Journal: Science immunology

Article Title: Distinct immunocyte-promoting and adipocyte-generating stromal components coordinate adipose-tissue immune and metabolic tenors

doi: 10.1126/sciimmunol.aaw3658

Figure Lengend Snippet: (A-D) Cytofluorimetric identification of IL-33-producing cells within eVAT of 8–10-week-old B6 mice using a polyclonal anti-IL-33 Ab. (A) Gating strategy to delineate cell fractions. (B) Representative dot-plots of control-Ab (top panels) or anti-IL-33 staining (bottom panels) of the cell fractions. (C) As per panel B except whole-body IL-33-deficient (II33−/−) mice were assessed. (D) Frequencies of IL-33+ cells in each cell fraction (left) and its contribution to total IL-33+ cells within the tissue (right). n≥7 from at least three experiments. (E-G) Confocal microscopic images of IL-33+ VmSCs. (E) The eVAT depot is circumferenced by a ring of high PDPN positivity, presumably the mesothelium. (F) IL-33+ cells locate in close proximity to CD31+ endothelial cells. (G) PDPN positivity outlines a large blood vessel surrounded by β3-tubulin+ nerves. Color code for Ab staining as indicated on top of each picture. PDPN, podoplanin; DNs, PDGFRα−Sca-1− cells; VmSCs, PDGFRα+Sca-1+ VAT mesenchymal stromal cells.

Article Snippet: Primary Abs were goat anti-mouse IL-33 (R&D Systems, #AF3526, 1:50 dilution), APC-conjugated Syrian Hamster anti-mouse PDPN (Biolegend, #AF3526, 1:100), or rabbit anti-GFP (Abcam, 1:1000), rabbit anti-β3-tubulin (Cell Signalling, #D6584, 1:50) and rat anti-mouse CD31 (BioLegend, #102512, 1:50).

Techniques: Staining

Journal: Science immunology

Article Title: Distinct immunocyte-promoting and adipocyte-generating stromal components coordinate adipose-tissue immune and metabolic tenors

doi: 10.1126/sciimmunol.aaw3658

Figure Lengend Snippet: (A) Two-dimensional tSNE plot of scRNA data from VAT (orange), muscle (green) and lymph node (blue) PDGFRα+Sca-1+ mSCs. VmSC subtypes 1–5 were delineated by k- means clustering; their fractional contributions to total VmSCs are indicated. (B) Heat-map showing genes differentially expressed between the VmSC subtypes (FDR < 5%). k-means clustered. (C) VmSCs dynamics (velocity field). The projected next cell state for each single cell is represented by an arrow and projected on the tSNE plot. (D) Same tSNE plot as in panel A overlain with heat-maps of the density of cells expressing various transcript markers. (E) Heat-map of transcript expression within the combined single-cell data of each VmSC subtype. (F-G) Representative dot-plots delineating the VmSC subtypes (left) and indicating their IL-33 expression levels (right) in PpargTdtIl33Egfp double-reporter mice. (G) Comparison of VmSC subtype frequencies obtained by scRNAseq versus flow cytometry. (H) Matrix of Spearman correlation coefficients in head-to-head comparisons of ≥2-fold differentially expressed genes from the scRNAseq and matching population (pop)-level RNAseq datasets. LN, lymph node.

Article Snippet: Primary Abs were goat anti-mouse IL-33 (R&D Systems, #AF3526, 1:50 dilution), APC-conjugated Syrian Hamster anti-mouse PDPN (Biolegend, #AF3526, 1:100), or rabbit anti-GFP (Abcam, 1:1000), rabbit anti-β3-tubulin (Cell Signalling, #D6584, 1:50) and rat anti-mouse CD31 (BioLegend, #102512, 1:50).

Techniques: Expressing, Comparison, Flow Cytometry

Journal: Science immunology

Article Title: Distinct immunocyte-promoting and adipocyte-generating stromal components coordinate adipose-tissue immune and metabolic tenors

doi: 10.1126/sciimmunol.aaw3658

Figure Lengend Snippet: (A) Representative plots (top), frequencies and numbers (bottom) of total VmSCs from mice of different ages. (B) Frequencies (top) and numbers (bottom) of total IL-33+ VmSCs. (C and D) Cytofluorometric dot-plots of VmSC subtypes (C) and corresponding fractions (top) and numbers (bottom) (D) from lean B6 males of the indicated ages. Pooled data from three independent experiments. Numbers of cells were normalized per tissue weight. One-way ANOVA analysis was performed to compare three or more groups. For all relevant plots, mean ± SEM. p-values: *, ≤0.05; **, ≤0.01; ***, ≤0.001; ****, ≤0.0001. SVF, stromal vascular fraction. Other abbreviations as per Fig 1.

Article Snippet: Primary Abs were goat anti-mouse IL-33 (R&D Systems, #AF3526, 1:50 dilution), APC-conjugated Syrian Hamster anti-mouse PDPN (Biolegend, #AF3526, 1:100), or rabbit anti-GFP (Abcam, 1:1000), rabbit anti-β3-tubulin (Cell Signalling, #D6584, 1:50) and rat anti-mouse CD31 (BioLegend, #102512, 1:50).

Techniques:

Journal: Science immunology

Article Title: Distinct immunocyte-promoting and adipocyte-generating stromal components coordinate adipose-tissue immune and metabolic tenors

doi: 10.1126/sciimmunol.aaw3658

Figure Lengend Snippet: (A) Frequencies (top) and numbers (bottom) of total IL-33+ VmSCs. (B) Cytofluorimetric dot-plots (left) and corresponding quantification of subtypes thereof (right) in gonadal (g)VAT and iSAT of lean male (M) and female (F) mice aged 18–20 weeks old. Pooled data from at least two independent experiments. (C) Three-dimensional PCA plot of the transcriptomes (population-level RNAseq) from coincidently prepared male and female VmSC subtypes from B6 mice 8–10 weeks old. (D) Esr1 (top) and Ar (bottom) transcripts levels for the individual VmSC subtypes from male (white bars) and female (black bars) mice aged 8–10 weeks old. Each dot represents an individual biological replicate from two or more pooled mice. (E) Correlation curves for numbers of IL-33+ VmSCs versus ST2+ Treg numbers in gVAT using meta-data from all male mice of various ages (6, 16, 18–20 and 32 weeks) and female mice 18–20 weeks old, i.e. all mice of normal physiologic state. In all cases, numbers of cells were normalized to tissue weight. r and p from Pearson’s correlation coefficient. All other statistics and abbreviations as per Fig. 1 and ​and55.

Article Snippet: Primary Abs were goat anti-mouse IL-33 (R&D Systems, #AF3526, 1:50 dilution), APC-conjugated Syrian Hamster anti-mouse PDPN (Biolegend, #AF3526, 1:100), or rabbit anti-GFP (Abcam, 1:1000), rabbit anti-β3-tubulin (Cell Signalling, #D6584, 1:50) and rat anti-mouse CD31 (BioLegend, #102512, 1:50).

Techniques:

Journal: Science immunology

Article Title: Distinct immunocyte-promoting and adipocyte-generating stromal components coordinate adipose-tissue immune and metabolic tenors

doi: 10.1126/sciimmunol.aaw3658

Figure Lengend Snippet: (A and B) Fractions (top) and numbers (bottom) of total IL-33+ VmSCs (A) and VmSC subtypes (B) subsequent to 4 or 16 weeks of high-fat feeding of 14-week-old B6 males. Data from at least two independent experiments. (C and D). Effects of IL-33 administration to 8–12-week-old B6 males. Frequency (top) and numbers (bottom) of total IL-33+ VmSCs (C) or of individual VmSC subtypes (D). Data shown corresponds to at least two pooled independent experiments. Numbers of cells were tissue weight-normalized in all cases. LFD, low-fat diet; HFD, high-fat diet; PBS, phosphate-buffered saline. All other statistics and abbreviations as per Figs. 1 and ​and55.

Article Snippet: Primary Abs were goat anti-mouse IL-33 (R&D Systems, #AF3526, 1:50 dilution), APC-conjugated Syrian Hamster anti-mouse PDPN (Biolegend, #AF3526, 1:100), or rabbit anti-GFP (Abcam, 1:1000), rabbit anti-β3-tubulin (Cell Signalling, #D6584, 1:50) and rat anti-mouse CD31 (BioLegend, #102512, 1:50).

Techniques: Saline

Journal: Science immunology

Article Title: Distinct immunocyte-promoting and adipocyte-generating stromal components coordinate adipose-tissue immune and metabolic tenors

doi: 10.1126/sciimmunol.aaw3658

Figure Lengend Snippet: (A and B) IL-33 was administered to wild-type B6 males aged 8–12 weeks. Frequencies (top) and numbers (bottom) of total (left) and ST2+ (right) Tregs (A) or ILC2s (B). (C) Confocal images (group of left panels) showing Tregs and IL-33-expressing cells within eVAT at low-magnification (left), corresponding delineation of adipocyte edges based on autofluorescence of DAPI channel (middle) and high-magnification of the squared indicated area (right). Distance quantification between Foxp3+ Tregs and the nearest IL-33- expressing cell (group of right panels) from whole eVAT tissue sections taken from male VAT-Treg TCR- transgenic mice at 7 (top) and 17 (bottom) weeks of age. Arrows in panel C depict Treg:IL-33+ cell proximity. Color code for Ab staining as indicated within the picture. (D, E and F) IL- 33 was injected into mice lacking ST2 expression specifically by Tregs vs wild-type littermate controls. Frequencies (top) and numbers (bottom) corresponding to total and KLRG1+ Tregs (D), ILC2s (E) and IL-33+ VmSCs (F). All numbers were calculated relative to total tissue weight. (G) Graphic scheme of the proposed VmSC:Treg negative regulatory loop model. All other abbreviations and statistics as per Figs. 1, ​,55 and ​and77.

Article Snippet: Primary Abs were goat anti-mouse IL-33 (R&D Systems, #AF3526, 1:50 dilution), APC-conjugated Syrian Hamster anti-mouse PDPN (Biolegend, #AF3526, 1:100), or rabbit anti-GFP (Abcam, 1:1000), rabbit anti-β3-tubulin (Cell Signalling, #D6584, 1:50) and rat anti-mouse CD31 (BioLegend, #102512, 1:50).

Techniques: Expressing, Transgenic Assay, Staining, Injection

Journal: Molecular Oncology

Article Title: Cancer‐associated fibroblasts educate normal fibroblasts to facilitate cancer cell spreading and T‐cell suppression

doi: 10.1002/1878-0261.13077

Figure Lengend Snippet: Localization of CEFs in gastric cancer and continuous generation of CEFs. (A) Paraffin sections of human gastric cancer specimens (case #1 and case #2) were immunostained with anti‐ASPN (red), anti‐α‐SMA (green), and anti‐Cytokeratin19 (white or magenta). Asterisks indicate blood vessels. Scale bar = 500 μm. The distribution of positive cells by each antibody was quantified using zen software. The fluorescence intensity was measured in the direction of invasion (white arrow) and plotted from proximal to distal (right panel). Dotted line indicates border between SM: submucosa and MP: muscle layer. In both cases, α‐SMA was stained in muscle layer. (B–E) Gastric cancer specimens (case#1) were immunostained with indicated antibodies. Confocal microscope images of similar regions in submucosa (SM) were shown. Scale bar = 50 μm. (F) Top: CEF‐3d indicates CEFs 3 days after generation. Bottom, left: mRNA levels of IL‐1β, IL‐33, and CXCL6 in CEFs 3 days after preparation (CEF‐3d) were compared with NFs by qRT‐PCR ( n = 3, ** P < 0.01 and *** P < 0.001). Right: CM of CEF‐3d did not disperse OCUM‐12 cells. The data in bar charts are presented as the mean ± SD. (G) L‐CEF: NF‐54 cells were treated with CAF‐CM for 7 days. L‐CEF‐7d indicates L‐CEFs 7 days after removal of CAF‐CM. Left; Representative image of OCUM‐12 cells incubated with L‐CEF‐7d‐CM. Right; Expression of CEF marker genes in L‐CEF‐7d was examined by western blot. (H) CEF 2 and CEF 3 (CEF‐educated fibroblasts): the second and the third generation of CEFs were prepared as indicated in the scheme. OCUM‐12 cells incubated with the NF‐CM, CEF 2 ‐CM, or CEF 3 ‐CM for 24 h. Representative images are shown. Disp + indicates cell dispersion. Scale bars = 200 μm. Right: Immunoblot analysis of CEF marker genes in CEF 2 and CEF 3 cells with indicated antibodies. (I) Cell dispersion was analyzed using Delaunay triangulation plot. The graph indicates the average size of whole triangles (* P < 0.05). Statistical significance was calculated using a one‐way ANOVA followed by Tukey's post hoc tests. The data in bar charts are presented as the mean ± SD.

Article Snippet: The following antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA): anti‐IDO (sc‐376413), anti‐IGF‐I (sc‐74116), anti‐IGFBP‐4 (sc‐517440), anti‐IGF‐IR (sc‐81464), anti‐Kynurenine (sc‐69890), anti‐Smad2/3 (sc‐6032), anti‐PAPP‐A (for immunoblotting, sc‐365226), from Cell Signaling Technology (Danvers, MA, USA): anti‐phospho‐AKT (9271S), E‐cadherin (3195), anti‐COX2 (12282), anti‐phospho‐ERK (9101S), anti‐ERK (9102), anti‐Phospho‐IGF‐1Rβ (3024), anti‐phospho‐IKBα (2859), anti‐phospho‐IKKa/B (2697), NF‐κB p65 Antibody Sampler Kit (4767), anti‐vimentin (3932S), anti‐phospho‐Smad2 (3108S), anti‐Snail (3879), anti‐phospho‐p38 (9211S), anti‐p38 (9212), from Sigma‐Aldrich (St. Louis, MO, USA): anti‐KYNU (HPA031686), anti‐LIF (HPA018844), anti‐α‐tubulin (T5168), from Abcam (Cambridge, UK): anti‐CXCL5+CXCL6 (EP13083) (ab198505), anti‐IL‐1B (ab9722), or from Proteintech (16806‐1‐AP, Chicago, IL, USA): anti‐CST1 (16025‐1‐AP), anti‐IL‐33 (66235‐1), anti‐KMO (10698‐1‐AP), and anti‐PAPP‐A (66962‐1‐Ig, for immunostaining).

Techniques: Software, Fluorescence, Staining, Microscopy, Quantitative RT-PCR, Incubation, Expressing, Marker, Western Blot