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: Biomolecules

Article Title: Cytotoxic Compounds of Two Demosponges ( Aplysina aerophoba and Spongia sp.) from the Aegean Sea

doi: 10.3390/biom11050723

Figure Lengend Snippet: Influence of 13 at different concentrations on ( A ) different colon carcinoma cell lines, on ( B ) SH-SY5Y neuroblastoma cell line, and on ( C ) primary human fibroblasts as non-cancerous control cells. Incubation time was 24 h followed by flow cytometric analysis (AnnV/PI staining). Percentage of cells alive was calculated compared to control (no treatment).

Article Snippet: Primary human foreskin fibroblasts were purchased from Promocell, Heidelberg, Germany.

Techniques: Incubation, Staining