CO, USA).

Techniques: In Vitro, Activation Assay, Isolation, Derivative Assay, Incubation, Control, Sandwich ELISA

Journal: Nature Communications

Article Title: circCsnk1g3- and circAnkib1-regulated interferon responses in sarcoma promote tumorigenesis by shaping the immune microenvironment

doi: 10.1038/s41467-022-34872-8

Figure Lengend Snippet: a Flow cytometry analysis for ratio between tumor cells and CD4 + T cell (left) or CD8 + T cells (right) in the subcutaneous sarcoma generated by control sarcoma cells (shSCR, n = 12 mice) or cells in which the expression of circCsnk1g3 was silenced (shcircCsnk1g3, n = 10 mice). Data are normalized to shSCR condition, over n = 3 independent experiments. b Schematic representation of the single-cell RNA-sequencing experiment. c t-SNE plot of the major cell populations identified. d t-SNE plot of lymphocyte subclusters. e Comparative proportions of lymphocytic cell identities in shSCR and shcircCsnk1g3 tumors ( n = 4 mice each group, totaling n = 1143 immune cells from shSCR tumors and n = 993 immune cells from shcircCsnk1g3 tumors). Data are reported as median, IQR (box), and 1.5 × IQR (whiskers). Dots represent individual mice. f Normalized expression of selected genes differentially expressed in CD4 + T cells from shSCR and shcircCsnk1g3 tumors, analyzed by single-cell RNA-sequencing. n = 273 and n = 186 CD4 + T cells from n = 4 shSCR and n = 4 shcircCsnk1g3 tumors, respectively. Mean expression values are plotted as dots and P values derived by Wilcoxon rank sum test with Bonferroni correction. g Immunofluorescence staining showing the distribution of CD3 + T lymphocytes at the border or in the inner tumor parenchyma upon silencing of circCsnk1g3 in the sarcoma cells. Representative pictures of the inner tumor and tumor border areas are shown. h Quantification of the total CD3 + T cells identified in tumor sections. Dots represent individual regions ( n = 171 shSCR, n = 109 shcircCsnk1g3) acquired from n ≥ 2 mice each group. i Weight of subcutaneous tumors generated in immunocompromised mice by control sarcoma cells (shSCR, n = 5 mice) and by sarcoma cells silenced for expression of circCsnk1g3 ( n = 5) or circAnkib1 ( n = 5). Weights normalized to shSCR condition. For all figures, data are reported as mean ± s.e.m., dots represent independent mice, and P values determined by unpaired Student’s t test, unless otherwise indicated. Source data are provided as Source Data File.

Article Snippet: Following blocking with a universal protein blocking reagent, serial sections were incubated with anti-CD3 primary antibody (clone SP7, 1:200 dilution) (#NB-600-1441, Novus Biologicals) for 2 hours at room temperature, washed with TBST buffer three times, and incubated with secondary anti-rabbit IgG H&L HRP-conjugated antibody (ab214880, Abcam, prediluted).

Techniques: Flow Cytometry, Generated, Control, Expressing, RNA Sequencing, Derivative Assay, Immunofluorescence, Staining