Journal: Genes

Article Title: Regulation of Smad2/3 Nuclear Exclusion by Follicle-Stimulating Hormone (FSH) in Chicken Follicular Granulosa Cells and Its Effect on FOXO3/4

doi: 10.3390/genes16030283

Figure Lengend Snippet: Primer pairs designed for quantitative real-time PCR analysis.

Article Snippet: Primary antibodies against Smad2 (Bioss, Wuhan, China, 1:100 dilution) and Smad3 (Bioss, Beijing, China, 1:100 dilution) were applied to the cells, and the cells were incubated at 4 °C for 12 h. Following phosphate buffer saline washes, the cells were treated with a fluorescently conjugated goat anti-rabbit IgG antibody (Epizyme Biotech, Shanghai, China, 1:1000 dilution).

Techniques: Real-time Polymerase Chain Reaction

Journal: Genes

Article Title: Regulation of Smad2/3 Nuclear Exclusion by Follicle-Stimulating Hormone (FSH) in Chicken Follicular Granulosa Cells and Its Effect on FOXO3/4

doi: 10.3390/genes16030283

Figure Lengend Snippet: The nuclear exclusion of Smad2/3 in the cultured granulosa cells (GCs) of the ovarian follicles. ( A ) Immunofluorescence detection of Smad2 subcellular localization in the GCs. Smad2 is shown in green, and nucleus is stained with 4′,6-diamidino-2-phenylindole (DAPI) (blue). ( B ) Immunofluorescence detection of Smad3 subcellular localization in the GCs. Smad3 is shown in green, and nucleus is stained with DAPI (blue). a. control, without any treatment; b. treatment of the cells with 10 ng/mL follicle-stimulating hormone (FSH) for 12 h; c. co-treatment of the cells with 10 ng/mL FSH and phosphatidylinositol 3-kinase (PI3K) inhibitor for 12 h. d. control, without any treatment; e. treatment of the cells with 50 μg/mL PI3K activator for 12 h; f. co-treatment of the cells with 50 μg/mL PI3K activator and 50 μg/mL alkaline phosphatase for 12 h. n = 3 independent experiments. Leica DMI8, 400×; scale bar = 5 μm.

Article Snippet: Primary antibodies against Smad2 (Bioss, Wuhan, China, 1:100 dilution) and Smad3 (Bioss, Beijing, China, 1:100 dilution) were applied to the cells, and the cells were incubated at 4 °C for 12 h. Following phosphate buffer saline washes, the cells were treated with a fluorescently conjugated goat anti-rabbit IgG antibody (Epizyme Biotech, Shanghai, China, 1:1000 dilution).

Techniques: Cell Culture, Immunofluorescence, Staining, Control

Journal: Genes

Article Title: Regulation of Smad2/3 Nuclear Exclusion by Follicle-Stimulating Hormone (FSH) in Chicken Follicular Granulosa Cells and Its Effect on FOXO3/4

doi: 10.3390/genes16030283

Figure Lengend Snippet: Smad2/3 phosphorylation in the cultured GCs of the ovarian follicles. ( A ) The Smad2 and p-Smad2 levels were determined by Western blotting analysis in the GCs without any treatment, treated with 10 ng/mL FSH for 12 h, and co-treated with 10 ng/mL FSH and 16 nM/mL PI3K inhibitor for 12 h. ( B ) The Smad3 and p-Smad3 levels were tested in the GCs without any treatment, treated with 10 ng/mL FSH for 12 h, and co-treated with 10 ng/mL FSH and 16 nM/mL PI3K inhibitor for 12 h. ( C ) The Smad2 and p-Smad2 levels were determined by Western blotting analysis in the GCs without any treatment, treated with 50 μg/mL PI3K activator for 12 h, and co-treated with 50 μg/mL PI3K activator and 50 μg/mL alkaline phosphatase for 12 h. ( D ) The Smad3 and p-Smad3 levels were tested in the GCs under the following conditions: without any treatment, treated with 50 μg/mL PI3K activator for 12 h, and co-treated with 50 μg/mL PI3K activator and 50 μg/mL alkaline phosphatase for 12 h. The data represent the mean ± SEM ( n = 3). Student’s t -test, ** p < 0.01 and * p < 0.05.

Article Snippet: Primary antibodies against Smad2 (Bioss, Wuhan, China, 1:100 dilution) and Smad3 (Bioss, Beijing, China, 1:100 dilution) were applied to the cells, and the cells were incubated at 4 °C for 12 h. Following phosphate buffer saline washes, the cells were treated with a fluorescently conjugated goat anti-rabbit IgG antibody (Epizyme Biotech, Shanghai, China, 1:1000 dilution).

Techniques: Cell Culture, Western Blot

Journal: Genes

Article Title: Regulation of Smad2/3 Nuclear Exclusion by Follicle-Stimulating Hormone (FSH) in Chicken Follicular Granulosa Cells and Its Effect on FOXO3/4

doi: 10.3390/genes16030283

Figure Lengend Snippet: Detection of Smad2/3 interference and overexpression vector transfection effect. ( A ) The mRNA levels of Smad2/3 were determined by qRT-PCR in GCs under interference and overexpression plasmid transfection and non-transfection conditions. The data represent mean ± SEM ( n = 5). Student’s t -test, ** p < 0.01 and * p < 0.05. ( B ) The protein levels of Smad2/3 were determined by Western blotting analysis in GCs under interference and overexpression plasmid transfection and non-transfection conditions. The data represent mean ± SEM ( n = 3). Student’s t -test, ** p < 0.01 and * p < 0.05.

Article Snippet: Primary antibodies against Smad2 (Bioss, Wuhan, China, 1:100 dilution) and Smad3 (Bioss, Beijing, China, 1:100 dilution) were applied to the cells, and the cells were incubated at 4 °C for 12 h. Following phosphate buffer saline washes, the cells were treated with a fluorescently conjugated goat anti-rabbit IgG antibody (Epizyme Biotech, Shanghai, China, 1:1000 dilution).

Techniques: Over Expression, Plasmid Preparation, Transfection, Quantitative RT-PCR, Western Blot

Journal: Genes

Article Title: Regulation of Smad2/3 Nuclear Exclusion by Follicle-Stimulating Hormone (FSH) in Chicken Follicular Granulosa Cells and Its Effect on FOXO3/4

doi: 10.3390/genes16030283

Figure Lengend Snippet: Effects of Smad2 on the forkhead box O 3 and 4 (FoxO3/4) phosphorylation in the cultured GCs of the ovarian follicles. The FOXO3/4 and p-FOXO3/4 levels were determined by Western blotting analysis in the GCs with Smad2 overexpression and interference. The two treatment groups were compared with the control group separately, but there was no comparative analysis between the treatment groups. The data represent mean ± SEM ( n = 3). Student’s t -test, ** p < 0.01 and * p < 0.05.

Article Snippet: Primary antibodies against Smad2 (Bioss, Wuhan, China, 1:100 dilution) and Smad3 (Bioss, Beijing, China, 1:100 dilution) were applied to the cells, and the cells were incubated at 4 °C for 12 h. Following phosphate buffer saline washes, the cells were treated with a fluorescently conjugated goat anti-rabbit IgG antibody (Epizyme Biotech, Shanghai, China, 1:1000 dilution).

Techniques: Cell Culture, Western Blot, Over Expression, Control

Journal: Journal of Inflammation Research

Article Title: Saccharomyces boulardii Alleviates Colitis by Regulating FXR-NLRP3 Mediated Macrophage Pyroptosis

doi: 10.2147/JIR.S504957

Figure Lengend Snippet: SbS administration activated FXR and inhibited NLRP3 expression in DSS-induced mice. ( A and B ) The mRNA levels of FXR and NLRP3 in the colon tissue were detected by RT-PCR. ( C ) The expression of colonic FGF15 was assessed by Immunofluorescence staining and visualized by fluorescence microscopy (red), the nucleus was counterstained with DAPI (blue) (scale bars: 100 µm). ( D and E ) Western blot analysis was applied to measure the expression of FXR, FGF15 and NLRP3, and the relative intensity was quantified by Image J software. All data were presented as mean ±SEM. *P < 0.05, ***P < 0.001, ****P<0.0001. ns, not significant.

Article Snippet: After being blocked with 10% skim milk or bovine serum albumin, membranes comprising proteins were incubated with primary anti-β-actin antibody (Cat#mAb3700, CST, 1:1000), anti-ZO-1 (Cat#A0659, ABclonal, 1:1000), anti-Claudin3 (Cat#83609, CST, 1:1000) antibodies, primary anti-FXR antibody (abs122163, Absin, 1:1200), anti-FGF15 (sc514647, Santa Cruze Biotechnology, 1:1000), anti-caspase-1 antibody (ab179515, Abcam, 1:1000), anti-GSDMD antibody (1:1000, ab209845, Abcam), anti-IL-1β antibody (#12242, Cell Signaling Technology,1:1000), and anti-IL-18 antibody (ab71495, Abcam, 1:1000) overnight at 4 °C.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Immunofluorescence, Staining, Fluorescence, Microscopy, Western Blot, Software

Journal: Journal of Inflammation Research

Article Title: Saccharomyces boulardii Alleviates Colitis by Regulating FXR-NLRP3 Mediated Macrophage Pyroptosis

doi: 10.2147/JIR.S504957

Figure Lengend Snippet: SbS administration ameliorated pyroptosis in THP-1 cells. ( A-D ) The expression level of FXR was measured by Western blot analysis in THP-1 cells ( A and B ) and RAW 264.7 cells ( C and D ) treated with SDB or SbS for 24 hours. ( E and F ) SDB or SbS was administrated for 24 hours in THP-1 cells before treated with 100ng/mL LPS for 4–6h and then 10μg/mL nigericin for 1 hour to induce pyroptosis of THP-1 cells. The mRNA levels of pro-inflammatory cytokines IL-1β ( E ) and IL-18 ( F ) in THP-1 cells were detected by real-time PCR. ( G-J ) The protein levels of FXR and the related indicators of pyroptosis pathway (NLRP3, caspase-1, GSDMD, IL-1β and IL-18) were measured by Western blot and the relative intensity was quantified by Image J software. All data were presented as mean ±SEM. *P < 0.05, **P < 0.01, ***P < 0.001. ns, not significant.

Article Snippet: After being blocked with 10% skim milk or bovine serum albumin, membranes comprising proteins were incubated with primary anti-β-actin antibody (Cat#mAb3700, CST, 1:1000), anti-ZO-1 (Cat#A0659, ABclonal, 1:1000), anti-Claudin3 (Cat#83609, CST, 1:1000) antibodies, primary anti-FXR antibody (abs122163, Absin, 1:1200), anti-FGF15 (sc514647, Santa Cruze Biotechnology, 1:1000), anti-caspase-1 antibody (ab179515, Abcam, 1:1000), anti-GSDMD antibody (1:1000, ab209845, Abcam), anti-IL-1β antibody (#12242, Cell Signaling Technology,1:1000), and anti-IL-18 antibody (ab71495, Abcam, 1:1000) overnight at 4 °C.

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

Journal: Journal of Inflammation Research

Article Title: Saccharomyces boulardii Alleviates Colitis by Regulating FXR-NLRP3 Mediated Macrophage Pyroptosis

doi: 10.2147/JIR.S504957

Figure Lengend Snippet: FXR inhibitor abolished the therapeutic effect of SbS. To induce pyroptosis in THP-1 cells, FXR inhibitor (GS) was administered to THP-1 cells for 18 hours, followed by the administration of SDB or SbS for 24 hours, 100ng/mL LPS for 4–6 hours, and 10μg/mL nigericin for 1 hour. ( A-C ) The protein levels of NLRP3, caspase-1, GSDMD, IL-1β and IL-18 in the pyroptosis pathway were measured by Western blot and the relative intensity was quantified by Image J software. All data were presented as mean ±SEM. *P < 0.05, **P < 0.01, ***P<0.001. ns, not significant.

Article Snippet: After being blocked with 10% skim milk or bovine serum albumin, membranes comprising proteins were incubated with primary anti-β-actin antibody (Cat#mAb3700, CST, 1:1000), anti-ZO-1 (Cat#A0659, ABclonal, 1:1000), anti-Claudin3 (Cat#83609, CST, 1:1000) antibodies, primary anti-FXR antibody (abs122163, Absin, 1:1200), anti-FGF15 (sc514647, Santa Cruze Biotechnology, 1:1000), anti-caspase-1 antibody (ab179515, Abcam, 1:1000), anti-GSDMD antibody (1:1000, ab209845, Abcam), anti-IL-1β antibody (#12242, Cell Signaling Technology,1:1000), and anti-IL-18 antibody (ab71495, Abcam, 1:1000) overnight at 4 °C.

Techniques: Western Blot, Software

Journal: Frontiers in Pharmacology

Article Title: Qinggan Yipi capsule ameliorates hepatic fibrosis in rats by down-regulating the TGF-β1/Smad2/3 signaling pathway and improving gut microbiota imbalance

doi: 10.3389/fphar.2025.1525914

Figure Lengend Snippet: Primers for quantitative polymerase chain reaction of liver tissue.

Article Snippet: Primary antibodies to Smad2 (19245S), Smad3 (3700S), p-Smad2 (5339S), p-Smad3 (9523S), α-SMA (18338S), β-Actin (9520S), and the secondary antibodies Anti-rabbit IgG (7074S) and Anti-mouse IgG (7076S) were bought from Cell Signaling Technology (Boston, United States).

Techniques: Real-time Polymerase Chain Reaction

Journal: Frontiers in Pharmacology

Article Title: Qinggan Yipi capsule ameliorates hepatic fibrosis in rats by down-regulating the TGF-β1/Smad2/3 signaling pathway and improving gut microbiota imbalance

doi: 10.3389/fphar.2025.1525914

Figure Lengend Snippet: Primers for quantitative polymerase chain reaction of HSCs.

Article Snippet: Primary antibodies to Smad2 (19245S), Smad3 (3700S), p-Smad2 (5339S), p-Smad3 (9523S), α-SMA (18338S), β-Actin (9520S), and the secondary antibodies Anti-rabbit IgG (7074S) and Anti-mouse IgG (7076S) were bought from Cell Signaling Technology (Boston, United States).

Techniques: Real-time Polymerase Chain Reaction

Journal: Frontiers in Pharmacology

Article Title: Qinggan Yipi capsule ameliorates hepatic fibrosis in rats by down-regulating the TGF-β1/Smad2/3 signaling pathway and improving gut microbiota imbalance

doi: 10.3389/fphar.2025.1525914

Figure Lengend Snippet: QgYp downregulates the TGF-β1/Smad2/3 signaling pathway and inhibits the activation of HSCs in rat liver. (A–C) Effects of QgYp on the expression of Smad2, Smad3, and TGF-β1 mRNA in the liver tissues of each group of rats (n = 6). (D–I) Effects of QgYp on the expression of the proteins TGF-β1, Smad2, p-Smad2, p-Smad3, and α-SMA in the liver tissues of each group of rats (n = 3). The mean ± standard deviation is used to express data. * P < 0.05, *** P < 0.001 vs. control group; # P < 0.05, ## P < 0.01, ### P < 0.001 vs. model group; Δ P < 0.05, ΔΔ P < 0.01 vs. COL group.

Article Snippet: Primary antibodies to Smad2 (19245S), Smad3 (3700S), p-Smad2 (5339S), p-Smad3 (9523S), α-SMA (18338S), β-Actin (9520S), and the secondary antibodies Anti-rabbit IgG (7074S) and Anti-mouse IgG (7076S) were bought from Cell Signaling Technology (Boston, United States).

Techniques: Activation Assay, Expressing, Standard Deviation, Control

Journal: Frontiers in Pharmacology

Article Title: Qinggan Yipi capsule ameliorates hepatic fibrosis in rats by down-regulating the TGF-β1/Smad2/3 signaling pathway and improving gut microbiota imbalance

doi: 10.3389/fphar.2025.1525914

Figure Lengend Snippet: QgYp can inhibit TGF-β1-induced activation of HSC-T6 cells and downregulate the TGF-β1/Smad2/3 pathway. (A) Effects of various TGF-β1 concentrations on the growth of HSC-T6 cells. Effects of different concentrations of QgYp-containing serum (B) and COL-containing serum (C) on 10 μg L -1 TGF-β1-induced HSC-T6 cells. (D–F) Effects of QgYp on the levels of the proteins Col I and a-SMA in TGF-β1-induced HSC-T6 cells. (G, H) Effects of QgYp on TGF-β1-induced HSC-T6 cell a-SMA and Col I mRNA expression. The results are presented as the mean ± standard deviation of three parallel experiments. * P < 0.05, *** P < 0.001 vs. control group; # P < 0.05, ## P < 0.01, ### P < 0.001 vs. TGF-β1-induced group.

Article Snippet: Primary antibodies to Smad2 (19245S), Smad3 (3700S), p-Smad2 (5339S), p-Smad3 (9523S), α-SMA (18338S), β-Actin (9520S), and the secondary antibodies Anti-rabbit IgG (7074S) and Anti-mouse IgG (7076S) were bought from Cell Signaling Technology (Boston, United States).

Techniques: Activation Assay, Expressing, Standard Deviation, Control

Journal: Frontiers in Pharmacology

Article Title: Qinggan Yipi capsule ameliorates hepatic fibrosis in rats by down-regulating the TGF-β1/Smad2/3 signaling pathway and improving gut microbiota imbalance

doi: 10.3389/fphar.2025.1525914

Figure Lengend Snippet: Effects of QgYp and COL on the TGF-β1/Smad2/3 pathway in activated HSC-T6 cells. (A–D) Effects of QgYp on the expression of the proteins TGF-βR1, p-Smad2, and p-Smad3 in TGF-β1-induced HSC-T6 cells. (E–G) Effects of QgYp on the mRNA expression of TGF-βR1, Smad2 and Smad3 in TGF-β1-induced HSC-T6 cells. The results are presented as the mean ± standard deviation of three parallel experiments. ** P < 0.01, *** P < 0.001 vs. control group; # P < 0.05, ### P < 0.001vs TGF-β1-induced group.

Article Snippet: Primary antibodies to Smad2 (19245S), Smad3 (3700S), p-Smad2 (5339S), p-Smad3 (9523S), α-SMA (18338S), β-Actin (9520S), and the secondary antibodies Anti-rabbit IgG (7074S) and Anti-mouse IgG (7076S) were bought from Cell Signaling Technology (Boston, United States).

Techniques: Expressing, Standard Deviation, Control