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: Molecules (Basel, Switzerland)

Article Title: F4/80 + Kupffer Cell-Derived Oncostatin M Sustains the Progression Phase of Liver Regeneration through Inhibition of TGF-β2 Pathway.

doi: 10.3390/molecules26082231

Figure Lengend Snippet: Figure 6. The effects of depletion of F4/80+ KCs post-PHx on the TGF-β2/smad2 pathway during liver regeneration. (A) KEGG signal pathway enrichment scatter map. (B) ELISA assay of serum TGF-β2 in post-PHx α-F4/80-treated mice. (C) Western blot of TGF-β2/smad2 pathway in livers from (B), n = 3–5; * p < 0.05, ** p < 0.01.

Article Snippet: After blocking with 0.5% BSA, the membranes were incubated with primary antibodies at 4 ◦C overnight and then with secondary antibodies for at least 2 h. The primary antibodies used were as follows: TGFβ2 (1:1000, R&D Systems, Wiesbaden, Germany); CyclinD1 (1:1000, proteintech, Wuhan, China); PCNA (1:2000, proteintech, Wuhan, China); VEGFR2 (1:1000, Cell Signaling Technology, Danvers, MA, USA); HNF-4α, GAPDH, and CD31 (1:500, Abcam, Cambridge, MA, USA); and VEGFR2, Smad2, Smad3, and phospho-Smad2/3 (1:1000, Cell Signaling Technology, Danvers, MA, USA).

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot

Journal: Molecules (Basel, Switzerland)

Article Title: F4/80 + Kupffer Cell-Derived Oncostatin M Sustains the Progression Phase of Liver Regeneration through Inhibition of TGF-β2 Pathway.

doi: 10.3390/molecules26082231

Figure Lengend Snippet: Figure 7. TGF-βRI treatment partially rescued the impaired proliferation of hepatocytes caused by depletion of F4/80+ KCs in post-PHx. (A) Phosphorylated and total protein levels of smad2 or smad3 in the post-PHx α-F4/80-treated groups in the absence or presence of TGF-βRI. Values represent the means ± SDs. (B) Liver/body weight ratios were analyzed in post-PHx α-F4/80-treated alone or that combined with TGF-βRI. (C) Representative photomicrographs of Ki67+ hepatocytes. Those relatively larger and rounder are considered hepatocytes. Quantification of the labeled cells at the lower left corner. (D) The protein levels of HNF-4α, PCNA, Cyclin D1, and GAPDH in livers (A). * p < 0.05, ** p < 0.01, n = 3.

Article Snippet: After blocking with 0.5% BSA, the membranes were incubated with primary antibodies at 4 ◦C overnight and then with secondary antibodies for at least 2 h. The primary antibodies used were as follows: TGFβ2 (1:1000, R&D Systems, Wiesbaden, Germany); CyclinD1 (1:1000, proteintech, Wuhan, China); PCNA (1:2000, proteintech, Wuhan, China); VEGFR2 (1:1000, Cell Signaling Technology, Danvers, MA, USA); HNF-4α, GAPDH, and CD31 (1:500, Abcam, Cambridge, MA, USA); and VEGFR2, Smad2, Smad3, and phospho-Smad2/3 (1:1000, Cell Signaling Technology, Danvers, MA, USA).

Techniques: Labeling

Journal: Molecules (Basel, Switzerland)

Article Title: F4/80 + Kupffer Cell-Derived Oncostatin M Sustains the Progression Phase of Liver Regeneration through Inhibition of TGF-β2 Pathway.

doi: 10.3390/molecules26082231

Figure Lengend Snippet: Figure 10. The effect of r-OSM reconstitution on hepatocyte proliferation and the TGF-β2/smad2 signaling pathway during the progression process of liver regeneration. (A) Representative photomicrographs and quantification of Ki67+ hepatocytes in α-F4/80 alone or combined with r-OSM-treated mice after PHx. (B) Expression of HNF-4α, PCNA, Cyclin D1, and GAPDH proteins in (A). (C) Expression of the TGF-β2/smad2 pathway-associated proteins in (A). n = 3–5, * p < 0.05, ** p < 0.01.

Article Snippet: After blocking with 0.5% BSA, the membranes were incubated with primary antibodies at 4 ◦C overnight and then with secondary antibodies for at least 2 h. The primary antibodies used were as follows: TGFβ2 (1:1000, R&D Systems, Wiesbaden, Germany); CyclinD1 (1:1000, proteintech, Wuhan, China); PCNA (1:2000, proteintech, Wuhan, China); VEGFR2 (1:1000, Cell Signaling Technology, Danvers, MA, USA); HNF-4α, GAPDH, and CD31 (1:500, Abcam, Cambridge, MA, USA); and VEGFR2, Smad2, Smad3, and phospho-Smad2/3 (1:1000, Cell Signaling Technology, Danvers, MA, USA).

Techniques: Expressing

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