Journal: Biology of Reproduction

Article Title: SMAD-dependent signaling mediates morphogenetic protein 6-induced stimulation of connective tissue growth factor in luteinized human granulosa cells †

doi: 10.1093/biolre/ioz108

Figure Lengend Snippet: BMP6 upregulates the expression of CTGF in hGL cells. A and B, SVOG cells were treated with a vehicle control or increasing concentrations (5, 20, or 100 ng/ml) of BMP6 for 3 h (A) or 6 h (B), and the mRNA (A) and protein (B) levels of CTGF were examined using RT-qPCR and western blotting, respectively. C and D, SVOG cells were treated with vehicle control or 30 ng/ml of BMP6 for 1, 3, 6, 12, or 24 h, and the mRNA (C) and protein (D) levels of CTGF were examined using RT-qPCR and western blotting, respectively. E and F, Primary hGL cells were treated with a vehicle control or increasing concentrations (5, 20, or 100 ng/ml) of BMP6 for 3 h (E) or 6 h (F), and the mRNA (E) and protein (F) levels of CTGF were examined using RT-qPCR and Western blot, respectively. The results are expressed as the mean ± SEM of at least three independent experiments. Values marked by different letters are significantly different (P < 0.05). B6, BMP6; Ctrl, control; hGL, primary human granulosa lutein.

Article Snippet: Antibodies and reagents Recombinant human BMP6 (#507-BP-020), recombinant human TGF-β1 (#240-B-010), dorsomorphin dihydrochloride (dorsomorphin) (#3093/10), DMH-1 (#4126/10), and SB431542 (#1614/10) were obtained from R&D Systems (Minneapolis, MN).

Techniques: Expressing, Quantitative RT-PCR, Western Blot

Journal: Biology of Reproduction

Article Title: SMAD-dependent signaling mediates morphogenetic protein 6-induced stimulation of connective tissue growth factor in luteinized human granulosa cells †

doi: 10.1093/biolre/ioz108

Figure Lengend Snippet: BMP6 does not affect the expression of CYR61 in hGL cells. (A and B) SVOG cells were treated with a vehicle control or increasing concentrations (5, 20, or 100 ng/ml) of BMP6 for 3 h (A) or 6 h (B), and the mRNA (A) and protein (B) levels of CYR61 were examined using RT-qPCR and western blotting, respectively. (C and D) SVOG cells were treated with vehicle control or 30 ng/ml of BMP6 for 1, 3, 6, 12, or 24 h, and the mRNA (C) and protein (D) levels of CYR61 were examined using RT-qPCR and western blotting, respectively. (E and F) Primary hGL cells were treated with a vehicle control or increasing concentrations (5, 20, or 100 ng/ml) of BMP6 for 3 h (E) or 6 h (F), and the mRNA (E) and protein (F) levels of CYR61 were examined using RT-qPCR and western blotting, respectively. The results are expressed as the mean ± SEM of at least three independent experiments. Values marked by different letters are significantly different (P < 0.05).

Article Snippet: Antibodies and reagents Recombinant human BMP6 (#507-BP-020), recombinant human TGF-β1 (#240-B-010), dorsomorphin dihydrochloride (dorsomorphin) (#3093/10), DMH-1 (#4126/10), and SB431542 (#1614/10) were obtained from R&D Systems (Minneapolis, MN).

Techniques: Expressing, Quantitative RT-PCR, Western Blot

Journal: Biology of Reproduction

Article Title: SMAD-dependent signaling mediates morphogenetic protein 6-induced stimulation of connective tissue growth factor in luteinized human granulosa cells †

doi: 10.1093/biolre/ioz108

Figure Lengend Snippet: BMP6 induces the phosphorylation of SMAD1/5/8 in SVOG cells. (A) SVOG cells were treated with a vehicle control or 30 ng/ml of BMP6 for 20, 40, or 60 min, and the phosphorylated protein levels of SMAD1/5/8 were examined using western blotting. (B) SVOG cells were pretreated with a vehicle control (DMSO), 5 μM dorsomorphin, or 5 μM DMH-1 for 60 min, and then treated with 30 ng/ml BMP6 for an additional 40 min. The phosphorylated protein levels of SMAD1/5/8 were examined using western blotting. (C) SVOG cells were treated with a vehicle control or 30 ng/ml BMP6 for 40 min, fixed in 4% paraformaldehyde in PBS, and examined for phosphorylated SMAD1/5/8 (red) and nuclear (DAPI in blue) immunofluorescence. (D) SVOG cells were treated with a vehicle control, 30 ng/ml of BMP6 (for 20, 40, or 60 min) or 5 ng/ml of TGF-β1 (for 20 min as a positive control) and the phosphorylated protein levels of SMAD2 and SMAD3 were examined using western blotting. (E) SVOG cells were pretreated with a vehicle control (DMSO), 5 μM dorsomorphin, or 5 μM DMH-1 for 60 min, and then treated with 30 ng/ml BMP6 for an additional 40 min. The phosphorylated protein levels of SMAD2 and SMAD3 were examined using western blotting. The results are expressed as the mean ± SEM of at least three independent experiments. Values marked by different letters are significantly different (P < 0.05).

Article Snippet: Antibodies and reagents Recombinant human BMP6 (#507-BP-020), recombinant human TGF-β1 (#240-B-010), dorsomorphin dihydrochloride (dorsomorphin) (#3093/10), DMH-1 (#4126/10), and SB431542 (#1614/10) were obtained from R&D Systems (Minneapolis, MN).

Techniques: Western Blot, Immunofluorescence, Positive Control

Journal: Biology of Reproduction

Article Title: SMAD-dependent signaling mediates morphogenetic protein 6-induced stimulation of connective tissue growth factor in luteinized human granulosa cells †

doi: 10.1093/biolre/ioz108

Figure Lengend Snippet: ALK2 or ALK3 is required for the BMP6-induced upregulation of CTGF expression in SVOG cells. (A and B) SVOG cells were pretreated with a vehicle control (DMSO), 5 μM dorsomorphin, or 5 μM DMH-1 for 60 min, and then treated with 30 ng/ml BMP6 for an additional 3 h (A) and 6 h (B), and the mRNA (A) and protein (B) levels of CTGF were examined using RT-qPCR and western blot, respectively. (C and D) SVOG cells were pretreated with a vehicle control (DMSO) or 5 μM SB431542 for 60 min, and then treated with 30 ng/ml BMP6 for an additional 3 h (C) and 6 h (D), and the mRNA (A) and protein (B) levels of CTGF were examined using RT-qPCR and western blot, respectively. (E and F) SVOG cells were transfected for 24 h with 25 nM of control siRNA (siCtrl), 25 nM of ALK2 siRNA (siALK2), 25 nM of ALK3 siRNA (siALK3), or 25 nM of siALK2 combined with 25 nM of siALK3, followed by treatment with vehicle control or 30 ng/ml of BMP6 for 40 min (E) and 3 h (F). The phosphorylated protein levels of SMAD1/5/8 (E) and mRNA levels of CTGF were examined using western blotting and RT-qPCR, respectively. The results are expressed as the mean ± SEM of at least three independent experiments. Values marked by different letters are significantly different (P < 0.05). ALK, activin receptor-like kinase.

Article Snippet: Antibodies and reagents Recombinant human BMP6 (#507-BP-020), recombinant human TGF-β1 (#240-B-010), dorsomorphin dihydrochloride (dorsomorphin) (#3093/10), DMH-1 (#4126/10), and SB431542 (#1614/10) were obtained from R&D Systems (Minneapolis, MN).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Transfection

Journal: Biology of Reproduction

Article Title: SMAD-dependent signaling mediates morphogenetic protein 6-induced stimulation of connective tissue growth factor in luteinized human granulosa cells †

doi: 10.1093/biolre/ioz108

Figure Lengend Snippet: SMAD-dependent signaling pathway is required for the BMP6-induced stimulation of CTGF in SVOG cells. (A) SVOG cells were transfected for 24 h with 25 nM of control siRNA (siCtrl) or 25 nM of SMAD4 siRNA (siSMAD4), followed by treatment with vehicle control or 30 ng/ml of BMP6 for 3 h. The mRNA levels of SMAD4 and CTGF were examined using RT-qPCR. (B) SVOG cells were transfected for 24 h with 25 nM of control siRNA (siCtrl) or 25 nM of SMAD4 siRNA (siSMAD4) followed by treatment with vehicle control or 30 ng/ml of BMP6 for 6 h. The protein levels of CTGF and SMAD4 were examined using western blotting. (C) SVOG cells were transfected for 24 h with 25 nM of control siRNA (siCtrl) or 25 nM of SMAD4 siRNA (siSMAD4), followed by treatment with vehicle control or 30 ng/ml of BMP6 for 3 h. The mRNA levels of SMAD4 and CYR61 were examined using RT-qPCR. (D) SVOG cells were transfected for 24 h with 25 nM of control siRNA (siCtrl) or 25 nM of SMAD4 siRNA (siSMAD4) followed by treatment with vehicle control or 30 ng/ml of BMP6 for 6 h. The protein levels of CYR61 and SMAD4 were examined using western blotting. The results are expressed as the mean ± SEM of at least three independent experiments. Values marked by different letters are significantly different (P < 0.05).

Article Snippet: Antibodies and reagents Recombinant human BMP6 (#507-BP-020), recombinant human TGF-β1 (#240-B-010), dorsomorphin dihydrochloride (dorsomorphin) (#3093/10), DMH-1 (#4126/10), and SB431542 (#1614/10) were obtained from R&D Systems (Minneapolis, MN).

Techniques: Transfection, Quantitative RT-PCR, Western Blot

Journal: Biology of Reproduction

Article Title: SMAD-dependent signaling mediates morphogenetic protein 6-induced stimulation of connective tissue growth factor in luteinized human granulosa cells †

doi: 10.1093/biolre/ioz108

Figure Lengend Snippet: CTGF mediates the BMP6-induced up-regulation of LOX expression in SVOG cells. SVOG cells were transfected for 24 h with 25 nM of control siRNA (siCtrl) or 25 nM of CTGF siRNA (siCTGF), followed by treatment with vehicle control or 30 ng/ml of BMP6 for 3 h. The mRNA levels of CTGF and LOX were examined using RT-qPCR. The results are expressed as the mean ± SEM of at least three independent experiments. Values marked by different letters are significantly different (P < 0.05). LOX, lysyl oxidase.

Article Snippet: Antibodies and reagents Recombinant human BMP6 (#507-BP-020), recombinant human TGF-β1 (#240-B-010), dorsomorphin dihydrochloride (dorsomorphin) (#3093/10), DMH-1 (#4126/10), and SB431542 (#1614/10) were obtained from R&D Systems (Minneapolis, MN).

Techniques: Expressing, Transfection, Quantitative RT-PCR

Journal: Biology of Reproduction

Article Title: SMAD-dependent signaling mediates morphogenetic protein 6-induced stimulation of connective tissue growth factor in luteinized human granulosa cells †

doi: 10.1093/biolre/ioz108

Figure Lengend Snippet: Proposed model for the effect of BMP6 on the expression levels CTGF and LOX in human granulosa-lutein cells. BMP6 binds to a complex containing type I (ALK2 and ALK3) and II receptors leading to the phosphorylation/activation of receptor-regulated SMAD (SMAD1/5/8), which binds to the common SMAD (SMAD4). This complex then translocates into the nucleus to promote the transcription of CTGF. The up-regulation of CTGF subsequently contributes to the increase in LOX expression in human granulosa-lutein cells.

Article Snippet: Antibodies and reagents Recombinant human BMP6 (#507-BP-020), recombinant human TGF-β1 (#240-B-010), dorsomorphin dihydrochloride (dorsomorphin) (#3093/10), DMH-1 (#4126/10), and SB431542 (#1614/10) were obtained from R&D Systems (Minneapolis, MN).

Techniques: Expressing, Activation Assay