murine tgfβ  (R&D Systems)

Journal: Cancer Cell

Article Title: Distinct spatiotemporal dynamics of CD8 + T cell-derived cytokines in the tumor microenvironment

doi: 10.1016/j.ccell.2023.12.010

Figure Lengend Snippet: Frequent IFNγ sensing in a syngeneic tumor model and relationship with reduced TGFβ sensing (A) Rag2 −/− mice were injected subcutaneously with a mixture of 10% OVA antigen expressing and 90% Ag − bystander NMM tumor cells, or with Ag − NMM tumor cells only, and, following tumor establishment, were treated with either PBS (control) or OT-1 CD8 + T cells, as indicated. Ag − bystander tumor cells were harvested for scRNA-seq analysis 44 h after treatment. (B) TNFα and IFNγ gene set scores, determined using the genes shown in a, for the T cell-exposed condition (green) and the two control conditions (T cell-exposed-Ag − bystander NMM tumor cells only tumors, and PBS treated tumors, shades of gray). Dots represent gene set scores of individual cells, violins represent densities of score distributions, white dots represent group medians. (C) Left panel: UMAP of NMM melanoma single cell data, as described in (A) and (B). Middle and right panels: a Milo model was fitted to the data to test for enrichment or depletion for any of the experimental conditions in neighborhoods of transcriptionally similar cells. Non-significantly imbalanced neighborhoods (Spatial FDR >0.05), as well as homogeneous neighborhoods, are colored white. (D) Left panel: heatmap of top 250 genes (rows) most strongly correlated (Spearman correlation) with enrichment for the T cell-exposed condition in cell state neighborhoods (columns) of transcriptionally similar cells. Depicted values are neighborhood averages. Neighborhoods are ordered according to compositional enrichment of cells from the T cell-exposed condition. Top panels show log fold change in differential abundance (logFC DA) for the indicated experimental condition relative to control condition. Right panel: heatmap showing bulk RNA-seq gene expression profiles of NMM cells exposed to indicated cytokines for the same genes as in the heatmap in the left panel, ordered identically. (E) As in (D), but for TGFβ responsive genes selected on bulk RNA-seq data. (F) Deconvolution mixing weights of neighborhoods in an independent bulk RNA-seq experiment. Neighborhoods ordered as in (D). Only the 6 out of 28 most highly selected reference profiles are shown, jointly comprising 94% of all assigned similarity. (G) Left: Increase in reconstruction error when the 17 reference profiles with TGFβ are omitted as compared to when all 28 profiles are included. Permutation testing was employed to test whether increase in reconstruction error could be explained by a lower number of reference profiles . Right: As left, but omitting the 17 reference profiles with IFNγ. (H) Model visualizing secondary effects of long range IFNγ sensing. In parallel to the mechanism in which long range IFNγ sensing leads to generation of, for instance, CXCL9/10/11 chemokine fields and subsequent increased immune cell infiltration, long range IFNγ sensing may result in secondary changes in the TME by decreasing TGFβ sensing. See also Figure S4 .

Article Snippet: Recombinant murine TGFβ , ebioscience , CAT#: 14-8342-80.

Techniques: Injection, Expressing, Control, RNA Sequencing, Gene Expression

Journal: Cancer Cell

Article Title: Distinct spatiotemporal dynamics of CD8 + T cell-derived cytokines in the tumor microenvironment

doi: 10.1016/j.ccell.2023.12.010

Figure Lengend Snippet:

Article Snippet: Recombinant murine TGFβ , ebioscience , CAT#: 14-8342-80.

Techniques: Virus, Retroviral, Recombinant, Membrane, Library Quantification, Sequencing, Mutagenesis, Bacteria, Software

Journal: Cancer Cell

Article Title: Distinct spatiotemporal dynamics of CD8 + T cell-derived cytokines in the tumor microenvironment

doi: 10.1016/j.ccell.2023.12.010

Figure Lengend Snippet: Frequent IFNγ sensing in a syngeneic tumor model and relationship with reduced TGFβ sensing (A) Rag2 −/− mice were injected subcutaneously with a mixture of 10% OVA antigen expressing and 90% Ag − bystander NMM tumor cells, or with Ag − NMM tumor cells only, and, following tumor establishment, were treated with either PBS (control) or OT-1 CD8 + T cells, as indicated. Ag − bystander tumor cells were harvested for scRNA-seq analysis 44 h after treatment. (B) TNFα and IFNγ gene set scores, determined using the genes shown in a, for the T cell-exposed condition (green) and the two control conditions (T cell-exposed-Ag − bystander NMM tumor cells only tumors, and PBS treated tumors, shades of gray). Dots represent gene set scores of individual cells, violins represent densities of score distributions, white dots represent group medians. (C) Left panel: UMAP of NMM melanoma single cell data, as described in (A) and (B). Middle and right panels: a Milo model was fitted to the data to test for enrichment or depletion for any of the experimental conditions in neighborhoods of transcriptionally similar cells. Non-significantly imbalanced neighborhoods (Spatial FDR >0.05), as well as homogeneous neighborhoods, are colored white. (D) Left panel: heatmap of top 250 genes (rows) most strongly correlated (Spearman correlation) with enrichment for the T cell-exposed condition in cell state neighborhoods (columns) of transcriptionally similar cells. Depicted values are neighborhood averages. Neighborhoods are ordered according to compositional enrichment of cells from the T cell-exposed condition. Top panels show log fold change in differential abundance (logFC DA) for the indicated experimental condition relative to control condition. Right panel: heatmap showing bulk RNA-seq gene expression profiles of NMM cells exposed to indicated cytokines for the same genes as in the heatmap in the left panel, ordered identically. (E) As in (D), but for TGFβ responsive genes selected on bulk RNA-seq data. (F) Deconvolution mixing weights of neighborhoods in an independent bulk RNA-seq experiment. Neighborhoods ordered as in (D). Only the 6 out of 28 most highly selected reference profiles are shown, jointly comprising 94% of all assigned similarity. (G) Left: Increase in reconstruction error when the 17 reference profiles with TGFβ are omitted as compared to when all 28 profiles are included. Permutation testing was employed to test whether increase in reconstruction error could be explained by a lower number of reference profiles . Right: As left, but omitting the 17 reference profiles with IFNγ. (H) Model visualizing secondary effects of long range IFNγ sensing. In parallel to the mechanism in which long range IFNγ sensing leads to generation of, for instance, CXCL9/10/11 chemokine fields and subsequent increased immune cell infiltration, long range IFNγ sensing may result in secondary changes in the TME by decreasing TGFβ sensing. See also Figure S4 .

Article Snippet: Ag − CFP + tumor cells were plated at 200,000 cells per well in 6-well plates for 24 h and were then treated with either human IFNγ (Invitrogen), human TNFα (Peprotech), murine IFNγ (Thermo Fisher), murine TNFα (Peprotech) or murine TGFβ (ebioscience) the indicated combination, or were treated with culture medium from T cell - tumor cell co-cultures (see below), at the indicated concentrations or dilutions.

Techniques: Injection, Expressing, Control, RNA Sequencing, Gene Expression

Journal: Cancer Cell

Article Title: Distinct spatiotemporal dynamics of CD8 + T cell-derived cytokines in the tumor microenvironment

doi: 10.1016/j.ccell.2023.12.010

Figure Lengend Snippet:

Article Snippet: Ag − CFP + tumor cells were plated at 200,000 cells per well in 6-well plates for 24 h and were then treated with either human IFNγ (Invitrogen), human TNFα (Peprotech), murine IFNγ (Thermo Fisher), murine TNFα (Peprotech) or murine TGFβ (ebioscience) the indicated combination, or were treated with culture medium from T cell - tumor cell co-cultures (see below), at the indicated concentrations or dilutions.

Techniques: Virus, Retroviral, Recombinant, Membrane, Library Quantification, Sequencing, Mutagenesis, Bacteria, Software

Journal: iScience

Article Title: HDAC1/2 inhibitor therapy improves multiple organ systems in aged mice

doi: 10.1016/j.isci.2023.108681

Figure Lengend Snippet: Influence of Cmpd60 on aging kidney (A) Representative western blot of tubular epithelial cells (TECs) treated with 20 ng/mL of recombinant TGF-β and with and without Cmpd60 (1 μM) for 72 h. Protein lysates of TECs blotted for anti-ZO-1, anti-E-cadherin, anti-αSMA, and β-actin. Cmpd60 suppresses markers for partial EMT, a hallmark of age-related renal fibrosis (n = 3)/group. (B) Schematic of aged mouse treatment regimen with Cmpd60 and analyses. (C) Relative histone H4 acetylation levels (H4K8Ac) assessed by western blot in renal tissue of aged mice with and without Cmpd60 treatment. Protein expression was normalized against H4 total and expressed as mean ± SEM. Mann Whitney test was used to determine statistical differences. ∗∗p < 0.01. (n = 5–6)/group. (D) PLS-DA analysis of aged mice treated with and without Cmpd60 (n = 5–6)/group. (E) Top GO terms of upregulated processes in aged mice treated with Cmpd60 (see also Table S2 ). (F) Representative histological images of picrosirius red staining in kidney of aged mice with and without Cmp60. (n = 5–6)/group. (G) Quantification of interstitial fibrosis determined by the percentage of positive picrosirius red staining/high power field, in mice treated with and without Cmpd60. Percentage of positive staining was assessed with ImageJ software. Data are expressed as mean ± SEM and the Mann Whitney test was used to determine statistical significance. ∗p < 0.05, (n = 5–6)/group.

Article Snippet: TECs were stimulated with 20ng/ml murine recombinant TGFβ (Prospec) for 72 hours in DMEM/F12 supplemented with 10% fetal calf serum, penicillin/streptomycin and 2mM L-glutamin.

Techniques: Western Blot, Recombinant, Expressing, MANN-WHITNEY, Staining, Software

Journal: Cell Stem Cell

Article Title: Dynamic and adaptive cancer stem cell population admixture in colorectal neoplasia

doi: 10.1016/j.stem.2022.07.008

Figure Lengend Snippet: Driver genes and pathways associated with variable stem cell molecular phenotype (A) Human genotype-stem cell phenotype correlation based on stem cell index distribution in TCGA tumors with different putative driver gene single-nucleotide variant (SNV) mutation genotypes, contrasted to normal tissue from same dataset (driver gene initials: A is APC, K is KRAS, P is p53, B is BRAF). (B) Comparison of mutation type and prevalence disrupting the Wnt pathway, MAPK and PIK3CA pathways, and the TGFβ superfamily in TCGA tumors subdivided into CBC- and RSC-predominant deciles. (C) Segregation of mouse and human lesions by CBC (x axis) and RSC (y axis) signature expression. Predominant (above median) expression signature in each tumor is defined by color (CBC in green and RSC in red), and the 10% most polarized CBC- or RSC-expressing tumors were segregated into CBC- and RSC-enriched deciles for comparison. (D) Gene set enrichment analysis of hallmark and select pathways in bulk transcriptome from human tumors (TCGA) and murine lesions (Glasgow dataset) segregated into CBC- and RSC-predominant deciles. Pathways shown have P FDR ≤ 0.25 apart from YAP in the mouse lesions and Fibroblast TGFβ response in the human tumors. (E) Correlation of key pathway expression signatures with CBC or RSC gene expression across a range of mouse models. Different genotypes are identified by different colors as determined by the key. Driver alleles initialization: A is Apc fl/+ , Apc Min is Apc Min , B is Braf V600E , K is Kras G12D , P is p53 fl/fl , T is Tgfβr1 fl/fl , N is Rosa26 N1icd/+ , Alk4 is Alk4 fl/fl .

Article Snippet: Mouse intestinal organoids (wild-type, AKPT, and KPN) were plated onto 24-well plates in Matrigel and cultured for 3 days before treatment with either recombinant murine IFN-γ (PeproTech, UK) at 1 ng/mL or recombinant murine TGFβ 1 (R&D, UK) at 5 ng/mL.

Techniques: Variant Assay, Mutagenesis, Expressing