Journal: Breast Cancer Research : BCR

Article Title: STAT5 is activated in macrophages by breast cancer cell-derived factors and regulates macrophage function in the tumor microenvironment

doi: 10.1186/s13058-021-01481-0

Figure Lengend Snippet: Tumor cell-derived GM-CSF activates STAT5 in macrophages. A qRT-PCR analysis for GM-CSF in TNBC (Hs578T and MDA-MB-231), ER + (MCF7) and HER2 + (BT-474) human breast cancer cells relative to expression in MCF-10A cells. B ELISA analysis for GM-CSF in CM collected from MCF-10A, MDA-MB-231, Hs578T, MCF7, and BT-474 cells. C ELISA analysis for GM-CSF in CM collected from 4T1 cells and B/B-stimulated HC11, HC11/R1, and HC11/R1-LM cells relative to EtOH controls. D Immunoblot analysis for pSTAT5, TSTAT5, and β-tubulin in BMDMs treated with No CM, tumor CM (4T1 or HC11/R1 BB), or tumor CM incubated for 1 h at 37 °C with 2.5 µg/mL neutralizing GM-CSF antibody (⍺GM-CSF Ab)

Article Snippet: STAT5 fl/fl DCM and STAT5 cKO DCM was subjected to mouse Type I Collagen ELISA (Novus Biologicals).

Techniques: Derivative Assay, Quantitative RT-PCR, Expressing, Enzyme-linked Immunosorbent Assay, Western Blot, Incubation

Journal: Breast Cancer Research : BCR

Article Title: STAT5 is activated in macrophages by breast cancer cell-derived factors and regulates macrophage function in the tumor microenvironment

doi: 10.1186/s13058-021-01481-0

Figure Lengend Snippet: STAT5 deletion in macrophages enhances tumor-promoting phenotype and impacts tumor cell migration and metastasis. A qRT-PCR analysis of genes of interest from RNA-seq associated with tumor-promoting pathways in rmGM-CSF or 4T1 CM-treated STAT5 fl/fl (blue) and STAT5 cKO (red) BMDMs. Unpaired t-test was used for statistical analysis. B Mouse Type 1 Collagen ELISA in STAT5 fl/fl unstimulated or 4T1 CM-stimulated STAT5 fl/fl and STAT5 cKO macrophage double CM (DCM). Data were analyzed using one-way ANOVA and Tukey’s multiple comparison test. C Representative immunoblot of pFAK, total FAK (FAK), and β-tubulin in 4T1 cells cultured alone or co-cultured with STAT5 fl/fl or STAT5 cKO BMDMs for 4 h. Densitometry analysis relative to loading control. D Migration analysis of 4T1 cells cultured alone or co-cultured with STAT5 fl/fl or STAT5 cKO BMDMs after 20 h. Cell counts relative to 4T1 alone in triplicate. E Kaplan–Meier curves of 4T1 cells co-injected with either STAT5 fl/fl (n = 8) or STAT5 cKO (n = 7) BMDMs in WT BALB/c mice. % Survival on Y-axes indicates proportion of mice reaching tumor size endpoint of 1cm 3 . F Quantified metastasis in H&E-stained lung sections. Lungs were sectioned at 3 different depths per mouse and analyzed for percent metastatic area per tissue section. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Scale bar: 50 μm

Article Snippet: STAT5 fl/fl DCM and STAT5 cKO DCM was subjected to mouse Type I Collagen ELISA (Novus Biologicals).

Techniques: Migration, Quantitative RT-PCR, RNA Sequencing, Enzyme-linked Immunosorbent Assay, Comparison, Western Blot, Cell Culture, Control, Injection, Staining

Journal: Molecular Metabolism

Article Title: Dysregulation of the Pdx1/Ovol2/Zeb2 axis in dedifferentiated β-cells triggers the induction of genes associated with epithelial–mesenchymal transition in diabetes

doi: 10.1016/j.molmet.2021.101248

Figure Lengend Snippet: Loss of β-cell identity is associated with alteration of islet microenvironment and TGFβ-dependent fibrosis . (A) Western blotting for phospho-Ser423/425 and γ-tubulin in islets from 12w Tg7 mice. (B) mRNA levels for TGFβ target genes in islets from 12w Tg7 and Wt mice obtained by qPCR (n = 6). (C) Fluorescence microscopy on pancreatic sections from Wt β−Tom and Tg7 β−Tom mice Red: tdTomato autofluorescence associated with β-cells. Scale bar: 40 μm. (D) Electron microscopy of 12w Wt and Tg7 islet preparations. Arrowhead indicates insulin granules. Arrows indicate the intercellular space between surrounding β-cells in Tg7 compared to control islets. Scale bar: 1 μm. Also shown is a zoom of the intercellular space between islet β-cells. (E) mRNA levels for ECM remodellers and collagen genes in islets from 12w Tg7 and Wt mice obtained by qPCR (n = 6). (F) Sirius red staining of pancreatic sections from 12w Tg7 mice and controls. (G) Collagen I content in islets from 12w Tg7 and Wt mice measured by ELISA. Islets from Tg7 mice were pre-treated with TGFβRI inhibitor Alk5i (1 uM) (n = 3–6). In panel G, one-way ANOVA with Sidak's multiple comparison test; otherwise, unpaired Student's t-test. Data are means ± SEM. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001.

Article Snippet: The supernatant was collected, kept on ice, and processed using a Mouse Collagen Type I (COL1) ELISA kit (Abbexa, Cambridge, UK) according to the manufacturer's instructions.

Techniques: Western Blot, Fluorescence, Microscopy, Electron Microscopy, Control, Staining, Enzyme-linked Immunosorbent Assay, Comparison

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: TNFR2 Deficiency Acts in Concert with Gut Microbiota to Precipitate Spontaneous Sex-biased CNS Demyelinating Autoimmune Disease

doi: 10.4049/jimmunol.1501664

Figure Lengend Snippet: (A) Female C57BL6/J 2D2 TNFR2−/− mice appear normal at birth and at early age; shown are representative 14 day old female TNFR2−/− and TNFR2+/+ mice. (B) 92% of female 2D2 TNFR2−/− mice spontaneously developed disease between 28–79 d of age. Representative affected 65 d old female 2D2 TNFR2−/− and unaffected age- and gender-matched 2D2 TNFR2+/+ mice are shown. (C) The cumulative incidence of 2D2 TNFR2−/− mice with clinical manifestations of disease with a score ≥ 2 (female 2D2 TNFR2−/−, (59/64); male 2D2 TNFR2−/−, (5/60); female 2D2 TNFR2+/+, (5/73); male 2D2 TNFR2+/+; 4/72). Remission of disease severity was not observed in any of the mice studied. (D) Soluble TNF (pg/ml) was determined in sera collected from 4–5 wk old, unaffected male and female 2D2 TNFR2+/+ and 2D2 TNFR2−/− mice by ELISA. The data are reported as mean ± SEM; n = 3 per group. (E) The cumulative incidence of 2D2 TNF−/− mice with a score ≥ 2 (female, 5/63; male, 4/55). Remission of disease severity was not observed in any of the mice studied. (F) Soluble TNFR1 (pg/ml) was determined by ELISA from sera collected from the unaffected mice described in D. The data are reported as mean ± SEM; n = 3. (G) Soluble TNFR2 (pg/ml) was determined by ELISA from sera collected from the unaffected 2D2 TNFR2+/+ mice described in D. The data are reported as mean ± SEM; n = 3. (H) Representative paraffin-embedded spinal cord sections from unaffected female 2D2 TNFR2−/− mice (top) or age and gender-matched affected 2D2 TNFR2−/− mice (bottom) stained with H&E, luxol fast blue (LFB), or Bielschowsky staining to visualize inflammatory cell infiltration, demyelination, and axonal damage, respectively. H&E stained infiltrates are evident in perivascular cuffs (arrow head) and the parenchyma (arrow). LFB show limited loss of myelin in the central white matter (arrow head), but extensive demyelination of peripheral white matter (arrow). Bielschowsky staining shows mild (arrow head) to severe (arrow) axonal loss. Scale bar = 200 μm. (I) Representative paraffin-embedded optic nerve sections from unaffected female 2D2 TNFR2−/− mice (top) or age and gender-matched affected 2D2 TNFR2−/− mice (bottom) stained with H&E, LFB, or Bielschowsky staining demonstrate profound inflammation (left) and demyelination (middle) and moderate axonal loss (right), respectively. A representative monocyte (arrow) and eosinophil infiltrate (arrowhead) are indicated. Scale bar = 50 μm. (J) T cell (anti-CD3), B cell (anti-B220), and macrophage/microglia (anti-CD68) infiltration into the spinal cord of unaffected (top) or age and gender-match affected (bottom) female 2D2 TNFR2−/− mice. Representative paraffin-embedded spinal cord sections are shown. Localization of cells in the perivascular cuffs of the spinal cord (arrowheads) and localization to the parenchyma (arrows). Scale bar = 200 μm. (K) T cell (anti-CD3), B cell (anti-B220), and macrophage/microglia (anti-CD68) infiltration into the optic nerves of unaffected (top) or age and gender-match affected (bottom) female 2D2 TNFR2−/− mice. Representative paraffin-embedded optic nerve sections are shown with representative T cells and macrophages/microglia indicated (arrowheads). Scale bar = 50 μm.

Article Snippet: ® ELISA kits (TNF and TNFR2) or the mouse TNFR1 DuoSet ® ELISA kit (R&D Systems, Minneapolis, MN).

Techniques: Enzyme-linked Immunosorbent Assay, Staining