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: iScience

Article Title: MicroRNA-452-5p regulates fibrogenesis via targeting TGF-β/SMAD4 axis in SCN5A-knockdown human cardiac fibroblasts

doi: 10.1016/j.isci.2024.110084

Figure Lengend Snippet: SCN5A knockdown promotes the expression of fibrogenic signaling (A) Upper panel: Knockdown of SCN5A gene in human cardiac fibroblasts by targeting SCN5A gene using shRNA lentivirus. Lower panel : fibroblast grown after SCN5A gene knockdown and morphology analyzed microscopically, Scale bar 350 μm (representative pictures shown). (B) The protein expression of Nav1.5 after the knockdown of the SCN5A gene in HCF represents a 50% decrease in Nav1.5 protein expression. Data are expressed as mean ± SEM, paired t-test, ∗∗ p < 0.01, n = 5 independent experiments. (C) Representatives immunoblot and quantitative analysis showing the expression of pro-Col1agen 1A1, α-SMA, and fibronectin in SCN5A knockdown and control HCF normalized with the internal control group. Data are expressed as mean ± SEM, paired t-test, ∗∗ p < 0.01, ∗∗∗ p < 0.001, n = 6 independent experiments. (D) There were higher soluble collagen-type 1 levels measured in a conditioned medium (serum-free) of SCN5A knockdown HCF than that from control cells Data are expressed as mean ± SEM, paired t-test, ∗∗∗ p < 0.001, n = 6 independent experiments. SCN5A shRNA: SCN5A knockdown HCF.

Article Snippet: The immortalized human cardiac fibroblasts (HCF, male, Innoprot, Derio, Spain) were cultured in fibroblast medium 2 (FM-2) with 10% heat-inactivated fetal bovine serum and antibiotics (penicillin 100 U/mL and streptomycin 100 μg/mL) in a 37°C incubator with 5% CO 2 .

Techniques: Expressing, shRNA, Western Blot

Journal: iScience

Article Title: MicroRNA-452-5p regulates fibrogenesis via targeting TGF-β/SMAD4 axis in SCN5A-knockdown human cardiac fibroblasts

doi: 10.1016/j.isci.2024.110084

Figure Lengend Snippet: The proposed mechanism of shielding potential of miR-452-5p in cardiac fibroblasts against SCN5A knockdown escalated cardiac fibrogenesis The expression level of miR-452-5p in normal HCF serves to restrain the activity of SMAD4 protein by binding to the 3ˊUTR of SMAD4 mRNA, therefore limiting its activity. However, in the case of SCN5A knockdown, the quantity of miR-452-5p is considerably reduced, impairing its capacity to bind with 3′UTR of SMAD4 mRNA and thereby increasing SMAD4 activity. The increase in SMAD4 activity causes the TGF-β to be overexpressed which activates the canonical TGF-β signaling pathway by increasing the phosphorylation of downstream signal transducers SMAD2 and SMAD3. Following phosphorylation, these form a heterodimer with SMAD4 and translocate into the nucleus, where they increase the expression of fibrogenesis-related genes such as pro-Collagen 1A1, fibronectin, and fibroblasts differentiation by overexpressing α-SMA, and increased cell migration leading to cumulative effect on fibrogenesis in SCN5A knockdown condition.

Article Snippet: The immortalized human cardiac fibroblasts (HCF, male, Innoprot, Derio, Spain) were cultured in fibroblast medium 2 (FM-2) with 10% heat-inactivated fetal bovine serum and antibiotics (penicillin 100 U/mL and streptomycin 100 μg/mL) in a 37°C incubator with 5% CO 2 .

Techniques: Expressing, Activity Assay, Protein Binding, Migration

Journal: iScience

Article Title: MicroRNA-452-5p regulates fibrogenesis via targeting TGF-β/SMAD4 axis in SCN5A-knockdown human cardiac fibroblasts

doi: 10.1016/j.isci.2024.110084

Figure Lengend Snippet:

Article Snippet: The immortalized human cardiac fibroblasts (HCF, male, Innoprot, Derio, Spain) were cultured in fibroblast medium 2 (FM-2) with 10% heat-inactivated fetal bovine serum and antibiotics (penicillin 100 U/mL and streptomycin 100 μg/mL) in a 37°C incubator with 5% CO 2 .

Techniques: Virus, Recombinant, Transfection, Membrane, Western Blot, Protein Extraction, Reverse Transcription, Qubit Protein Assay, Collagen Assay, Enzyme-linked Immunosorbent Assay, Extraction, Plasmid Preparation, Software, RNA Sequencing Assay

Journal: International Journal of Molecular Medicine

Article Title: Atorvastatin attenuates TGF-β1-induced fibrogenesis by inhibiting Smad3 and MAPK signaling in human ventricular fibroblasts

doi: 10.3892/ijmm.2020.4607

Figure Lengend Snippet: Effect of ATV on TGF-β1-induced proliferation of hVFs. (A) Cells were exposed to different concentrations of ATV, and then the viability of the cells was assessed using a CCK-8 assay. (B) hVFs were pretreated with various concentrations of ATV (0, 2, 5 and 10 µ M) for 3 h, and then exposed to 5 ng/ml TGF-β1 for 24 h. The proliferation of hVFs was then assessed by CCK-8 assay. All data are presented as the mean ± standard error of mean of three independent experiments and described as a percentage of the control group. * P<0.05, ** P<0.01 vs. untreated control group. # P<0.05 vs. TGF-β1 only-treated group. hVFs, human ventricular fibroblasts; ATV, atorvastatin; TGF-β1, transforming growth factor-β1; NS, not significant; CCK-8, Cell Counting Kit-8.

Article Snippet: Adult hVFs (cat. no. 6310) and complete medium for their culture (fibroblast medium-2; FM-2; cat. no. 2331) were obtained from ScienCell Research Laboratories, Inc. Antibodies against α-SMA, matrix metal-loproteinase-2 (MMP-2), collagen I and collagen III were purchased from ProteinTech Group, Inc. Antibodies against phosphorylated (p)-Smad3, total (t)-Smad3, p-ERK1/2, t-ERK1/2, p-JNK, t-JNK, p-p38 and t-p38 were purchased from Cell Signaling Technology, Inc. Anti-GAPDH antibody and Cell Counting Kit-8 (CCK-8) were purchased from Beyotime Institute of Biotechnology.

Techniques: CCK-8 Assay, Control, Cell Counting

Journal: International Journal of Molecular Medicine

Article Title: Atorvastatin attenuates TGF-β1-induced fibrogenesis by inhibiting Smad3 and MAPK signaling in human ventricular fibroblasts

doi: 10.3892/ijmm.2020.4607

Figure Lengend Snippet: ATV inhibits the trans-differentiation of hVFs into myofibroblasts. Cells were exposed to ATV (10 µ M) for 3 h and then co-treated with 5 ng/ml TGF-β1 for a further 24 h. (A) Cells were then immunostained with anti-α-SMA antibody (green) and stained with DAPI (nuclei; blue). Magnification, ×200. (B) The α-SMA mRNA level was assessed by reverse transcription-quantitative PCR assay. (C) The protein expression of α-SMA was measured by western blotting. (D) Quantitative analysis of α-SMA protein level in hVFs. The relative value was normalized to GAPDH expression. The data are presented as the mean ± standard error of mean of three independent experiments. ** P<0.01, *** P<0.001 vs. untreated control group. # P<0.05 vs. TGF-β1 only-treated group. ATV, atorvastatin; TGF-β1, transforming growth factor-β1; hVFs, human ventricular fibroblasts; α-SMA, α-smooth muscle actin.

Article Snippet: Adult hVFs (cat. no. 6310) and complete medium for their culture (fibroblast medium-2; FM-2; cat. no. 2331) were obtained from ScienCell Research Laboratories, Inc. Antibodies against α-SMA, matrix metal-loproteinase-2 (MMP-2), collagen I and collagen III were purchased from ProteinTech Group, Inc. Antibodies against phosphorylated (p)-Smad3, total (t)-Smad3, p-ERK1/2, t-ERK1/2, p-JNK, t-JNK, p-p38 and t-p38 were purchased from Cell Signaling Technology, Inc. Anti-GAPDH antibody and Cell Counting Kit-8 (CCK-8) were purchased from Beyotime Institute of Biotechnology.

Techniques: Staining, Reverse Transcription, Real-time Polymerase Chain Reaction, Expressing, Western Blot, Control

Journal: International Journal of Molecular Medicine

Article Title: Atorvastatin attenuates TGF-β1-induced fibrogenesis by inhibiting Smad3 and MAPK signaling in human ventricular fibroblasts

doi: 10.3892/ijmm.2020.4607

Figure Lengend Snippet: ATV inhibits TGF-β1-stimulated ECM production in hVFs. Fibroblasts were pretreated with ATV (10 µ M) for 3 h and then co-stimulated with 5 ng/ml TGF-β1 for a further 24 h. (A) The mRNA levels of MMP-2, type I collagen and type III collagen were assessed by reverse transcription-quantitative PCR. (B) The protein expression levels of MMP-2, collagen I and collagen III were detected by western blotting. (C) Quantitative analysis of the western blot results was performed by evaluating the protein band densities with Image Pro-Plus version 6.0 software. The relative value was normalized to GAPDH expression. All data are expressed as the mean ± standard error of mean of three independent experiments. ** P<0.01, *** P<0.001 vs. untreated control group. # P<0.05 vs. TGF-β1 only-treated group. ATV, atorvastatin; TGF-β1, transforming growth factor-β1; ECM, extracellular matrix; hVFs, human ventricular fibroblasts; MMP-2, matrix metalloproteinase-2.

Article Snippet: Adult hVFs (cat. no. 6310) and complete medium for their culture (fibroblast medium-2; FM-2; cat. no. 2331) were obtained from ScienCell Research Laboratories, Inc. Antibodies against α-SMA, matrix metal-loproteinase-2 (MMP-2), collagen I and collagen III were purchased from ProteinTech Group, Inc. Antibodies against phosphorylated (p)-Smad3, total (t)-Smad3, p-ERK1/2, t-ERK1/2, p-JNK, t-JNK, p-p38 and t-p38 were purchased from Cell Signaling Technology, Inc. Anti-GAPDH antibody and Cell Counting Kit-8 (CCK-8) were purchased from Beyotime Institute of Biotechnology.

Techniques: Reverse Transcription, Real-time Polymerase Chain Reaction, Expressing, Western Blot, Software, Control

Journal: International Journal of Molecular Medicine

Article Title: Atorvastatin attenuates TGF-β1-induced fibrogenesis by inhibiting Smad3 and MAPK signaling in human ventricular fibroblasts

doi: 10.3892/ijmm.2020.4607

Figure Lengend Snippet: ATV inhibits TGF-β1-induced Smad3 and MAPK signaling activation in hVFs. Fibroblasts were pre-incubated with ATV (10 µ M) for 3 h and then co-treated with 5 ng/ml TGF-β1 for 30 min. The protein levels of (A) p-Smad3 and t-Smad3, (B) p-ERK1/2 and t-ERK1/2, (C) p-p38 and t-p38, and (D) p-JNK and t-JNK were detected using western blotting. The relative density was expressed as the ratio of p-protein to the corresponding t-protein. Values represent the mean ± standard error of mean of three independent experiments. ** P<0.01, *** P<0.001 vs. untreated control group. # P<0.05 vs. TGF-β1 only-treated group. ATV, atorvastatin; TGF-β1, transforming growth factor-β1; MAPK, mitogen-activated protein kinase; hVFs, human ventricular fibroblasts; p-, phosphorylated; t-, total; ERK1/2, extracellular signal-regulated kinase 1/2; JNK, c-Jun N-terminal kinase.

Article Snippet: Adult hVFs (cat. no. 6310) and complete medium for their culture (fibroblast medium-2; FM-2; cat. no. 2331) were obtained from ScienCell Research Laboratories, Inc. Antibodies against α-SMA, matrix metal-loproteinase-2 (MMP-2), collagen I and collagen III were purchased from ProteinTech Group, Inc. Antibodies against phosphorylated (p)-Smad3, total (t)-Smad3, p-ERK1/2, t-ERK1/2, p-JNK, t-JNK, p-p38 and t-p38 were purchased from Cell Signaling Technology, Inc. Anti-GAPDH antibody and Cell Counting Kit-8 (CCK-8) were purchased from Beyotime Institute of Biotechnology.

Techniques: Activation Assay, Incubation, Western Blot, Control

Journal: International Journal of Molecular Medicine

Article Title: Atorvastatin attenuates TGF-β1-induced fibrogenesis by inhibiting Smad3 and MAPK signaling in human ventricular fibroblasts

doi: 10.3892/ijmm.2020.4607

Figure Lengend Snippet: Schematic diagram demonstrating how atorvastatin inhibits TGF-β1-induced fibrogenesis in hVFs. TGF-β1 treatment can induce activation of the Smad3 and MAPK signaling pathways, cause an increase in ECM synthesis and promote the fibrogenesis in hVFs. Atorvastatin plays a protective role and can prevent TGF-β1-induced cardiac fibrosis by inhibiting the activation of Smad3 and MAPK signaling pathways in hVFs. MAPK, mitogen-activated protein kinase; hVFs, human ventricular fibroblasts; ECM, extracellular matrix; p-, phosphorylated; ERK1/2, extracellular signal-regulated kinase 1/2; JNK, c-Jun N-terminal kinase; TGF-β1, transforming growth factor-β1.

Article Snippet: Adult hVFs (cat. no. 6310) and complete medium for their culture (fibroblast medium-2; FM-2; cat. no. 2331) were obtained from ScienCell Research Laboratories, Inc. Antibodies against α-SMA, matrix metal-loproteinase-2 (MMP-2), collagen I and collagen III were purchased from ProteinTech Group, Inc. Antibodies against phosphorylated (p)-Smad3, total (t)-Smad3, p-ERK1/2, t-ERK1/2, p-JNK, t-JNK, p-p38 and t-p38 were purchased from Cell Signaling Technology, Inc. Anti-GAPDH antibody and Cell Counting Kit-8 (CCK-8) were purchased from Beyotime Institute of Biotechnology.

Techniques: Activation Assay, Protein-Protein interactions