Journal: PLOS ONE

Article Title: MiR-132-3p suppresses peritoneal fibrosis induced by peritoneal dialysis via targeting TGF- β 1/Smad2/3 signaling pathway

doi: 10.1371/journal.pone.0301540

Figure Lengend Snippet: Sequences of the primers for q-PCR.

Article Snippet: The primary antibodies used were as follows: Smad2 (A00090-1, Boster, 1:200 dilution; China); Smad3 (BM3919, Boster, 1:200 dilution; China); α-SMA (BM0002, Boster, 1:200 dilution; China); TGF- β 1 (MA00019, Boster, 1:200 dilution; China).

Techniques:

Journal: PLOS ONE

Article Title: MiR-132-3p suppresses peritoneal fibrosis induced by peritoneal dialysis via targeting TGF- β 1/Smad2/3 signaling pathway

doi: 10.1371/journal.pone.0301540

Figure Lengend Snippet: (A) Area semi-quantitative statistical analysis by immunohistochemistry; (B-E)Expression of TGF- β 1, smad2, smad3,col1a1 col3a1and α-SMA by immunohistochemistry. All data are presented as mean ± SD. n = 6 for each group. * p < 0.05; ** p < 0.01; *** p < 0.001; between the indicated groups.

Article Snippet: The primary antibodies used were as follows: Smad2 (A00090-1, Boster, 1:200 dilution; China); Smad3 (BM3919, Boster, 1:200 dilution; China); α-SMA (BM0002, Boster, 1:200 dilution; China); TGF- β 1 (MA00019, Boster, 1:200 dilution; China).

Techniques: Immunohistochemistry, Expressing

Journal: PLOS ONE

Article Title: MiR-132-3p suppresses peritoneal fibrosis induced by peritoneal dialysis via targeting TGF- β 1/Smad2/3 signaling pathway

doi: 10.1371/journal.pone.0301540

Figure Lengend Snippet: Quantitative real time-PCR was used to measure the mRNA levels of(A)TGF- β1 ;(B)α-SMA;(C)Smad2;(D)Smad3. All data are presented as mean ± SD. n = 4 for each group. * p < 0.05; ** p < 0.01; *** p < 0.001 and **** p < 0.0001 between the indicated groups.

Article Snippet: The primary antibodies used were as follows: Smad2 (A00090-1, Boster, 1:200 dilution; China); Smad3 (BM3919, Boster, 1:200 dilution; China); α-SMA (BM0002, Boster, 1:200 dilution; China); TGF- β 1 (MA00019, Boster, 1:200 dilution; China).

Techniques: Real-time Polymerase Chain Reaction

Journal: PLOS ONE

Article Title: MiR-132-3p suppresses peritoneal fibrosis induced by peritoneal dialysis via targeting TGF- β 1/Smad2/3 signaling pathway

doi: 10.1371/journal.pone.0301540

Figure Lengend Snippet: (A and D) Immunofluorescence was used to measure the protein expression levels of(B)TGF- β 1;(C)Smad3;(E) α -SMA;(F)Smad2. All data are presented as mean ± SD. n = 3 for each group. * p < 0.05; ** p < 0.01; *** p < 0.001 and **** p < 0.0001 between the indicated groups.

Article Snippet: The primary antibodies used were as follows: Smad2 (A00090-1, Boster, 1:200 dilution; China); Smad3 (BM3919, Boster, 1:200 dilution; China); α-SMA (BM0002, Boster, 1:200 dilution; China); TGF- β 1 (MA00019, Boster, 1:200 dilution; China).

Techniques: Immunofluorescence, 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

Journal: Molecular Medicine Reports

Article Title: Astaxanthin ameliorates renal interstitial fibrosis and peritubular capillary rarefaction in unilateral ureteral obstruction

doi: 10.3892/mmr.2019.9970

Figure Lengend Snippet: ASX inhibits UUO-induced activation of the TGF-β1/Smad signaling pathway. Protein expression levels of (A) TGF-β1, (B) Smad2, (C) Smad3, (D) Smad4 and (E) Smad7 in murine renal tissues were assessed by western blotting. The data are presented as the means ± standard deviation (n=6). **P<0.01, ***P<0.001. ASX, Astaxanthin; p, phosphorylated; TGF-β1, transforming growth factor β1; UUO, unilateral ureteral obstruction.

Article Snippet: After blocking with 5% skimmed milk for 1 h at room temperature, the membranes were probed with primary antibodies against collagen I (1:2,000; cat. no. 14695-1-AP; ProteinTech Group, Inc.), TSP-1 (1:500; cat. no. 18304-1-AP; ProteinTech Group, Inc.), VEGF-A (1:1,000; cat. no. 19003-1-AP; ProteinTech Group, Inc.), TGF-β1 (1:500; cat. no. 21898-1-AP; ProteinTech Group, Inc.), phosphorylated (p)-Smad2 (1:1,000; cat. no. 3108; Cell Signaling Technology, Inc., Danvers, MA, USA), Smad2 (1:3,000; cat. no. 12570-1-AP; ProteinTech Group, Inc.), fibronectin (1:1,000; cat. no. 15613-1-AP; ProteinTech Group, Inc.), α-smooth muscle actin (α-SMA) (1:500; cat. no. 55135-1-AP; ProteinTech Group, Inc.), Smad3 (1:1,000; cat. no. bs-3484R; BIOSS, Beijing, China), p-Smad3 (1:1,000; cat. no. bsm-52205R; BIOSS), Smad4 (1:1,000; cat. no. bs-0585R; BIOSS), Smad7 (1:1,000; cat. no. bs-23328R; BIOSS) and β-actin (1:500; cat. no. bsm-33036M; BIOSS) overnight at 4°C.

Techniques: Activation Assay, Expressing, Western Blot, Standard Deviation

Journal: Molecular Medicine Reports

Article Title: Astaxanthin ameliorates renal interstitial fibrosis and peritubular capillary rarefaction in unilateral ureteral obstruction

doi: 10.3892/mmr.2019.9970

Figure Lengend Snippet: ASX suppresses TGF-β1-induced expression of profibrogenic factors via inactivation of the Smad signaling pathway. Protein expression levels of (A) collagen I, (B) α-SMA, (C) Smad2, (D) Smad3, (E) Smad4 and (F) Smad7 in NRK-52E cells were assessed by western blotting. The data are presented as the means ± standard deviation (n=6). *P<0.05, **P<0.01, ***P<0.001. ASX, Astaxanthin; NS, not significant; p, phosphorylated; TGF-β1, transforming growth factor β1; UUO, unilateral ureteral obstruction.

Article Snippet: After blocking with 5% skimmed milk for 1 h at room temperature, the membranes were probed with primary antibodies against collagen I (1:2,000; cat. no. 14695-1-AP; ProteinTech Group, Inc.), TSP-1 (1:500; cat. no. 18304-1-AP; ProteinTech Group, Inc.), VEGF-A (1:1,000; cat. no. 19003-1-AP; ProteinTech Group, Inc.), TGF-β1 (1:500; cat. no. 21898-1-AP; ProteinTech Group, Inc.), phosphorylated (p)-Smad2 (1:1,000; cat. no. 3108; Cell Signaling Technology, Inc., Danvers, MA, USA), Smad2 (1:3,000; cat. no. 12570-1-AP; ProteinTech Group, Inc.), fibronectin (1:1,000; cat. no. 15613-1-AP; ProteinTech Group, Inc.), α-smooth muscle actin (α-SMA) (1:500; cat. no. 55135-1-AP; ProteinTech Group, Inc.), Smad3 (1:1,000; cat. no. bs-3484R; BIOSS, Beijing, China), p-Smad3 (1:1,000; cat. no. bsm-52205R; BIOSS), Smad4 (1:1,000; cat. no. bs-0585R; BIOSS), Smad7 (1:1,000; cat. no. bs-23328R; BIOSS) and β-actin (1:500; cat. no. bsm-33036M; BIOSS) overnight at 4°C.

Techniques: Expressing, Western Blot, Standard Deviation

Journal: The Journal of Biological Chemistry

Article Title: Increased cAMP Levels Modulate Transforming Growth Factor-?/Smad-induced Expression of Extracellular Matrix Components and Other Key Fibroblast Effector Functions

doi: 10.1074/jbc.M109.038620

Figure Lengend Snippet: Increased cAMP levels inhibit Smad3/4-dependent transcription without alteration of Smad3 phosphorylation and nuclear translocation in response to TGF-β in HDF. A, subconfluent cells were transfected with a Smad-dependent promoter/reporter construct (CAGA)9-luc, together with expression vectors for Smad3 or the corresponding empty vector in a medium supplemented with 1% FCS. 6 h after transfection, forskolin (20 μm) or Bt2cAMP (db-cAMP) (1 mm) was added in the presence (+) or absence of TGF-β (10 ng/ml). Incubations were continued for 20 h after which the reporter gene activity was determined. Results of a representative experiment performed on duplicate samples are shown as relative luciferase activity. B, subconfluent HDF were treated with TGF-β (10 ng/ml) in the presence or absence of forskolin (20 μm) or Bt2 cAMP (1 mm) left untreated. After 30 min, cells were lysed, and samples were processed for Western immunoblotting with specific antibodies for phospho-Smad3 and α-tubulin. C, subconfluent HDF in eight-well tissue chambers were incubated with forskolin (20 μm) or Bt2cAMP (1 mm) together with TGF-β (10 ng/ml). After 30 min, cells were fixed and double-stained with a Smad2/3 antibody (red signal) and with 4′,6-diamidino-2-phenylindole (DAPI) to visualize cell nuclei (blue signal) followed by fluorescence microscopy (scale bar, 25 μm).

Article Snippet: Endogenous Smad2/3 content was detected by incubation for 2 h at room temperature with a monoclonal anti-Smad2/3 antibody (1:250, BD Transduction Laboratories, Palo Alto, CA).

Techniques: Translocation Assay, Transfection, Construct, Expressing, Plasmid Preparation, Activity Assay, Luciferase, Western Blot, Incubation, Staining, Fluorescence, Microscopy