Journal: European journal of immunology

Article Title: Sca-1 + cardiac fibroblasts promote development of heart failure

doi: 10.1002/eji.201847583

Figure Lengend Snippet: Sca-1+ cardiac fibroblasts are potent GM-CSF-producers during cardiac inflammation. Cardiac fibroblasts were harvested from wild type BALB/c mice with EAM on days 0, 14 or 21 for flow cytometry analysis. (A) Representative flow cytometry plots depict gating strategies for cardiac fibroblast subsets. (B) Flow cytometric quantification of the numbers of cardiac fibroblast subsets per mg cardiac tissue at different time points after EAM induction. (n = 4 for EAM day 0 group; n = 6 for EAM day 14 group; n = 7 for EAM day 21 group; CF cardiac fibroblast) (C) Demonstration of cell number changes in cardiac fibroblast subsets after EAM induction. (D) Representative flow cytometry= plots showing GM-CSF staining of cardiac fibroblast subsets on day 21 of EAM. (E and F) Representative t-SNE clustering plots of (E) cardiac fibroblast subsets and GM-CSF-positive cardiac fibroblasts or (F) all GM-CSF-positive cardiac cells on day 21 of EAM using t-SNE dimensionality reduction algorithm plugin in FlowJo 10.4.2 (FlowJo, LLC). (G) Relative frequency of different cell populations out of all GM-CSF-positive cells in the hearts on day 21 of EAM. (H) Frequency and number of GM-CSF-positive Sca-1+ cardiac fibroblasts per mg cardiac tissue in immunized mice (n = 9) versus mock-immunized mice (n = 10) on day 21 of EAM. (I) Frequency and number of GM-CSF-positive Sca-1+ cardiac fibroblasts per mg cardiac tissue in immunized WT mice and Il17ra−/− mice on day 21 of EAM (n = 10 per group). Data are representative of three independent experiments (A–I). Groups were compared using one-way ANOVA followed by Tukey’s post-test (B) or Student’s t-test (H–I). *p < 0.05; **p < 0.01; ***p < 0.001.

Article Snippet: Human primary cardiac fibroblasts We acquired human primary cardiac fibroblasts from Cell Applications, Inc (San Diego, CA).

Techniques: Flow Cytometry, Staining

Journal: European journal of immunology

Article Title: Sca-1 + cardiac fibroblasts promote development of heart failure

doi: 10.1002/eji.201847583

Figure Lengend Snippet: Sca-1+ cardiac fibroblasts are potent producers of different cytokines and chemokines and are plastic in response to environment changes. (A) The mRNA expression levels of Csf2, Ccl2, Cxcl10, Ccl11 and Postn in na¨ıve mouse heart homogenates, EAM day 21 mouse heart homogenates and FACS-sorted Sca-1+ and Sca-1− cardiac fibroblasts from EAM day 21 mouse hearts (n =5 per group; n.d. not detected; WH = whole heart homogenate). Expression levels were analyzed by qPCR (2− ΔΔ Ct values relative to Gapdh expression levels and=na¨ıve whole heart transcripts). (B) The mRNA expression levels of Csf2, Ccl2, Cxcl10 and Ccl11 in in vitro cultured Sca-1+ cardiac fibroblasts stimulated with IL-17A, IL-13, IFN-γ, or unstimulated (control). Expression levels were analyzed by qPCR (2−ΔΔCt values relative to Gapdh expression levels and unstimulated controls). (C) Representative histogram of mCherry (CCL2) expression in cultured Sca-1+ cardiac fibroblasts under Th17 conditions or unstimulated. (D) Schematic of the experiment procedures for all data showing in E–F. (E) Expression levels of Ccl2, Csf2 and Ccl11 mRNA in cells harvested on day 3 or day 6. Expression levels were analyzed by qPCR (2−͉ ΔΔCt values relative to Gapdh expression levels and unstimulated controls). (F) Protein concentrations of secreted CCL11 and GM-CSF in the culture supernatant harvested on day 3 or day 6 were measured by quantitative sandwich ELISA. Data are shown as mean + SD of five mice per group (A) or of technical triplicates (B, E–F). Data are representative of three indepen dent experiments (A–F). Groups were compared using one-way ANOVA followed by Tukey’s post-test (A, E–F) or Student’s t-test (B). **p < 0.01; ***p < 0.001.

Article Snippet: Human primary cardiac fibroblasts We acquired human primary cardiac fibroblasts from Cell Applications, Inc (San Diego, CA).

Techniques: Expressing, In Vitro, Cell Culture, Control, Sandwich ELISA

Journal: European journal of immunology

Article Title: Sca-1 + cardiac fibroblasts promote development of heart failure

doi: 10.1002/eji.201847583

Figure Lengend Snippet: IL-17A induces GM-CSF production in Sca-1+ cardiac fibroblasts through synergistic activation of NF-κB and NFAT2. (A–C) Sca-1+ cardiac fibroblasts were MACS-sorted from naïve WT mouse hearts and cultured under Th17 conditions (IL-17A TNF-α), Th2 conditions (IL-13) or unstimulated (control) for two days (A) or for multiple time points (B–C). (A) Representative flow cytometry plots showing phosphorylated p-65 (pp65)-positive and/or NFAT2-positive Sca-1+ cardiac fibroblasts and representative histogram showing their GM-CSF expression. (B) Immunoblots showing the detection of total or phosphorylated Stat6, Stat3, p65 and Erk1/2 in whole cell lysate. (C) Immunoblots showing the detection of p65, Stat6, Parp1 (nuclear positive control) and pro-Caspase 3 (nuclear negative control) in the nuclear fraction of Sca-1 cardiac fibroblasts with/without stimulation. Data are representative of three independent experiments (A–C).

Article Snippet: Human primary cardiac fibroblasts We acquired human primary cardiac fibroblasts from Cell Applications, Inc (San Diego, CA).

Techniques: Activation Assay, Cell Culture, Control, Flow Cytometry, Expressing, Western Blot, Positive Control, Negative Control

Journal: European journal of immunology

Article Title: Sca-1 + cardiac fibroblasts promote development of heart failure

doi: 10.1002/eji.201847583

Figure Lengend Snippet: IL-17A signaling ablation leads to less GM-CSF-positive Sca-1+ cardiac fibroblasts and protects mice from post-MI heart failure and death. (A) Representative flow cytometry plots showing GM-CSF- and CCL2-positive Sca-1+ cardiac fibroblasts in infarcted and sham-operated mouse hearts on day 2 post-MI. Cells were pre-gated on viable CD45−CD31−Sca-1+ cardiac cells. (B) Flow cytometric quantification of GM-CSF+, CCL2+ and GM-CSF+CCL2+ Sca-1+ cardiac fibroblasts per mg cardiac tissue of infarcted or sham-operated WT mice (n =3 per group). (C) Kaplan-Meier survival curve of WT mice (n = 8) and Il17ra−/− mice (n = 17) on day 28 post-MI. (D) images Representative of Masson’s trichrome stained histology slides of the cross-sections at the middle of the hearts from WT and Il17ra−/− mice showing the expansion of the infarct area and the dilation of the left ventricles on day 28 post-MI. (Scalebar, 1mm) (E) Calculated total infarct sizes of WT mice (n = 6) and Il17ra−/− mice (n 14) on day 28 post-MI. (F) Flow cytometric quantification of GM-CSF-positive Sca-1+ cardiac fibroblasts per mg cardiac tissue in WT and Il17ra−/−=mice on day 2 post-MI (n = 5 per group). Data are representative of three independent experiments (A–F). Groups were compared using Student’s t-test (B, E–F) or Long-rank (Mantel-Cox) test (C). *p < 0.05; **p < 0.01.

Article Snippet: Human primary cardiac fibroblasts We acquired human primary cardiac fibroblasts from Cell Applications, Inc (San Diego, CA).

Techniques: Flow Cytometry, Staining

Journal: European journal of immunology

Article Title: Sca-1 + cardiac fibroblasts promote development of heart failure

doi: 10.1002/eji.201847583

Figure Lengend Snippet: GM-CSF-positive Sca-1+ cardiac fibroblasts express periostin post-MI and specific IL-17RA ablation in periostin+ cardiac fibroblasts protects mice from post-MI heart failure and death. (A) Representative flow cytometry plots showing Sca-1+ and GM-CSF+Sca-1+ cardiac fibroblasts and representative histograms of their GFP (periostin) expression in infarcted WT mouse hearts (gray shadowed), sham (black dashed line) and infarcted (black solid line) PostnCre/eGFP mouse hearts on day 2 post-MI. Cells were pre-gated on viable CD45−CD31−CD29+ cells. (B) Representative images of Masson’s trichrome stained histology slides of the cross-sections at the middle of the hearts from sham and infarcted PostnCre, infarcted Il17rafl/fl and infarcted PostnCreIl17rafl/fl mice on day 7 post-MI (PostnCreIl17rafl/fl mice were labeled in this Figure as PostnIL−17RAKO) showing the expansion of the infarct area and the dilation of the left ventricle of the infarcted mouse hearts. (Scalebar, 1mm) (C) Calculated total infarct sizes of infarcted Il17rafl/fl mice (n = 8) and PostnCreIl17rafl/fl mice (n =9) on day 7 post-MI. (D) Kaplan-Meier survival analysis of infarcted Il17rafl/fl mice (n = 31) and PostnCreIl17rafl/fl mice (n = 11) on day 28 post-MI. (E) Flow cytometric quantification of GM-CSF-positive Sca-1+ cardiac fibroblasts per mg cardiac tissue in PostnCre and PostnCreIl17rafl/fl mice on day 4 post-MI (n = 3 per group). (F-G) Flow cytometric quantification of the frequency (F) and the number (G) of Ly6Chi monocytes per mg cardiac tissue in PostnCre and PostnCreIl17rafl/fl mice on day 4 post-MI (n = 3 per group). “PostnIL−17RAKO” stands for “PostnCreIl17rafl/fl mice”. “CFs” stands for “cardiac fibroblasts”. Data are representative of three independent = experiments (A–G). Groups were compared using Student’s t-test (C, E–G) or Long-rank (Mantel-Cox) test (D). *p < 0.05, **p < 0.01.

Article Snippet: Human primary cardiac fibroblasts We acquired human primary cardiac fibroblasts from Cell Applications, Inc (San Diego, CA).

Techniques: Flow Cytometry, Expressing, Staining, Labeling

Journal: European journal of immunology

Article Title: Sca-1 + cardiac fibroblasts promote development of heart failure

doi: 10.1002/eji.201847583

Figure Lengend Snippet: GM-CSF-expressing cardiac fibroblast subset was found in human cardiac fibroblasts and exhibits similar functional plasticity. (A) Representative flow cytometry plots showing the gating strategies for cultured human primary cardiac fibroblasts. (B) Representative t-SNE clustering plots of three human cardiac fibroblast subsets using t-SNE dimensionality reduction algorithm plugin in FlowJo 10.4.2 (FlowJo, LLC). (C) Representative expression heat maps of cell surface markers, CD29, CD73, CD105, CD90, PDGFRα, cKit, DDR2 and GM-CSF expression in human cardiac fibroblasts using t-SNE dimensionality reduction algorithm plugin in FlowJo 10.4.2 (FlowJo, LLC). (D–E) The mRNA expression levels of CCL2, CSF2, CCL11, CCL24 and CCL26 of human primary cardiac fibroblasts cultured under IL-17A, IL-13 or unstimulated (control) for three days (D) or washed and recultured again with/without change of stimuli for another three days (E). The mRNA expression levels were analyzed by qPCR (2−ΔΔ Ct values relative to HPRT1 expression levels and unstimulated controls). Data are shown as mean SD of technical triplicates and are representative of three independent experiments (D–E). Groups were compared using Student’s t test (D) or +using one-way ANOVA followed by Tukey’s post-test (E). **p < 0.01; ***p < 0.001.

Article Snippet: Human primary cardiac fibroblasts We acquired human primary cardiac fibroblasts from Cell Applications, Inc (San Diego, CA).

Techniques: Expressing, Functional Assay, Flow Cytometry, Cell Culture, Control

Journal: European journal of immunology

Article Title: Sca-1 + cardiac fibroblasts promote development of heart failure

doi: 10.1002/eji.201847583

Figure Lengend Snippet: Human cardiac fibroblasts from heart failure patients produce GM-CSF and CCL2. (A) Representative flow cytometry plots of the concatenated sample showing gating strategy for human CD45−CD31−CD29+PDGFRα+ cardiac fibroblasts and flow cytometry plots showing GMCSF and CCL2 expressions for PDGFRα+ and PDGFRα− cardiac fibroblasts from the heart biopsies of myocarditis or ischemic heart failure patients. (B) Representative flow cytometry plots showing GM-CSF and CCL2 expression by PDGFRα+ cardiac fibroblasts from heart biopsies of five myocarditis heart failure patients. (C) Representative flow cytometry plots showing GM-CSF and CCL2 expression by PDGFRα+ cardiac fibroblasts from heart biopsies of five ischemic heart failure patients. Data are from 5 myocarditis heart failure patients and 5 ischemic heart failure patients.

Article Snippet: Human primary cardiac fibroblasts We acquired human primary cardiac fibroblasts from Cell Applications, Inc (San Diego, CA).

Techniques: Flow Cytometry, Expressing

Journal: International Journal of Ophthalmology

Article Title: Effect of zymosan on the expression and function of the gap-junction protein connexin 43 in human corneal fibroblasts

doi: 10.18240/ijo.2021.03.02

Figure Lengend Snippet: A: After exposure of HCFs to various concentrations of zymosan, the expression of Cx43 was examined by Western blot analysis; B: The immunoblots were subjected to densitometric analysis in order to determine the band intensity. The error bars represent the standard deviation. aP<0.05 (Dunnett's multiple comparison test) versus the control (no zymosan).

Article Snippet: Culture of Human Corneal Fibroblasts HCFs were purchased from ScienCell Research Laboratories (cat. no. #6520; Carlsbad, CA, USA) and cultured in MEM containing 10% FBS (5% CO 2 , 37°C).

Techniques: Expressing, Western Blot, Standard Deviation, Comparison, Control

Journal: International Journal of Ophthalmology

Article Title: Effect of zymosan on the expression and function of the gap-junction protein connexin 43 in human corneal fibroblasts

doi: 10.18240/ijo.2021.03.02

Figure Lengend Snippet: A: After the exposure of HCFs to zymosan (600 µg/mL) for the indicated time, the expression of Cx43 was examined by Western blot analysis; B: The immunoblots were subjected to densitometric analysis in order to determine the band intensity. The error bars represent the standard deviation. aP<0.05 (Dunnett's multiple comparison test) versus the control (no zymosan).

Article Snippet: Culture of Human Corneal Fibroblasts HCFs were purchased from ScienCell Research Laboratories (cat. no. #6520; Carlsbad, CA, USA) and cultured in MEM containing 10% FBS (5% CO 2 , 37°C).

Techniques: Expressing, Western Blot, Standard Deviation, Comparison, Control

Journal: International Journal of Ophthalmology

Article Title: Effect of zymosan on the expression and function of the gap-junction protein connexin 43 in human corneal fibroblasts

doi: 10.18240/ijo.2021.03.02

Figure Lengend Snippet: After HCFs were incubated with or without zymosan (600 µg/mL) for 24h, the Cx43 mRNA level in the cells was then determined by qRT-PCR analysis. The error bars represent the standard deviation. aP<0.05 (Student's t-test) versus the control (no zymosan).

Article Snippet: Culture of Human Corneal Fibroblasts HCFs were purchased from ScienCell Research Laboratories (cat. no. #6520; Carlsbad, CA, USA) and cultured in MEM containing 10% FBS (5% CO 2 , 37°C).

Techniques: Incubation, Quantitative RT-PCR, Standard Deviation, Control

Journal: International Journal of Ophthalmology

Article Title: Effect of zymosan on the expression and function of the gap-junction protein connexin 43 in human corneal fibroblasts

doi: 10.18240/ijo.2021.03.02

Figure Lengend Snippet: A: After HCFs were treated with PD98059, SB203580, or JNK inhibitor II (at 1, 3, and 10 µmol/L), respectively, in the absence or presence of zymosan (600 µg/mL), the expression of Cx43 was then examined by Western blot analysis; B: Densitometric analysis was performed for the immunoblots to determine the band intensity. The error bars represent the standard deviation. aP<0.05 (Dunnett's test) versus the control (no zymosan); bP<0.05 (Dunnett's test) versus the corresponding control (zymosan only).

Article Snippet: Culture of Human Corneal Fibroblasts HCFs were purchased from ScienCell Research Laboratories (cat. no. #6520; Carlsbad, CA, USA) and cultured in MEM containing 10% FBS (5% CO 2 , 37°C).

Techniques: Expressing, Western Blot, Standard Deviation, Control

Journal: International Journal of Ophthalmology

Article Title: Effect of zymosan on the expression and function of the gap-junction protein connexin 43 in human corneal fibroblasts

doi: 10.18240/ijo.2021.03.02

Figure Lengend Snippet: A: HCFs were treated with IKK2 inhibitor IV (1, 3, and 10 µmol/L) in the absence or presence of zymosan (600 µg/mL). The expression of Cx43 was then examined by Western blot analysis. B: The immunoblots were subjected to densitometric analysis in order to determine the band intensity. The error bars represent the standard deviation. aP<0.05 (Dunnett's test) versus the control (no zymosan). bP<0.05 (Dunnett's test) versus the corresponding control (zymosan only).

Article Snippet: Culture of Human Corneal Fibroblasts HCFs were purchased from ScienCell Research Laboratories (cat. no. #6520; Carlsbad, CA, USA) and cultured in MEM containing 10% FBS (5% CO 2 , 37°C).

Techniques: Expressing, Western Blot, Standard Deviation, Control

Journal: International Journal of Ophthalmology

Article Title: Effect of zymosan on the expression and function of the gap-junction protein connexin 43 in human corneal fibroblasts

doi: 10.18240/ijo.2021.03.02

Figure Lengend Snippet: HCFs were treated with the ERK inhibitor PD98059, JNK inhibitor II, or IKK2 inhibitor IV (10 µmol/L), respectively, in the absence or presence of zymosan (600 µg/mL). A scrape-loading assay with Lucifer yellow was used to measure the GJIC activity. A: Representative fluorescence microscopy images of fixed cells from different treatments (scale bar, 100 µm); B: The maximum distances from the scrape to dye fluorescence were quantified to show the GJIC activity. The error bars represent the standard deviation. aP<0.05 (Dunnett's test) versus the control (no zymosan). bP<0.05 (Dunnett's test) versus the corresponding control (zymosan only).

Article Snippet: Culture of Human Corneal Fibroblasts HCFs were purchased from ScienCell Research Laboratories (cat. no. #6520; Carlsbad, CA, USA) and cultured in MEM containing 10% FBS (5% CO 2 , 37°C).

Techniques: Activity Assay, Fluorescence, Microscopy, Standard Deviation, Control