Journal: Experimental and Therapeutic Medicine

Article Title: MicroRNA-146a attenuates isoproterenol-induced cardiac fibrosis by inhibiting FGF2

doi: 10.3892/etm.2022.11433

Figure Lengend Snippet: miR-146a-5p directly targets FGF2 and FGF2 knockdown decreases collagen I and α-SMA expression levels in CFs. (A) StarBase database showed the potential binding sites between FGF2 and miR-146a-5p. (B) Luciferase reporter assay was used to determine the binding ability between FGF2 and miR-146a-5p in 293 cells. * P<0.05 vs. NC mimics. (C) RT-qPCR and (D) western blotting were used to assess the mRNA and protein levels of FGF2 in ISO-treated CFs transfected with NC or miR-146a-5p mimics or inhibitor or inhibitor. (E) RT-qPCR and (F) western blotting showed mRNA and protein levels of FGF2 in CFs transfected with siNC or siFGF2. (G) RT-qPCR analysis showed collagen I and α-SMA mRNA levels in ISO-treated CFs transfected with siNC or siFGF2. * P<0.05 vs. siNC. miR, microRNA; FGF2, fibroblast growth factor 2; α-SMA, α-smooth muscle actin; NC, negative control; CF, cardiac fibroblast; RT-q, reverse transcription-quantitative; ISO, isoproterenol; si, small interfering; WT, wild-type; MUT, mutant.

Article Snippet: After blocking with 5% non-fat milk for 2 h at room temperature, the membrane was incubated with primary antibodies against FGF2 (1:1,000; cat. no. PB0619; Boster Biological Technology, Ltd.) and GADPH (1:1,000; cat. no. ab9485; Abcam) overnight at 4 ̊C.

Techniques: Knockdown, Expressing, Binding Assay, Luciferase, Reporter Assay, Quantitative RT-PCR, Western Blot, Transfection, Negative Control, Reverse Transcription, Mutagenesis

Journal: Experimental and Therapeutic Medicine

Article Title: MicroRNA-146a attenuates isoproterenol-induced cardiac fibrosis by inhibiting FGF2

doi: 10.3892/etm.2022.11433

Figure Lengend Snippet: miR-146a-5p suppresses ISO-treated cardiac fibrosis by targeting FGF2. (A) RT-qPCR and (B) western blotting were performed to determine the mRNA and protein levels of FGF2 in ISO-treated CFs transfected with NC or miR-146a-5p mimics or miR-146a-5p mimics + pcDNA3.1/FGF2. (C) RT-qPCR and (D) Cell Counting Kit-8 assay determined collagen I and α-SMA expression levels, as well as viability, in ISO-treated CFs transfected with NC or miR-146a-5p mimics or miR-146a-5p mimics + pcDNA3.1/FGF2. * P<0.05. miR, microRNA; ISO, isoproterenol; FGF2, fibroblast growth factor 2; RT-q, reverse transcription-quantitative; CF, cardiac fibroblast; NC, negative control; OD, optical density.

Article Snippet: After blocking with 5% non-fat milk for 2 h at room temperature, the membrane was incubated with primary antibodies against FGF2 (1:1,000; cat. no. PB0619; Boster Biological Technology, Ltd.) and GADPH (1:1,000; cat. no. ab9485; Abcam) overnight at 4 ̊C.

Techniques: Quantitative RT-PCR, Western Blot, Transfection, Cell Counting, Expressing, Reverse Transcription, Negative Control

Journal: PLoS ONE

Article Title: EphA2-Induced Angiogenesis in Ewing Sarcoma Cells Works through bFGF Production and Is Dependent on Caveolin-1

doi: 10.1371/journal.pone.0071449

Figure Lengend Snippet: A. bFGF expression was analyzed in CAV1 knocked-down models showing that bFGF expression was affected constantly in all models. The graph shows the quantification of the OD volume determined from the intensity of the bands from the RT-PCR images showing the statistically significant reduction in bFGF in CAV1 knocked-down cells; bars, SD (*P≤0.05 n = 3) B. Immunoblot showing bFGF reduction in the TC71 model either in total protein extract or CM. Actin blot and ponceau staining was used as loading control. Quantitative data measuring relative intensity of the bands is indicated below each lane (n = 3) C. Quantification of endothelial cell migration. CM was used as chemoattractant for endothelial cells. A neutralizing antibody -N. Ab- (2.5 µg/ml and 5 µg/ml) and a recombinant protein -R. Prot- (10 ng/ml); bars, SD (*P<0.002– when compared with parental CM – and *P = 0.0005– when compared with clone n = 3) were used to block and promote migration respectively. RPMI media alone was used as negative control.

Article Snippet: Neutralizing antibody against bFGF (Anti-FGF2/basic FGF #05-117) and human recombinant FGF-2/basic (FGF2 #01-106) were from Millipore and were used at mentioned concentrations.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Staining, Control, Migration, Recombinant, Blocking Assay, Negative Control

Journal: Cells

Article Title: Pro-Angiogenic and Osteogenic Effects of Adipose Tissue-Derived Pericytes Synergistically Enhanced by Nel-like Protein-1

doi: 10.3390/cells10092244

Figure Lengend Snippet: NELL-1 increases FGF2-AKT-eNOS pathway signaling. ( A ) Heatmap showing temporal expression patterns of angiogenesis-related mRNAs identified using pericytes treated with NELL-1 (100 ng/mL or 800 ng/mL). Z -scores of normalized read counts are indicated by the colored bars. Red: high expression; green: low expression. ( B ) Validation of selected differentially expressed mRNAs in pericytes treated with NELL-1 (800 ng/mL) via qRT-PCR. β-actin was used as a normalization control. Data were obtained from 3 independent experiments. Error bars represent mean ± SEM. ( C ) Western blot analysis of FGF2 pathway-related proteins after 72 h of stimulation with 800 ng/mL NELL-1. Data were obtained from 3 independent experiments. Error bars represent mean ± SEM. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: The primary antibodies against FGF2 (cat. no. sc-271930, 1:1000), GAPDH (cat.no. sc-47724, 1:3000) were purchased from Santa Cruz Biotechnology, Inc. Antibodies against phospho-PLCγ (cat.no.

Techniques: Expressing, Biomarker Discovery, Quantitative RT-PCR, Control, Western Blot