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: Microbial cell factories

Article Title: Secretion of tumoricidal human tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) by recombinant Lactococcus lactis: optimization of in vitro synthesis conditions.

doi: 10.1186/s12934-018-1028-2

Figure Lengend Snippet: Fig. 9 Selectivity of hsTRAIL-induced death to cancer cells. HCT116 cells and human cardiac fibroblasts were incubated for 48 h with increasing concentration of hsTRAIL present in supernatant from broth culture of L. lactis (hsTRAIL+). As controls in experimental setup were used: corresponding volumes of supernatants from broth culture of L. lactis (hsTRAIL−)—negative control; corresponding concentrations of recombinant human TRAIL (Peprotech)—positive control. Viability of cancer cells and non-malignant, cardiac fibroblasts was assessed by MTS assay. Results are presented as % of viability of cells incubated in standard culture medium only. Secreted hsTRAIL remained non-cytotoxic to fibroblasts (Fig. 9a), while decreased viability of cancer cells in a dose-dependent manner (Fig. 9b). Table below—concentration of hsTRAIL [ng/ml] in specified volume of supernatant. The bars indicate the mean value ± SD of three independent experiments, each performed in triplicates. Statistical significance was calculated using two-way ANOVA with Tukey’s multiple comparison post-test. *p < 0.05, **p < 0.01, ***p < 0.001

Article Snippet: Human primary proliferating cardiac fibroblasts were obtained from Cell Applications, Inc. (San Diego, CA).

Techniques: Incubation, Concentration Assay, Negative Control, Recombinant, Positive Control, MTS Assay, Comparison

Journal: Scientific Reports

Article Title: TRPC3-GEF-H1 axis mediates pressure overload-induced cardiac fibrosis

doi: 10.1038/srep39383

Figure Lengend Snippet: ( a–c ) Effect of Pyr3 (1 μM) on induction of fibrosis-related mRNAs (CTGF and TGF-β1 or -β2) by MS in human iPSC-derived cardiomyocytes (n = 3). Cells were subjected to 20% static-MS for 3 h. GAPDH and 18 S ribosomal RNA were used as internal controls. ( d–g ) Effects of knockdown ( d ) or pharmacological inhibition ( e ) of TRPC3 on CTGF mRNA expression ( d,e ) and α-SMA protein expression ( f,g ) induced by TGF-β2 stimulation in human cardiac fibroblasts (n = 3). Error bars, s.e.m. *P < 0.05, **P < 0.01. ( h ) Schema for the mechanism of TRPC3-mediated cardiac fibrosis induced by pressure overload. TRPC3 mediates MS-induced expression of profibrotic factors (CTGF and TGF-β) in cardiomyocytes and TGF-β-induced fibrotic responses of cardiac fibroblasts, partially through Nox2-dependent GEF-H1 activation.

Article Snippet: For protein knockdown, cells were transfected with siRNAs (100 nM) using Lipofectamine 2000 for 72 h. Primary human cardiac fibroblasts were purchased from Lonza and cultured according to manufacturer’s instruction.

Techniques: Derivative Assay, Inhibition, Expressing, Activation 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

Journal: GeroScience

Article Title: Rapamycin increases oxidative metabolism and enhances metabolic flexibility in human cardiac fibroblasts

doi: 10.1007/s11357-018-0030-2

Figure Lengend Snippet: Progerin expression induces a glycolytic phenotype which is reversed by rapamycin. Measurements of basal respiration (a), glucose uptake (b), and extracellular acidification (ECAR) (c) in human cardiac fibroblasts (HCFs) expressing lamin A or progerin maintained with or without 1 nM rapamycin. Oxygen consumption rates were determined using a Seahorse bioanalyzer. Oxygen consumption and extracellular acidification rates were normalized to cell number. Data presented as mean ± SD (n = 2–3, in triplicates). The differences are shown to be statistically significant with p < 0.05 in comparison to the late passage + rapamycin values

Article Snippet: Cell culture and cell culture reagents Human cardiac fibroblast cells (ScienCell Inc. Carlsbad, CA) and HGPS patient-derived fibroblasts (Coriell Institute, Camden, NJ) were cultivated in minimal essential medium (MEM) supplemented with 10% fetal bovine serum, 1% l -glutamine, 1% MEM vitamins, and 1% MEM non-essential amino acids according to standard culture protocol for lifespan analysis of human diploid fibroblasts (Cristofalo and Charpentier 1980 ), with or without the addition of 1 nM rapamycin.

Techniques: Expressing, Comparison

Journal: GeroScience

Article Title: Rapamycin increases oxidative metabolism and enhances metabolic flexibility in human cardiac fibroblasts

doi: 10.1007/s11357-018-0030-2

Figure Lengend Snippet: Late passage human fibroblasts are relatively glycolytic. Seahorse bioanalyzer measurements of basal respiration (a), glucose uptake (b), and extracellular acidification (c) in early and late passage (> 90% lifespan completed) human cardiac fibroblasts. The values were normalized to cell numbers in each well. Samples were measured in quintuplicate and results are representative of at least two independent experiments ± SD. The bars with an asterisk represent values that are significantly (*, p < 0.05) differ from the late passage ones

Article Snippet: Cell culture and cell culture reagents Human cardiac fibroblast cells (ScienCell Inc. Carlsbad, CA) and HGPS patient-derived fibroblasts (Coriell Institute, Camden, NJ) were cultivated in minimal essential medium (MEM) supplemented with 10% fetal bovine serum, 1% l -glutamine, 1% MEM vitamins, and 1% MEM non-essential amino acids according to standard culture protocol for lifespan analysis of human diploid fibroblasts (Cristofalo and Charpentier 1980 ), with or without the addition of 1 nM rapamycin.

Techniques:

Journal: GeroScience

Article Title: Rapamycin increases oxidative metabolism and enhances metabolic flexibility in human cardiac fibroblasts

doi: 10.1007/s11357-018-0030-2

Figure Lengend Snippet: Distinct rates of pyruvate and glutamine/palmitate oxidation by early and late passage human fibroblasts and the effect of rapamycin. a The rates of pyruvate-mediated respiration were calculated as the percentage of inhibition of oxygen consumption by specific pyruvate transporter inhibitor UK5099. The rates of oxygen consumption obtained via sequential addition of first the glutamine and palmitate oxidation inhibitors, BPTES and etomoxir, and then UK5099 represent the capacity of pyruvate oxidation. b The rates of glutamine/palmitate oxidation were determined in the presence of corresponding inhibitors BPTES and etomoxir, while the capacity was determined following first the addition of pyruvate inhibitor UK5099 and then BPTES/etomoxir. c, d Effect of rapamycin on capacity of late passage cells to oxidize pyruvate and glutamine/palmitate in late passage fibroblasts maintained in the presence or absence of 1 nM rapamycin. Values are generated as in b through the addition of inhibitors and calculating the percent inhibition of oxygen consumption. In all cases, graphs represent the values of one of at least two independent experiments measured in triplicates and presented as mean ± SD; *p < 0.05 vs late passage

Article Snippet: Cell culture and cell culture reagents Human cardiac fibroblast cells (ScienCell Inc. Carlsbad, CA) and HGPS patient-derived fibroblasts (Coriell Institute, Camden, NJ) were cultivated in minimal essential medium (MEM) supplemented with 10% fetal bovine serum, 1% l -glutamine, 1% MEM vitamins, and 1% MEM non-essential amino acids according to standard culture protocol for lifespan analysis of human diploid fibroblasts (Cristofalo and Charpentier 1980 ), with or without the addition of 1 nM rapamycin.

Techniques: Inhibition, Generated

Journal: GeroScience

Article Title: Rapamycin increases oxidative metabolism and enhances metabolic flexibility in human cardiac fibroblasts

doi: 10.1007/s11357-018-0030-2

Figure Lengend Snippet: Rapamycin improves metabolic flexibility in human fibroblast cells expressing progerin. Measurements of the capacity for oxidizing pyruvate (a) or glutamine/palmitate (b) in late passage fibroblasts maintained in the presence or absence of 1 nM rapamycin. Measurements were made as in Fig. ​Fig.2.2. The data were normalized to cell number in each well. Data presented as mean ± SD (n = 2, in triplicates). The differences are shown to be statistically significant with p < 0.05 in comparison to the late passage values

Article Snippet: Cell culture and cell culture reagents Human cardiac fibroblast cells (ScienCell Inc. Carlsbad, CA) and HGPS patient-derived fibroblasts (Coriell Institute, Camden, NJ) were cultivated in minimal essential medium (MEM) supplemented with 10% fetal bovine serum, 1% l -glutamine, 1% MEM vitamins, and 1% MEM non-essential amino acids according to standard culture protocol for lifespan analysis of human diploid fibroblasts (Cristofalo and Charpentier 1980 ), with or without the addition of 1 nM rapamycin.

Techniques: Expressing, Comparison

Journal: GeroScience

Article Title: Rapamycin increases oxidative metabolism and enhances metabolic flexibility in human cardiac fibroblasts

doi: 10.1007/s11357-018-0030-2

Figure Lengend Snippet: Rapamycin prevents premature senescence induced by galactose. Human cardiac fibroblasts maintained with or without 1 nM rapamycin were transferred to glucose-free medium supplemented with either 10 or 20 mM galactose at a population doubling of 27.5. Cumulative population doublings were calculated as described in “Materials and methods” section

Article Snippet: Cell culture and cell culture reagents Human cardiac fibroblast cells (ScienCell Inc. Carlsbad, CA) and HGPS patient-derived fibroblasts (Coriell Institute, Camden, NJ) were cultivated in minimal essential medium (MEM) supplemented with 10% fetal bovine serum, 1% l -glutamine, 1% MEM vitamins, and 1% MEM non-essential amino acids according to standard culture protocol for lifespan analysis of human diploid fibroblasts (Cristofalo and Charpentier 1980 ), with or without the addition of 1 nM rapamycin.

Techniques: