Journal: International Journal of Molecular Sciences

Article Title: Characterization and Clinical Relevance of Endometrial CAFs: Correlation between Post-Surgery Event and Resistance to Drugs

doi: 10.3390/ijms24076449

Figure Lengend Snippet: Detailed information on the antibodies and primers used in the study.

Article Snippet: Fibroblasts Antibody (TE-7) , NOVUS , NBP2-50082.

Techniques: Sequencing, Recombinant, Flow Cytometry

Journal: Scientific Reports

Article Title: Macrophages promote a profibrotic phenotype in orbital fibroblasts through increased hyaluronic acid production and cell contractility

doi: 10.1038/s41598-019-46075-1

Figure Lengend Snippet: Macrophages do not significantly alter spontaneous lipid vesicle formation in 3D-cultured orbital fibroblasts. (a) Representative Oil-Red-O (ORO) staining of lipid-containing vesicles in orbital fibroblasts in 3D culture for 7 days: HO2 without macrophages (left) and CO6 with macrophages (right, arrow), at a 1:1 fibroblast:macrophage ratio. Scale bar, 25 μm. (b) Macrophages did not alter spontaneous lipid vesicle formation in 3D culture. GO (HOs) or control (COs) fibroblasts (0.74 × 10 5 cells/ml) were cultured with macrophages at a 1:1 fibroblast:macrophage ratio in the presence of 10% serum for 7 days. The fraction of ORO positive cells is shown as mean ± SEM (n = 3, no statistical difference between with and without macrophages). (c) ORO positive cells in control fibroblast lines, CO4 and CO7, did not significantly increase as the number of macrophages increased (mean ± SEM; n = 4, p = 0.118 (CO4) and p = 0.123 (CO7), Kruskal-Wallis test).

Article Snippet: To confirm the efficiency of the blocking antibody, HA levels were measured in fibroblasts cultured in serum-free medium with 20 μM IGF-1 (R&D Systems, MN, USA) to stimulate HA production, with or without IGF-1R blocking antibody.

Techniques: Cell Culture, Staining, Control

Journal: Scientific Reports

Article Title: Macrophages promote a profibrotic phenotype in orbital fibroblasts through increased hyaluronic acid production and cell contractility

doi: 10.1038/s41598-019-46075-1

Figure Lengend Snippet: Macrophages stimulate hyaluronic acid (HA) production by orbital fibroblasts. (a) Control (COs) and GO-derived (HOs) orbital fibroblasts (0.74 × 10 5 cells/ml) were cultured in collagen gels in serum-free medium with macrophages at different ratios, and HA levels were measured from pooled medium and gel digest after 3 days (mean ± SEM; COs: CO4, CO5, CO6, CO7; HOs: HO1, HO2, HO4, HO6(R); 3–4 independent experiments each). * p < 0.05, ** p < 0.01, *** p < 0.001, Kruskal-Wallis test. Stars directly above HOs bars indicate the comparison between COs and HOs in that fibroblast:macrophage ratio group, Mann-Whitney test. (b) Macrophage-induced HA production is independent of IGF-1R signalling. HO2 cells were cultured with macrophages at 1:2 ratio or in the presence of 20 μM IGF-1, and IGF-1 signalling was blocked by adding IGF-1R blocking antibody 5 μg/ml in medium and 10 μg/ml in gel mix (n = 3, * p < 0.05, Kruskal-Wallis test).

Article Snippet: To confirm the efficiency of the blocking antibody, HA levels were measured in fibroblasts cultured in serum-free medium with 20 μM IGF-1 (R&D Systems, MN, USA) to stimulate HA production, with or without IGF-1R blocking antibody.

Techniques: Control, Derivative Assay, Cell Culture, Comparison, MANN-WHITNEY, Blocking Assay

Journal: Scientific Reports

Article Title: Macrophages promote a profibrotic phenotype in orbital fibroblasts through increased hyaluronic acid production and cell contractility

doi: 10.1038/s41598-019-46075-1

Figure Lengend Snippet: Macrophages promote orbital fibroblasts contractility independently from αSMA levels. (a) Orbital fibroblasts (1.48 × 10 5 cells/ml) were cultured with macrophages (2.22 × 10 5 cells/ml) in serum-free medium for 7 days. Gel contraction is shown as mean ± SEM. Control (COs) and GO (HOs) fibroblasts are each averaged from 3 lines (COs: CO4, CO6, CO7; HOs: HO1, HO2, HO4) with n = 3 and triplicates in each experiment. * p < 0.05, ** p < 0.01, Mann-Whitney test. (b) Representative western blots and quantification of relative αSMA protein levels for the same 3 COs and 3 HOs cell lines after 5~7 days culture in the presence of 10% serum in monolayers (2D) and in gels (3D), and in serum-free medium in gels (SF) (n ≥ 3; ** p < 0.01, Kruskal-Wallis test and post-hoc). Full-length blots are presented in Supplementary Fig. . (c) Representative western blot and quantification of αSMA levels showed effective αSMA knockdown by siRNA in HO2 (GO) orbital fibroblasts (n = 4). Full-length blots are presented in Supplementary Fig. . (d) Downregulating αSMA significantly decreased the contractility of HO2 fibroblasts in the presence of 10% serum (n = 3 and triplicates in each experiment, Day 7 p < 0.001, Mann-Whitney test). (e) Downregulating αSMA did not decrease macrophage-stimulated contraction of HO2 orbital fibroblasts in serum-free medium (n = 4 and triplicates in each experiment, Day 7 p = 0.215, Mann-Whitney test).

Article Snippet: To confirm the efficiency of the blocking antibody, HA levels were measured in fibroblasts cultured in serum-free medium with 20 μM IGF-1 (R&D Systems, MN, USA) to stimulate HA production, with or without IGF-1R blocking antibody.

Techniques: Cell Culture, Control, MANN-WHITNEY, Western Blot, Knockdown

Journal: Scientific Reports

Article Title: Macrophages promote a profibrotic phenotype in orbital fibroblasts through increased hyaluronic acid production and cell contractility

doi: 10.1038/s41598-019-46075-1

Figure Lengend Snippet: Macrophages increased F-actin levels and αSMA incorporation in actin fibers. (a) Representative images of αSMA and F-actin staining in HO2 fibroblasts following 4 days culture in gels in the presence of 10% serum (10%FBS), and in serum-free medium without (SF) and with macrophages (SF + mΦ). Scale bar, 25 µm. Merge images show αSMA (green), F-actin (red), and DAPI (blue). (b) αSMA distribution patterns in HO2 fibroblasts (>100 cells, n = 3). (c , d) αSMA ( c ) and F-actin ( d ) levels in HO2 cells in gels in the presence of 10% serum, and in serum-free medium with/without macrophages; shown are fluorescence intensity measurements from maximum intensity projections normalized to the value of serum-free condition (n = 4, * p < 0.05, Mann-Whitney test).

Article Snippet: To confirm the efficiency of the blocking antibody, HA levels were measured in fibroblasts cultured in serum-free medium with 20 μM IGF-1 (R&D Systems, MN, USA) to stimulate HA production, with or without IGF-1R blocking antibody.

Techniques: Staining, Fluorescence, MANN-WHITNEY

Journal: Scientific Reports

Article Title: Macrophages promote a profibrotic phenotype in orbital fibroblasts through increased hyaluronic acid production and cell contractility

doi: 10.1038/s41598-019-46075-1

Figure Lengend Snippet: Macrophages stimulate protrusive activity of orbital fibroblasts. (a) Representative images of F-actin staining of HO2 cells in gels without (left) and with (right) macrophages in serum-free medium. Shown are maximal intensity projections of deconvolved image stacks. Scale bar, 50 µm. (b) Fibroblast protrusive activity with/without macrophage co-culture: shown is the average number of protrusions per cell, as determined using F-actin staining (n = 4, * p < 0.05, Mann-Whitney test).

Article Snippet: To confirm the efficiency of the blocking antibody, HA levels were measured in fibroblasts cultured in serum-free medium with 20 μM IGF-1 (R&D Systems, MN, USA) to stimulate HA production, with or without IGF-1R blocking antibody.

Techniques: Activity Assay, Staining, Co-Culture Assay, MANN-WHITNEY

Journal: Scientific Reports

Article Title: Macrophages promote a profibrotic phenotype in orbital fibroblasts through increased hyaluronic acid production and cell contractility

doi: 10.1038/s41598-019-46075-1

Figure Lengend Snippet: TGF-β and PI3K pathways differentially regulate macrophages’ effect on orbital fibroblasts fibrotic phenotype. (a) Representative images of ORO-stained HO2 fibroblasts (0.74 × 10 5 cells/ml) co-cultured with macrophages (0.74 × 10 5 cells/ml) in 3D for 7 days in the presence of DMSO control (0.1% DMSO), TGF-β (SB431542, 10 μM) and PI3K (LY294002, 10 μM) inhibitors. (Scale bar, 25 μm). (b) TGF-β (SB431542, 10 μM) and PI3K (LY294002, 10 μM) inhibitors did not alter lipid vesicle formation in HO2 cells co-cultured with macrophages. HO2 fibroblasts (0.74 × 10 5 cells/ml) were cultured in gels with macrophages (0.74 × 10 5 cells/ml) for 7 days and the proportion of cells containing lipid vesicles was counted after Oil-Red-O staining (Mean ± SEM, n = 4, p = 0.343 (SB131542) and p = 0.2 (LY294002), Mann-Whitney test). (c) TGF-β and PI3K inhibition prevented macrophage-mediated HA production. HO2 fibroblasts (0.74 × 10 5 cells/ml) were co-cultured with macrophages at 1:2 fibroblast:macrophage ratio in gels with/without inhibitors and HA levels were measured after 3 days (n = 3, * p < 0.05, ** p < 0.01, Kruskal-Wallis test and post-hoc). (d) LDH assay showed minimal cytotoxicity of SB131542 and LY294002 after 3 days in 1:2 HO2:macrophage co-culture 3D gels. Shown is calculated toxicity normalized to 100% toxicity control (n = 2). (e) HO2 cells (1.48 × 10 5 cells/ml) were co-cultured with macrophages (2.22 × 10 5 cells/mL) in collagen gels in serum-free medium for 7 days for contraction assay, with/without TGF-β or PI3K inhibitors. (Mean ± SEM, n = 4, * p < 0.05, Mann-Whitney test).

Article Snippet: To confirm the efficiency of the blocking antibody, HA levels were measured in fibroblasts cultured in serum-free medium with 20 μM IGF-1 (R&D Systems, MN, USA) to stimulate HA production, with or without IGF-1R blocking antibody.

Techniques: Staining, Cell Culture, Control, MANN-WHITNEY, Inhibition, Lactate Dehydrogenase Assay, Co-Culture Assay, Contraction Assay

Journal: Frontiers in Veterinary Science

Article Title: Life-Threatening Mediastinal Hematoma Formation After Removal of the Hemodialysis Catheter in a Boxer: A Case Report

doi: 10.3389/fvets.2021.691472

Figure Lengend Snippet: Bedside ultrasound of the thorax. Left paracostal view of a 4-year old female Boxer with mediastinal hematoma. Located dorsally to the heart, a hyperechoic well-demarcated intrathoracic structure is pictured.

Article Snippet: The bedside ultrasound (TE7 ACE, Mindray Medical Germany GmbH, Darmstadt, DE) of the chest ( ) revealed moderate hypovolemia as well as a hyperechoic well-demarcated intrathoracic structure, located dorsally of the heart.

Techniques:

Journal: Cell metabolism

Article Title: Human skeletal muscle CD90 + fibro-adipogenic progenitors are associated with muscle degeneration in type 2 diabetic patients

doi: 10.1016/j.cmet.2021.10.001

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: TE-7 (mouse) , Merck Millipore , Cat# CBL271, RRID:AB_93449.

Techniques: Recombinant, Blocking Assay, Staining, Imaging, Sample Prep, Software, Flow Cytometry, RNA Sequencing Assay, Expressing

Journal: Cell metabolism

Article Title: Human skeletal muscle CD90 + fibro-adipogenic progenitors are associated with muscle degeneration in type 2 diabetic patients

doi: 10.1016/j.cmet.2021.10.001

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: TE-7 (mouse) , Merck Millipore , Cat# CBL271, RRID:AB_93449.

Techniques: Recombinant, Blocking Assay, Staining, Imaging, Sample Prep, Software, Flow Cytometry, RNA Sequencing Assay, Expressing

Journal: JCI Insight

Article Title: Synaptopodin is upregulated by IL-13 in eosinophilic esophagitis and regulates esophageal epithelial cell motility and barrier integrity

doi: 10.1172/jci.insight.96789

Figure Lengend Snippet: (A) Schematic structure of the SYNPO isoforms; black and white rectangles represent exons and introns, respectively. SYNPO-long and SYNPO-short represent the common protein isoforms 1 and 2, respectively. The SYNPO regions detected by RT-PCR are indicated by dotted lines; SYNPO 1–2 and SYNPO 3–4 represent common PCR products for indicated isoforms. (B) An image capture from the USCS browser shows aligned RNA-sequencing reads on the SYNPO gene in TE-7 cells either untreated or treated with IL-13 for 24 hours. Black rectangles represent exons. The indicated P value comparing expression under these conditions was calculated by DESeq analysis (59). Note that reads are detected only on the exons corresponding to SYNPO-long and SYNPO-short isoforms. (C) Expression of SYNPO isoforms by TE-7 cells induced by IL-13 was assessed by RT-PCR. *P < 0.05, t test. (D) Expression of SYNPO was assessed by RNA sequencing in TE-7 cells subjected to gene silencing control (scrambled) and STAT6 knockdown (STAT6KD) pool. SYNPO expression in RPKM is shown. ****P < 0.0001 for 24-hour time point, DESeq analysis. (E) An image capture from the USCS browser shows aligned RNA-sequencing reads on the SYNPO gene in control TE-7 cells and STAT6KD pool from D. Note that both isoforms are affected by the knockdown. The indicated P value comparing expression under these conditions was calculated by DESeq analysis (59). (F) Expression of SYNPO isoforms (mean ± SD) was assessed by RT-PCR. ****P < 0.001, Holm-Sidak method. (G) Expression of SYNPO was analyzed in primary esophageal epithelial cells stimulated with IL-13. Combined data from cells isolated from 3 independent biopsies are shown. *P < 0.05, t test. For C, F, and G, expression was normalized to expression of GAPDH. For box-and-whisker plots, the box represents 50th percentile of the data, whiskers show minimum and maximum values, and the line in the box represents the median. For B and E, the read depth is of the same scale within each panel.

Article Snippet: The squamous esophageal epithelial cell line TE-7 (a gift from Pierre Hainaut, Institute for Advanced Biosciences, INSERM 1209, UMR CNRS 5309, Université Grenoble-Alpes, Grenoble, France), which was originally selected from human esophageal tumors ( 57 ), was maintained in RPMI-1640 media (Invitrogen) supplemented with 5% FCS.

Techniques: Reverse Transcription Polymerase Chain Reaction, RNA Sequencing, Expressing, Control, Knockdown, Isolation, Whisker Assay

Journal: JCI Insight

Article Title: Synaptopodin is upregulated by IL-13 in eosinophilic esophagitis and regulates esophageal epithelial cell motility and barrier integrity

doi: 10.1172/jci.insight.96789

Figure Lengend Snippet: (A) Representative immunofluorescence images of SYNPO (green) in the esophageal epithelial TE-7 cell line. (B) Primary esophageal epithelial cells isolated from biopsies. DNA was labeled by Hoechst (blue), and actin was stained with phalloidin (red). The merged image shows partial colocalization of SYNPO and actin (yellow). Original magnification: ×60 (A); ×40 (B); scale bars: 10 μM.

Article Snippet: The squamous esophageal epithelial cell line TE-7 (a gift from Pierre Hainaut, Institute for Advanced Biosciences, INSERM 1209, UMR CNRS 5309, Université Grenoble-Alpes, Grenoble, France), which was originally selected from human esophageal tumors ( 57 ), was maintained in RPMI-1640 media (Invitrogen) supplemented with 5% FCS.

Techniques: Immunofluorescence, Isolation, Labeling, Staining

Journal: JCI Insight

Article Title: Synaptopodin is upregulated by IL-13 in eosinophilic esophagitis and regulates esophageal epithelial cell motility and barrier integrity

doi: 10.1172/jci.insight.96789

Figure Lengend Snippet: Under normal conditions (NL) esophageal epithelium is characterized by strong intercellular contacts, and barrier integrity is preserved. In the homeostatic esophagus, SYNPO is expressed at the basal layer of the esophageal epithelium and interacts with actin filaments. SYNPO is transcribed at low levels, and its promoter is decorated by low levels of activating epigenetic marks. In EoE, IL-13 is secreted by tissue-resident lymphocytes, causing STAT-6–dependent transcriptional activation of SYNPO in esophageal epithelium accompanied by elevated levels of activating epigenetic marks in the promoter of SYNPO gene. Basal zone hyperplasia and expansion of SYNPO expression into the suprabasal layers of the esophagus are evident. Subsequently, epithelial barrier integrity is altered and eosinophils penetrate esophageal mucosa. Treatment of EoE patients with anti–IL-13 antibody or knocking down STAT6 in esophageal epithelial cells counteracts IL-13–dependent transcriptional and epigenetic changes of SYNPO. Note that cell nuclei are omitted for clarity.

Article Snippet: The squamous esophageal epithelial cell line TE-7 (a gift from Pierre Hainaut, Institute for Advanced Biosciences, INSERM 1209, UMR CNRS 5309, Université Grenoble-Alpes, Grenoble, France), which was originally selected from human esophageal tumors ( 57 ), was maintained in RPMI-1640 media (Invitrogen) supplemented with 5% FCS.

Techniques: Activation Assay, Expressing