Journal: Frontiers in Cardiovascular Medicine

Article Title: MiR-208b/miR-21 Promotes the Progression of Cardiac Fibrosis Through the Activation of the TGF-β1/Smad-3 Signaling Pathway: An in vitro and in vivo Study

doi: 10.3389/fcvm.2022.924629

Figure Lengend Snippet: Primers used for RT-qPCR.

Article Snippet: After being transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, United States), the total protein was sealed with 5% dried skim milk at room temperature for 1 h. After blocking, the samples were treated with primary antibodies against TGF-β1 (1:1000, ab92486) (Abcam, United States), Smad-3 (1:1000, bs-3484R) (Bioss, China), and β-actin (1:500, 4970S) (CST, United States) at 4°C for 12 h. After the membrane was rinsed with TBST (Solarbio, China), the secondary HRP-conjugated antibody was added and incubated in the dark at room temperature for 1 h. Next, after TBST washing, the bands were visualized using the ECL kit (Solarbio, China).

Techniques:

Journal: Frontiers in Cardiovascular Medicine

Article Title: MiR-208b/miR-21 Promotes the Progression of Cardiac Fibrosis Through the Activation of the TGF-β1/Smad-3 Signaling Pathway: An in vitro and in vivo Study

doi: 10.3389/fcvm.2022.924629

Figure Lengend Snippet: Expression of serum indices in AMI patients at different time periods.

Article Snippet: After being transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, United States), the total protein was sealed with 5% dried skim milk at room temperature for 1 h. After blocking, the samples were treated with primary antibodies against TGF-β1 (1:1000, ab92486) (Abcam, United States), Smad-3 (1:1000, bs-3484R) (Bioss, China), and β-actin (1:500, 4970S) (CST, United States) at 4°C for 12 h. After the membrane was rinsed with TBST (Solarbio, China), the secondary HRP-conjugated antibody was added and incubated in the dark at room temperature for 1 h. Next, after TBST washing, the bands were visualized using the ECL kit (Solarbio, China).

Techniques: Expressing

Journal: Frontiers in Cardiovascular Medicine

Article Title: MiR-208b/miR-21 Promotes the Progression of Cardiac Fibrosis Through the Activation of the TGF-β1/Smad-3 Signaling Pathway: An in vitro and in vivo Study

doi: 10.3389/fcvm.2022.924629

Figure Lengend Snippet: Temporal changes in serum indices in AMI patients. (A) The expression levels of miR-208b and miR-21 change with time; (B) the expression levels of TGF-β1 and Smad-3 change with time; (C) the expression level of cTnT changes with time; (D) the expression level of CK-MB changes with time. CK-MB, creatine kinase isoenzyme; cTnT, cardiac calcium protein T; TGF-β1, transforming growth factor-β1.

Article Snippet: After being transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, United States), the total protein was sealed with 5% dried skim milk at room temperature for 1 h. After blocking, the samples were treated with primary antibodies against TGF-β1 (1:1000, ab92486) (Abcam, United States), Smad-3 (1:1000, bs-3484R) (Bioss, China), and β-actin (1:500, 4970S) (CST, United States) at 4°C for 12 h. After the membrane was rinsed with TBST (Solarbio, China), the secondary HRP-conjugated antibody was added and incubated in the dark at room temperature for 1 h. Next, after TBST washing, the bands were visualized using the ECL kit (Solarbio, China).

Techniques: Expressing

Journal: Frontiers in Cardiovascular Medicine

Article Title: MiR-208b/miR-21 Promotes the Progression of Cardiac Fibrosis Through the Activation of the TGF-β1/Smad-3 Signaling Pathway: An in vitro and in vivo Study

doi: 10.3389/fcvm.2022.924629

Figure Lengend Snippet: Comparison of the expression of each index under different hypoxia conditions in vitro . (A) Expression of miR-208b and miR-21 in the hypoxic treatment group at 6 h, 24 h, 48 h, and 72 h detected by RT-qPCR; (B) expression of TGF-β1 and Smad-3 in the hypoxic treatment group at 6 h, 24 h, 48 h, and 72 h detected by RT-qPCR. RT-qPCR, reverse transcription-quantitative polymerase chain reaction; TGF-β1, transforming growth factor-β1; * P < 0.05 compared with the control; ** P < 0.01 compared with the control.

Article Snippet: After being transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, United States), the total protein was sealed with 5% dried skim milk at room temperature for 1 h. After blocking, the samples were treated with primary antibodies against TGF-β1 (1:1000, ab92486) (Abcam, United States), Smad-3 (1:1000, bs-3484R) (Bioss, China), and β-actin (1:500, 4970S) (CST, United States) at 4°C for 12 h. After the membrane was rinsed with TBST (Solarbio, China), the secondary HRP-conjugated antibody was added and incubated in the dark at room temperature for 1 h. Next, after TBST washing, the bands were visualized using the ECL kit (Solarbio, China).

Techniques: Expressing, In Vitro, Quantitative RT-PCR, Real-time Polymerase Chain Reaction

Journal: Frontiers in Cardiovascular Medicine

Article Title: MiR-208b/miR-21 Promotes the Progression of Cardiac Fibrosis Through the Activation of the TGF-β1/Smad-3 Signaling Pathway: An in vitro and in vivo Study

doi: 10.3389/fcvm.2022.924629

Figure Lengend Snippet: The correlation between miR-208b/miR-21 and TGF-β1/Smad-3. (A) Positive correlation between miR-208b and TGF-β1; (B) positive correlation between miR-208b and Smad-3; (C) positive correlation between miR-21 and TGF-β1; (D) positive correlation between miR-21 and Smad-3. TGF-β1, transforming growth factor-β1.

Article Snippet: After being transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, United States), the total protein was sealed with 5% dried skim milk at room temperature for 1 h. After blocking, the samples were treated with primary antibodies against TGF-β1 (1:1000, ab92486) (Abcam, United States), Smad-3 (1:1000, bs-3484R) (Bioss, China), and β-actin (1:500, 4970S) (CST, United States) at 4°C for 12 h. After the membrane was rinsed with TBST (Solarbio, China), the secondary HRP-conjugated antibody was added and incubated in the dark at room temperature for 1 h. Next, after TBST washing, the bands were visualized using the ECL kit (Solarbio, China).

Techniques:

Journal: Frontiers in Cardiovascular Medicine

Article Title: MiR-208b/miR-21 Promotes the Progression of Cardiac Fibrosis Through the Activation of the TGF-β1/Smad-3 Signaling Pathway: An in vitro and in vivo Study

doi: 10.3389/fcvm.2022.924629

Figure Lengend Snippet: Comparison of the expression of TGF-β1 and Smad-3 in H9C2 cells following transfection with miR-208b mimics and miR-208b inhibitors. (A) Comparison of the expression of TGF-β1, Smad-3, and miR-208b detected by RT-qPCR in each group; (B) Representative blot images; (C) Comparison of the expression of TGF-β1 and Smad-3 detected by western blot analysis in each group. miR-208b +, miR-208b overexpression group; miR- 208b-, miR-208b inhibition group; TGF-β1, transforming growth factor-β1; * P < 0.05 compared with the blank group; ** P < 0.01 compared with the blank group.

Article Snippet: After being transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, United States), the total protein was sealed with 5% dried skim milk at room temperature for 1 h. After blocking, the samples were treated with primary antibodies against TGF-β1 (1:1000, ab92486) (Abcam, United States), Smad-3 (1:1000, bs-3484R) (Bioss, China), and β-actin (1:500, 4970S) (CST, United States) at 4°C for 12 h. After the membrane was rinsed with TBST (Solarbio, China), the secondary HRP-conjugated antibody was added and incubated in the dark at room temperature for 1 h. Next, after TBST washing, the bands were visualized using the ECL kit (Solarbio, China).

Techniques: Expressing, Transfection, Quantitative RT-PCR, Western Blot, Over Expression, Inhibition

Journal: Frontiers in Cardiovascular Medicine

Article Title: MiR-208b/miR-21 Promotes the Progression of Cardiac Fibrosis Through the Activation of the TGF-β1/Smad-3 Signaling Pathway: An in vitro and in vivo Study

doi: 10.3389/fcvm.2022.924629

Figure Lengend Snippet: Comparison of the expression of TGF-β1 and Smad-3 in H9C2 cells following transfection with miR-21 mimics and miR-21 inhibitors. (A) Comparison of the expression of TGF-β1, Smad-3, and miR-21 detected by RT-qPCR in each group; (B) Representative blot images; (C) Comparison of the expression of TGF-β1 and Smad-3 detected by western blot analysis in each group. miR-21 +, miR-21 overexpression group; miR- 21-, miR-21 inhibition group; TGF-β1, transforming growth factor-β1; * P < 0.05 compared with the blank group; ** P < 0.01 compared with the blank group.

Article Snippet: After being transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, United States), the total protein was sealed with 5% dried skim milk at room temperature for 1 h. After blocking, the samples were treated with primary antibodies against TGF-β1 (1:1000, ab92486) (Abcam, United States), Smad-3 (1:1000, bs-3484R) (Bioss, China), and β-actin (1:500, 4970S) (CST, United States) at 4°C for 12 h. After the membrane was rinsed with TBST (Solarbio, China), the secondary HRP-conjugated antibody was added and incubated in the dark at room temperature for 1 h. Next, after TBST washing, the bands were visualized using the ECL kit (Solarbio, China).

Techniques: Expressing, Transfection, Quantitative RT-PCR, Western Blot, Over Expression, Inhibition

Journal: Clinical and translational medicine

Article Title: FKBP51 increases the tumour-promoter potential of TGF-beta.

doi: 10.1186/2001-1326-3-1

Figure Lengend Snippet: Figure 2 FKBP51 interacts with the general transcriptional co-activator p300 and the TGF-β transcription factor Smad2/3. Left; FKBP51 co immunoprecipitates with p300. Right; p300 co immunoprecipitates with FKBP51. Total cell lysates were prepared by SAN melanoma cells transfected with FKBP51/Flag. Cell lysates were immunoprecipitated with anti-Kat3B/p300 (IP p300) or anti-Flag (IP FKBP51). Immunoprecipitated and total lysates were then subjected to Western blot with anti-FKBP51, anti-p300 or anti-Smad 2/3. Smad 2/3 co immunoprecipitate with either p300 (left) and FKBP51 (right).

Article Snippet: Primary antibodies against FKBP51 (F-13; goat polyclonal; Santa Cruz Biotechnology, CA, USA); Smad 2/3 (H465, rabbit polyclonal; Santa Cruz Biotechnology); SPARC (H-90, rabbit polyclonal; Santa Cruz Biotechnology); N-Cadherin (5D5, mouse monoclonal; Abcam, Cambridge, UK); G3PDH (D16H11, rabbit monoclonal; Cell Signaling, Danvers, USA); were used diluted.

Techniques: Transfection, Immunoprecipitation, Western Blot

Journal: Clinical and translational medicine

Article Title: FKBP51 increases the tumour-promoter potential of TGF-beta.

doi: 10.1186/2001-1326-3-1

Figure Lengend Snippet: Figure 5 Mechanism proposed for FKBP51 enhancement of TGF-β pro-oncogenic signal. Left, FKBP51 facilitates Smad recruitment to coactivators. Right, FKBP51 takes part to the transcriptional complex formed by P300 and Smad 2,3 Increase in FKBP51, as it occurs in melanoma, generates an auto regulatory loop of TGF-β signaling, which in turn promotes tumour progression.

Article Snippet: Primary antibodies against FKBP51 (F-13; goat polyclonal; Santa Cruz Biotechnology, CA, USA); Smad 2/3 (H465, rabbit polyclonal; Santa Cruz Biotechnology); SPARC (H-90, rabbit polyclonal; Santa Cruz Biotechnology); N-Cadherin (5D5, mouse monoclonal; Abcam, Cambridge, UK); G3PDH (D16H11, rabbit monoclonal; Cell Signaling, Danvers, USA); were used diluted.

Techniques:

Journal: Redox biology

Article Title: Mitoquinone ameliorates pressure overload-induced cardiac fibrosis and left ventricular dysfunction in mice.

doi: 10.1016/j.redox.2019.101100

Figure Lengend Snippet: Fig. 5. MitoQ blunts activation of myocardial TGF-β1 and profibrogenic signaling in response to pressure overload. A: mRNA expression levels of Agt, Pdgf, TGF-β1 and Edn1; B: mRNA expression levels of Nox4, Ctgf, Acta2, and collagen (Col1a1, Col1a2 and Col3a1); C: Western blotting of TGF-β1, NOX4, phosphorylated SMAD2 (p-SMAD2), and total SMAD2; and D: Quantification of normalized TGF-β1 (left), NOX4 (middle) and p-SMAD2/total SMAD2 (right) protein expression. *: P < 0.05 vs. Sham. **: P < 0.01 vs. Sham. #: P < 0.05 vs. AAC. ##: P < 0.01 vs. AAC. n = 4–6 each group. Agt: angiotensinogen; Pdgf: platelet-derived growth factor; Edn1: endothelin; Nox4: NADPH oxidase 4; Ctgf: connective tissue growth factor; Acta: actin assembly-inducing protein. AAC: ascending aortic constriction.

Article Snippet: Antibodies against SMAD2 (mAb #8685), p-SMAD2 (mAb # 8828), TGF-β1 (pAb #3711) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Activation Assay, Expressing, Western Blot, Derivative Assay

Journal: Redox biology

Article Title: Mitoquinone ameliorates pressure overload-induced cardiac fibrosis and left ventricular dysfunction in mice.

doi: 10.1016/j.redox.2019.101100

Figure Lengend Snippet: Fig. 6. MitoQ attenuates NOX4-induced oxidative stress and SMAD2 signaling pathway in TGF-β-treated cardiac fibroblasts (CF). A: Representative confocal images of MitoSox stained CF (left) and quantification of ROS measurement (right); B: mRNA expression of TGF-β1, Nox4, Ctgf, Acta2 and Col1a2 in CF; C: Representative western blotting of NOX4, phosphorylated SMAD2 (p-SMAD2), and total SMAD2; D: Quantification of normalized NOX4 and p-SMAD2/total SMAD2 protein ex- pression; E: mRNA expression of Nrf2 and Nqo1 in CF; and F: Nrf2 protein expression in AAC myocardium *: P < 0.05 vs. Control. #: P < 0.05 vs. TGF-β (A-E) or AAC (F). n = 3 each group for CF (A-E) and n = 6 each group for mouse (F). NOX4: NADPH oxidase 4; Ctgf: connective tissue growth factor; Acta: actin assembly- inducing protein; Col1: collagen type I alpha 2 chain; Nrf2: nuclear factor erythroid 2-related factor 2; Nqo1: NAD(P)H quinone dehydrogenase 1. AAC: ascending aortic constriction.

Article Snippet: Antibodies against SMAD2 (mAb #8685), p-SMAD2 (mAb # 8828), TGF-β1 (pAb #3711) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Staining, Expressing, Western Blot, Control

Journal: JCI Insight

Article Title: TGF- β promotes fibrosis after severe acute kidney injury by enhancing renal macrophage infiltration

doi: 10.1172/jci.insight.123563

Figure Lengend Snippet: Peritoneal macrophages from Tgfbr2fl/fl (WT) and LysM-Cre Tgfbr2fl/fl (macrophage TGF-βRII–/–; MφTGF-βRII–/–) mice 3 days after peritoneal thioglycollate administration were used. (A) TGF-βRII levels were markedly lower in TGF-βRII–/– macrophages than in WT macrophages. ***P < 0.001, n = 3 in each group. Data were expressed as mean ± SEM. (B) Macrophages were stimulated with 2 ng/ml recombinant TGF-β for 30 minutes. TGF-βRII deletion led to decreases in TGF-β–stimulated phosphorylation of Smad2 and Smad3, an indication of TGF-βRII deficiency. p-Smad3, phospho-Smad3; p-Smad2, phospho-Smad2; FAK, focal adhesion kinase.

Article Snippet: Rat anti-mouse F4/80 (MCA497R, a marker of macrophages) and CD3 (MCA1477, a marker of T lymphocytes) were purchased from AbD Serotec (now Bio-Rad); mouse anti–mannose receptor (CD206, MAB25341) and mouse anti–4-HNE (a marker of oxidative stress, 198960) were from R&D Systems; rabbit anti–TGF-βRII (SC-400) and goat anti-human CTGF (SC-14939) were from Santa Cruz Biotechnology; rabbit anti-human Smad2 (600-401-A59), phospho-Smad2 (600-401-K09S), and phospho-Smad3 (600-401-919) as well as rabbit anti-murine collagen type I (600-401-103-01) were from Rockland Immunochemicals; mouse anti–α-SMA (A5228, a marker of myofibroblasts) was from Sigma-Aldrich; and rabbit anti–TGF-β1 (NBP1-80289) and mouse anti–TGF-β2 (Mab612-SP) were from Novus Biologicals.

Techniques: Recombinant