Journal: Oxidative Medicine and Cellular Longevity

Article Title: Hdac8 Inhibitor Alleviates Transverse Aortic Constriction-Induced Heart Failure in Mice by Downregulating Ace1

doi: 10.1155/2022/6227330

Figure Lengend Snippet: PCI34051 alleviates cardiac fibrosis in transverse aortic constriction (TAC) mice through the TGF- β 1-Smad2/3 pathway. (a, b) Picrosirius red staining of mouse cardiac tissues; representative images and quantification are shown. Scale bar = 50 μ m. The expression levels of the fibrosis marker genes Col1a1 (c), Fn1 (d), Acta2 (e), and Tgfb1 (f) were determined using quantitative real-time polymerase chain reaction ( n = 5–6 per group). (g) The cardiac expression levels of Fn1, Acta2, Tgfb1, p-Smad2/3, and Smad2/3 were analyzed using western blotting. Actb was used as a loading control. Representative blots are shown. (h–k) Quantification of Fn1, Acta2, Tgfb1, and p-Smad2/3 to Smad2/3 ( n = 5–6 per group).

Article Snippet: Anti-phospho-Smad2/3 (8828), anti-Smad2/3 (3102), anti-Rela (8242), and anti-Nfkbia (4812) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Staining, Expressing, Marker, Real-time Polymerase Chain Reaction, Western Blot

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Hdac8 Inhibitor Alleviates Transverse Aortic Constriction-Induced Heart Failure in Mice by Downregulating Ace1

doi: 10.1155/2022/6227330

Figure Lengend Snippet: PCI34051 alleviates pulmonary congestion and fibrosis in transverse aortic constriction (TAC) mice. (a) The ratio of lung weight-to-bodyweight (LW/BW) in the sham+vehicle, sham+PCI34051 (10 mg/kg bodyweight/day), TAC+vehicle, and TAC+PCI34051 (3, 10, or 30 mg/kg bodyweight/day) groups ( n = 5–6 per group) was determined. Representative images of mouse lungs (B). (b) Representative images of pulmonary tissues stained with hematoxylin and eosin. Scale bar = 50 μ m. (c) Representative images of pulmonary tissues stained with Picrosirius red. Scale bar = 50 μ m. (d) Pulmonary fibrosis was quantified using ImageJ software. (e–h) The mRNA levels of Col1a1 , Fn1 , Acta2 , and Tgfb1 were evaluated using quantitative real-time polymerase chain reaction ( n = 5–6 per group). (i–m) Representative western blots and quantification of Fn1, Acta2, Tgfb1, p-Smad2/3, and Smad2/3 levels in the pulmonary tissues ( n = 5–6 per group).

Article Snippet: Anti-phospho-Smad2/3 (8828), anti-Smad2/3 (3102), anti-Rela (8242), and anti-Nfkbia (4812) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Staining, Software, Real-time Polymerase Chain Reaction, Western Blot

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Hdac8 Inhibitor Alleviates Transverse Aortic Constriction-Induced Heart Failure in Mice by Downregulating Ace1

doi: 10.1155/2022/6227330

Figure Lengend Snippet: PCI34051 or Hdac8 knockdown mitigates TGF- β 1-induced fibrosis in rat cardiac fibroblasts. (a–e) Rat cardiac fibroblasts were pre-treated with TGF– β 1 (10 ng/mL) for 1 h and cultured in the presence of vehicle or PCI34051 (10 μ M) for 8 h. mRNA levels of Hdac8 , Col1a1 , Fn1 , Acta2 , and Tgfb1 were evaluated using quantitative real-time polymerase chain reaction (qRT-PCR) ( n = 4–6 per group). (f–k) Representative immunoblots and quantification of Hdac8, Fn1, Acta2, Tgfb1, p-Smad2/3, and Smad2/3 levels in rat cardiac fibroblasts ( n = 6 per group). Actb was used as a loading control. (l–p) Rat cardiac fibroblasts were transfected with control or short-interfering RNA against Hdac8 and treated with TGF- β 1 (10 ng/mL) for 9 h. The mRNA levels of Hdac8 , Fn1 , Acta2 , Tgfb1 , and Ace1 were determined using qRT-PCR ( n = 5 per group). (q–w) Representative immunoblots and quantification of Hdac8, Ace1, Fn1, Acta2, Tgfb1, p-Smad2/3, and Smad2/3 levels in rat cardiac fibroblasts ( n = 4 per group). Actb was used as a loading control.

Article Snippet: Anti-phospho-Smad2/3 (8828), anti-Smad2/3 (3102), anti-Rela (8242), and anti-Nfkbia (4812) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Cell Culture, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Transfection, Small Interfering RNA

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Hdac8 Inhibitor Alleviates Transverse Aortic Constriction-Induced Heart Failure in Mice by Downregulating Ace1

doi: 10.1155/2022/6227330

Figure Lengend Snippet: Suppression of HDAC8 attenuates fibrosis, inflammation, cardiac dysfunction, and pulmonary congestion in heart failure. Schematic diagram depicting the Hdac8 selective inhibitor- (PCI34051-) mediated or Hdac8 short-interfering RNA-mediated inhibition of transverse aortic constriction- (TAC-) induced heart failure. TAC-mediated pressure overload upregulates the expression of Hdac8 and Ace1 and downregulates the expression of Ace2 in the heart and lungs. In cardiomyocytes, overexpression of Hdac8 upregulates the expression of Ace1 and downregulates the expression of Ace2. PCI34051 treatment or Hdac8 knockdown mitigates TGF- β 1-induced upregulation of p-Smad2/3, Col1a1, Fn1, and Acta2 in vitro. PCI34051 regulates the TNF α -mediated or Ace1 overexpression-mediated expression of Rela and Nfkbia. PCI34051 alleviates cardiac hypertrophy and dysfunction, inflammation, fibrosis, and pulmonary congestion in heart failure.

Article Snippet: Anti-phospho-Smad2/3 (8828), anti-Smad2/3 (3102), anti-Rela (8242), and anti-Nfkbia (4812) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Small Interfering RNA, Inhibition, Expressing, Over Expression, In Vitro

Journal: International Journal of Biological Sciences

Article Title: Soluble Receptor for Advanced Glycation End-products regulates age-associated Cardiac Fibrosis

doi: 10.7150/ijbs.56379

Figure Lengend Snippet: RAGE deficiency induces age-associated TGF-β signaling. (A) Relative expression of Tgfb1 gene. Young WT n=4, Young Rage-/- n=4-5, Middle-age (MA) WT n=4, MA Rage-/- n=4, Old WT n=5, Old Rage-/- n=6. (B) (Left panel) Representative images of LV sections stained with an anti-Smad2-3 antibody. Bar, 20 µm. (Right panel) Quantification of Smad2/3 signal. Young WT n=5, Young Rage-/- n=5, MA WT n=4, MA Rage-/- n=4, Old WT n=4, Old Rage-/- n=5. (C) (Left panel) Representative images of LV sections stained with an anti-TFGbetaR1 antibody. Bar, 20 µm. (Right panel) Quantification of TFGbetaR1 signal. Young WT n=5, Young Rage-/- n=5, MA WT n=4, MA Rage-/- n=4, Old WT n=4, Old Rage-/- n=5. Each dot represents a mouse; mean ± SD are shown (*, P<0.05; **, P<0.01; ***, P<0.001; 2-way ANOVA plus Bonferroni post-hoc test for multiple comparisons).

Article Snippet: One hundred thousand cells were collected, washed with PBS, fixed and permeabilized with BD Cytofix/Cytoperm™ fixation/permeabilization Kit (#554714, BD Bioscience) and stained with primary antibodies for TGFbetaR1 (1 µg/10 6 cells, ABF17-I, Sigma-Aldrich) and P-Smad2-3 (1:800, #8828, Cell Signaling Technology) for 30 minutes at rt.

Techniques: Expressing, Staining

Journal: International Journal of Biological Sciences

Article Title: Soluble Receptor for Advanced Glycation End-products regulates age-associated Cardiac Fibrosis

doi: 10.7150/ijbs.56379

Figure Lengend Snippet: Recombinant sRAGE reduces TGFbetaR1 expression and inhibits Smad2/3 signaling in hcFbs. HcFbs were stimulated with nothing (Ctrl) or the indicated concentrations of sRAGE for 4, 8 or 12 h. TGF-β1 (10 ng/ml) was used as positive control when indicated. (A) TGF-β1 released in the supernatant of cells determined by ELISA; n=6. (B) TFGbetaR1 expression determined by FACS analysis; n=3-8. (C) Quantification of P-Smad2-3 expression determined by FACS analysis 4 h after stimulation; n=3. (D) (Left panel) Representative immunofluorescence images for P-Smad2-3 (green) 4 h after stimulation; nuclei were stained with Hoechst (blue). Bar, 50 μm. (Right panel) Quantification of nuclear P-Smad2-3 expression; n=3 Each dot represents a biological replicate; mean ± SD are shown (*, P<0.05; **, P<0.01; ***, P<0.001; t-test or 1-way ANOVA plus Bonferroni post-hoc test for multiple comparisons between Ctrl and sRAGE at the same time point; dotted line: t-test between Ctrl and TGF-β1).

Article Snippet: One hundred thousand cells were collected, washed with PBS, fixed and permeabilized with BD Cytofix/Cytoperm™ fixation/permeabilization Kit (#554714, BD Bioscience) and stained with primary antibodies for TGFbetaR1 (1 µg/10 6 cells, ABF17-I, Sigma-Aldrich) and P-Smad2-3 (1:800, #8828, Cell Signaling Technology) for 30 minutes at rt.

Techniques: Recombinant, Expressing, Positive Control, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Staining

Journal: Molecular Vision

Article Title: Nintedanib inhibits TGF-β-induced myofibroblast transdifferentiation in human Tenon’s fibroblasts

doi:

Figure Lengend Snippet: Nintedanib suppresses Smad2/3, p38MAPK, and ERK1/2 activation induced by transforming growth factor β1 (TGF-β1) at later timepoints. Human Tenon’s fibroblasts (HTFs) were treated with TGF-β1 (5 ng/ml) in the presence or absence of SB203580 or nintedanib (1 μM) for 24 h. TGF-β1 and p-Smad2/3 ( A ), p-p38MAPK and p-ERK1/2 ( B ) mRNA and protein expression were analyzed by quantitative reverse transcription polymerase chain reaction (RT-PCR) and western blot, respectively, and normalized to total Smad2/3, ERK1/2, or GAPDH. Data are presented as the means ± standard deviations (SDs; n = 3) of independent repeated experiments (* p <0.01;**** p <0.0001 compared with the control or TGF-β1 group).

Article Snippet: Protein samples were separated on sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) gels and transferred to polyvinylidene fluoride (PVDF) membranes (Thermo Scientific), which were then probed overnight with primary antibodies against human α smooth muscle actin (α-SMA; ab5694, 1:1,000, Abcam, Cambridge, UK), TGF-β1 (sc-130348, 1:1,000, Santa Cruz Biotechnology, Dallas, TX), Smad2 (#5339, 1:1,000, Cell Signaling Technology, Danvers, MA), Smad3 (#9523, 1:1,000, Cell Signaling Technology), phospho-Smad2/3 (#8828, 1:1,000, Cell Signaling Technology), p38MAPK (#8690, 1:1,000, Cell Signaling Technology), phospho-p38MAPK (#4511, 1:1,000, Cell Signaling Technology), ERK1/2 (#4695, 1:1,000, Cell Signaling Technology), phospho-ERK1/2 (#4370, 1:1,000, Cell Signaling Technology), Snail (#3879, 1:1,000, Cell Signaling Technology), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; ab9485, 1:1,000, Abcam), followed by incubating with horseradish peroxidase–conjugated secondary antibodies (sc-2375, 1:2,000, Santa Cruz Biotechnology) for 1 h at room temperature.

Techniques: Activation Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: Journal of Cancer

Article Title: Identification of a Novel Compound That Suppresses Breast Cancer Invasiveness by Inhibiting Transforming Growth Factor-β Signaling via Estrogen Receptor α

doi: 10.7150/jca.7202

Figure Lengend Snippet: N-23 suppresses TGF-β signaling by inhibiting TGF-β-dependent recruitment of Smad3 to the PAI-1 promoter. ( A ) N-23 does not affect pSmad protein levels. MCF-7 cells were treated with or without TGF-β (1 ng/mL), E2 (10 -8 M), or N-23 (10 -6 M). Western blotting was used to examine the levels of pSmad2, pSmad3, Smad2, Smad3, and β-Actin. ( B ) N-23 inhibits TGF-β-dependent recruitment of Smad3 to the PAI-1 promoter. MCF-7 cells were cultured in the presence or absence of TGF-β (1 ng/mL), E2 (10 -8 M), or N-23 (10 -6 M). A ChIP assay was performed with control IgG or anti-Smad3 antibodies. Immunoprecipitated DNA was examined using real-time RT-PCR and primers specific for the PAI-1 promoter. Samples were normalized to the amount of input DNA. ** indicates p < 0.01 and * indicates p < 0.05.

Article Snippet: The antibodies used in this study were: anti-Smad2/3 (BD), Smad3 (Abcam), phospho-Smad2 (Cell Signaling), phospho-Smad3 (BIOSOURCE) and β-actin (Sigma) antibodies.

Techniques: Western Blot, Cell Culture, Immunoprecipitation, Quantitative RT-PCR

Journal: Journal of Enzyme Inhibition and Medicinal Chemistry

Article Title: Pentabromopseudilin: a myosin V inhibitor suppresses TGF- β activity by recruiting the type II TGF- β receptor to lysosomal degradation

doi: 10.1080/14756366.2018.1465416

Figure Lengend Snippet: MyoVa depletion enhances T β RII turnover and mitigates TGF- β /Smad signalling. A549 cells were infected with the lentivirus carrying control (Ctr shRNA) or MyoVa shRNA constructs (MyoVa shRNA#1 and MyoVa shRNA#2). Three days post-infection, total protein was extracted and subjected to Western blot using anti-MyoVa, anti-T β RII or anti- β -actin antibodies. (A) Two MyoVa shRNAs abolish MyoVa protein production and reduced the T β RII protein levels. (B) Effects of MyoVa on TGF- β -induced Smad2/3 phosphorylation. A549 cells harbouring MyoVa shRNA#1 or control shRNA were stimulated with 5, 10, 20, 50 and 100 pM of TGF- β for 30 min. The amount of pSmad2/3 obtained from the lysates of cells in the absence (Lanes 8–12) or presence (Lanes 1–7) of MyoVa was analysed through Western blotting using anti-pSmad2/3, anti-Smad2/3 and anti- β -actin were used as loading control. (C) MyoVa depletion inhibits TGF- β -induced fibronectin, PAI-1, and N-cadherin expression. Control and MyoVa-silenced A549 cells were treated with 5, 10, 20, 50 and 100 pM of TGF- β for 48 h. The protein abundance of cells in the absence (Lanes 8–12) or presence (Lanes 1–7) of MyoVa was analysed through Western blotting using appropriate antibodies. Right graphs illustrate quantitative analyses of ECL (mean ± SD) from at least three independent experiments; ** p < .01.

Article Snippet: Rabbit polyclonal antibodies against pSmad2/3 (#8828), Smad2/3 (#8685), PAI-1 (#11907), Flag tag (#8146), Lamp-1 (sc-9091) and MyoVa (#3402) were purchased from Cell Signalling (Boston, MA).

Techniques: Infection, shRNA, Construct, Western Blot, Expressing

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Qingjie Fuzheng Granule Inhibited the Migration and Invasion of Colorectal Cancer Cells by Regulating the lncRNA ANRIL/let-7a/TGF- β 1/Smad Axis

doi: 10.1155/2020/5264651

Figure Lengend Snippet: The effect of QFG treatment on the expression of proteins in the TGF- β 1/Smad signaling pathway in HCT-8 and HCT116 cells. HCT-8 and HCT116 cells were incubated with QFG (0, 0.5, 1, or 2 mg/mL) for 24 h. (a) Protein expression of TGF- β 1, p-Smad2/3, Smad2/3, Smad4, N-cadherin, and E-cadherin, as determined by western blotting. The internal control used was β -actin. Images are representative of three independent experiments. (b) Relative densitometric analysis of the above protein is displayed. Δ P < 0.05, and ∗ P < 0.01 vs. untreated cells.

Article Snippet: Then, the proteins were probed overnight at 4°C using primary antibodies against TGF- β 1 (Cat no: 3711), phosphorylated (p-) Smad2/3 (Cat no: 8828), Smad2/3 (Cat no: 8685), and Smad4 (Cat no: 38454) (all from Cell Signaling Technology, Inc., Beverly, MA, USA; all diluted 1 : 1,000); against N-cadherin (Cat no: ab18203; diluted 1 : 1,000) and E-cadherin (Cat no: ab1416; diluted 1 : 2,000) (both from Abcam, Cambridge, UK); and against β -actin (Cat no: 66009-1-Ig; Proteintech Group Inc., Chicago, IL, USA; diluted 1 : 5,000).

Techniques: Expressing, Incubation, Western Blot