Journal: bioRxiv

Article Title: Sterol-O acyltransferase 1 is inhibited by gga-miR-181a-5p and gga-miR-429-3p through the TGFβ pathway in endodermal epithelial cells of Japanese quail

doi: 10.1101/569806

Figure Lengend Snippet: The validations of selected miRNAs to chicken TGFBR1 3’UTR. (A) The scheme of the constructed luciferase plasmid. (B) The relative luciferase expressions after miRNA mimic transfection. Transient transfection was conducted on 293T cells (3*10 4 cells/ well). The pmirGLO-WT-3’UTR plasmid (100 ng) was used, and co-treated with 5 nM miRNA mimics or siRNA (negative control, NC). Firefly and Renilla luminescence were detected after transfection for 24 hours. N= nine per group. Data were expressed as mean ± S.E.M. Transfection with pmirGLO-WT-3’UTR only was set as 1. All groups were compared with NC groups. Statistical significance was determined by one-way ANOVA. Dunnett’s multiple comparisons test was used to evaluate differences between means (*P ≤ 0.05). (C) The sequence alignment of gga-miR-181a-5p and gga-miR-429-3p with the binding sites of the chicken TGFBR1 3’UTR.

Article Snippet: TGFBR1 was detected with rat anti-mouse TGFBR1 primary antibody (1:300, sc-101574, Santa Cruz, Dallas, Texas, USA) and followed by incubation with anti-rat IgG HRP-linked secondary antibody (1:5000, bs-0293G-HRP, Bioss, Woburn, MA, USA).

Techniques: Construct, Luciferase, Plasmid Preparation, Transfection, Negative Control, Sequencing, Binding Assay

Journal: bioRxiv

Article Title: Sterol-O acyltransferase 1 is inhibited by gga-miR-181a-5p and gga-miR-429-3p through the TGFβ pathway in endodermal epithelial cells of Japanese quail

doi: 10.1101/569806

Figure Lengend Snippet: Target gene expressions after transient transfection for 48 hours using gga-miR-181a-5p or gga-miR-429-3p. The expressions of SOAT1 and TGFBR1 were analyzed by real-time PCR after 48 hours of miRNAs transfection. Data were expressed as mean ± S.E.M. N= nine per group. Control group was set as 1. All groups were compared with NC groups. Statistical significance was determined by one-way ANOVA. Dunnett’s multiple comparisons test was used to evaluate differences between means (*P≤0.05).

Article Snippet: TGFBR1 was detected with rat anti-mouse TGFBR1 primary antibody (1:300, sc-101574, Santa Cruz, Dallas, Texas, USA) and followed by incubation with anti-rat IgG HRP-linked secondary antibody (1:5000, bs-0293G-HRP, Bioss, Woburn, MA, USA).

Techniques: Transfection, Real-time Polymerase Chain Reaction

Journal: bioRxiv

Article Title: Sterol-O acyltransferase 1 is inhibited by gga-miR-181a-5p and gga-miR-429-3p through the TGFβ pathway in endodermal epithelial cells of Japanese quail

doi: 10.1101/569806

Figure Lengend Snippet: TGFBR1 is one of the direct target gene of gga-miR-181a-5p and gga-miR-429-3p. The relative luciferase expressions after miRNA mimics transfection. (A) Scheme of potential binding sites of gga-miR-181a-5p and gga-miR-429-3p on the wild- or mutated-type of chicken TGFBR1 3’UTR. (B) The transient transfection was conducted on 293T cells (3*10 4 cells/ well). The pmirGLO-WT-3’UTR plasmid and pmirGLO-MU-3’UTR plasmid (100 ng/ well) were used and co-treated with 5 nM miRNA mimics. siRNA served as the negative control (NC). Firefly and Renilla luminescence were detected after transfection for 24 hours. N=10 to 14 per group. Data were expressed as mean ± S.E.M. All groups were compared with the NC group. Statistical significance was determined by one-way ANOVA. Dunnett’s multiple comparison test was used to evaluate differences between means. A significant difference (*P≤0.05 or **P≤0.01) was indicated.

Article Snippet: TGFBR1 was detected with rat anti-mouse TGFBR1 primary antibody (1:300, sc-101574, Santa Cruz, Dallas, Texas, USA) and followed by incubation with anti-rat IgG HRP-linked secondary antibody (1:5000, bs-0293G-HRP, Bioss, Woburn, MA, USA).

Techniques: Luciferase, Transfection, Binding Assay, Plasmid Preparation, Negative Control

Journal: bioRxiv

Article Title: Sterol-O acyltransferase 1 is inhibited by gga-miR-181a-5p and gga-miR-429-3p through the TGFβ pathway in endodermal epithelial cells of Japanese quail

doi: 10.1101/569806

Figure Lengend Snippet: The SOAT1 and TGFBR1 protein levels after transfections with gga-miR-181a-5p or gga-miR-429-3p. EECs were transfected by miRNAs mimic for 48 hours and extracted for western blotting analysis. Total density of SOAT1 or TGFBR1 were normalized by total density of β-actin. Data were expressed as mean ± S.E.M. N = five to nine per group. C = group of no transfection in EECs, Mock = group of transfections with reagent only, NC = negative control group, the group of transfections with AllStars Negative Control siRNA. Others = groups of miRNAs transfections (5 nM or 30 nM miRNA mimics). Control group was set as 1. All groups were compared with NC groups. Statistical significance was determined by one-way ANOVA. Dunnett’s multiple comparison test was used to evaluate differences between means. A significant difference (*P≤0.05 or **P≤0.01) was indicated.

Article Snippet: TGFBR1 was detected with rat anti-mouse TGFBR1 primary antibody (1:300, sc-101574, Santa Cruz, Dallas, Texas, USA) and followed by incubation with anti-rat IgG HRP-linked secondary antibody (1:5000, bs-0293G-HRP, Bioss, Woburn, MA, USA).

Techniques: Transfection, Western Blot, Negative Control

Journal: bioRxiv

Article Title: Sterol-O acyltransferase 1 is inhibited by gga-miR-181a-5p and gga-miR-429-3p through the TGFβ pathway in endodermal epithelial cells of Japanese quail

doi: 10.1101/569806

Figure Lengend Snippet: The possible relationship between miRNAs, TGFβ signaling pathway and SOAT1 expressions. The TGFβ signaling pathway is activated when the ligand (e.g., TGFβ1) binds to TGFBR2, TGFBR2 phosphorylates TGFBR1. The signal transmitter Smad2/3 is phosphorylated by TGFBR1 and joins with phosphorylated Smad4 to form SMAD complex in cytoplasm. The SMAD complex then enters nucleus to target to transcription factor binding site to affect SOAT1 gene expression. However, the gga-miR-181a-5p and gga-miR-429-3p both have the inhibitory ability on TGFBR1, and then decrease the SOAT1 expression. Although the gga-miR-133a-5p is found to attenuate SOAT1 expression, the effect is independent of the TGFβ signaling pathway.

Article Snippet: TGFBR1 was detected with rat anti-mouse TGFBR1 primary antibody (1:300, sc-101574, Santa Cruz, Dallas, Texas, USA) and followed by incubation with anti-rat IgG HRP-linked secondary antibody (1:5000, bs-0293G-HRP, Bioss, Woburn, MA, USA).

Techniques: Binding Assay, Expressing

Journal: The European respiratory journal

Article Title: The transforming growth factor-beta/Smad2,3 signalling axis is impaired in experimental pulmonary hypertension.

doi: 10.1183/09031936.00138206

Figure Lengend Snippet: FIGURE 3. Localisation of: a, b) transforming growth factor-b receptor (TFGBR)-1; c, d) TGFBR-2; e, f) Smad2; g, h) Smad3; and i, j) Smad4 in the airways of lungs from

Article Snippet: Elastin and haematoxylin and eosin staining, and immunofluorescence were performed on 3 mm tissue sections [18], with antibodies against smooth muscle actin (1:850; Sigma, Taufkirchen, Germany), von Willebrand factor (vwf; 1:800; Dako, Hamburg, Germany), TGFBR-1 (R-20), TGFBR-2 (H-567) and proliferating cell nuclear antigen (PCNA; all at 1:100; Santa Cruz), Smad3 and Smad4 (both at 1:50; Upstate), Smad2 (R&D Systems) and phospho-Smad2 (Cell Signaling Technology).

Techniques:

Journal: The European respiratory journal

Article Title: The transforming growth factor-beta/Smad2,3 signalling axis is impaired in experimental pulmonary hypertension.

doi: 10.1183/09031936.00138206

Figure Lengend Snippet: FIGURE 4. Localisation of: a–d) transforming growth factor-b receptor (TGFBR)-1; e–h) TGFBR-2; i–l) Smad4; m–o) Smad3; and p–r) Smad2 in pulmonary resistance

Article Snippet: Elastin and haematoxylin and eosin staining, and immunofluorescence were performed on 3 mm tissue sections [18], with antibodies against smooth muscle actin (1:850; Sigma, Taufkirchen, Germany), von Willebrand factor (vwf; 1:800; Dako, Hamburg, Germany), TGFBR-1 (R-20), TGFBR-2 (H-567) and proliferating cell nuclear antigen (PCNA; all at 1:100; Santa Cruz), Smad3 and Smad4 (both at 1:50; Upstate), Smad2 (R&D Systems) and phospho-Smad2 (Cell Signaling Technology).

Techniques:

Journal: International journal of oncology

Article Title: MicroRNA-98-5p inhibits proliferation and metastasis in non-small cell lung cancer by targeting TGFBR1.

doi: 10.3892/ijo.2018.4610

Figure Lengend Snippet: Figure 6. TGFBR1 is a target gene of miR‑98‑5p. (A) TGFBR1 is predicted to be a target of miR‑98‑5p. (B and C) Changes in mRNA and protein levels of TGFBR1 following transfection with miR‑98‑5p mimic in A549 cells. (D and E) Changes in mRNA and protein levels of TGFBR1 following transfection with miR‑98‑5p inhibitor in GLC‑82 cells. (F) TGFBR1 3’‑UTR luciferase reporter assays in A549 cells. TGFBR1, transforming growth factor beta receptor 1. **P<0.01 and ***P<0.001 vs. miR‑control.

Article Snippet: For immunohistochemistry, the sections were respectively incubated with primary rat anti-mouse antibody Ki-67 (9449; CST; dilution: 1:400), MMP-9 (13667S; CST; dilution: 1:325) and TGFBR1 (70R-36484; Fitzgerald Industries International; dilution: 1:200), and then treated with secondary antibody biotinylated goat anti-rat immunoglobulin (Abcam, Cambridge, MA, USA).

Techniques: Transfection, Luciferase

Journal: International journal of oncology

Article Title: MicroRNA-98-5p inhibits proliferation and metastasis in non-small cell lung cancer by targeting TGFBR1.

doi: 10.3892/ijo.2018.4610

Figure Lengend Snippet: Figure 7. miR‑98‑5p inhibits tumor growth in vivo. (A) Representative images of tumors after different treatments at day 21. (B) Inhibition of tumor growth in the subcutaneous xenograft tumor model of A549 cells after the different treatments. (C) Weight of tumors after different treatments at day 21. (D) H&E evaluation and immunohistochemistry analysis of Ki‑67, MMP‑9 and TGFBR1 in tumor sections after different treatments. Magnification, x200. TGFBR1, transforming growth factor beta receptor 1. **P<0.01 vs. control.

Article Snippet: For immunohistochemistry, the sections were respectively incubated with primary rat anti-mouse antibody Ki-67 (9449; CST; dilution: 1:400), MMP-9 (13667S; CST; dilution: 1:325) and TGFBR1 (70R-36484; Fitzgerald Industries International; dilution: 1:200), and then treated with secondary antibody biotinylated goat anti-rat immunoglobulin (Abcam, Cambridge, MA, USA).

Techniques: In Vivo, Inhibition, Immunohistochemistry, Control