Journal: Frontiers in Molecular Biosciences

Article Title: CCN2/CTGF tip the balance of growth factors towards TGF-β2 in primary open-angle glaucoma

doi: 10.3389/fmolb.2023.1045411

Figure Lengend Snippet: Ciliary body morphology and pSmad1/5/8 levels on P1 of wildtype and βB1-CTGF6 mice. (A) Semithin sections of wildtype and βB1-CTGF6 mice at P1. n = 3 (B) Immunohistochemical staining of pSmad1/5/8 (green) in the ciliary body of wildtype and βB1-CTGF6 mice at P1. Fluorescence intensity of pSmad1/5/8 (green) was markedly decreased in the ciliary epithelium of transgenic βB1-CTGF6 mice, compared to age-matched wildtype littermates. Nuclei were stained with Dapi (blue). OCE: outer ciliary epithelium; ICE: inner ciliary epithelium; Ir: Iris; L: lens; n = 5.

Article Snippet: Afterwards sections were incubated with the primary antibody as follows: goat anti-BMP-4 (1:50, Santa Cruz Biotechnology; RRID:AB_2243391), goat anti- BMP-7 (1:50, Santa Cruz Biotechnology; RRID:AB_2227926), rabbit anti-Gremlin (1:50, Santa Cruz Biotechnology; RRID:AB_2279266), rabbit anti-pSmad1/5/8 (1:100, Cell Signaling Technology, Danvers, MA, United States; RRID:AB_331671), rabbit anti-pSmad2 (1:50, Cell Signaling Technology; RRID:AB_390732) and rabbit anti-TGF-β2 (1:50, Cell Signaling Technology) at 4°C overnight.

Techniques: Immunohistochemical staining, Staining, Fluorescence, Transgenic Assay

Journal: Frontiers in Molecular Biosciences

Article Title: CCN2/CTGF tip the balance of growth factors towards TGF-β2 in primary open-angle glaucoma

doi: 10.3389/fmolb.2023.1045411

Figure Lengend Snippet: BMP signaling in the anterior eye segment of βB1-CTGF1 mice. (A, B) pSmad1/5/8 immunoreactivity (green) in the anterior chamber angle of 2-month-old βB1-CTGF1 mice and wildtype littermates. Immunoreactivity of pSmad1/5/8 was reduced in the TM of transgenic mice, compared to wildtype mice. Nuclei were stained with Dapi (blue); n = 5. (C) Western blot analysis revealed a significantly reduced phosphorylation of pSmad1/5/8 in the anterior eye segment of βB1-CTGF1 mice, compared to wildtype littermates (n = 4) Mean value of wildtype animals (control) was set at 1. Total protein stained with Coomassie was used to normalize protein synthesis. Data represented as mean ± SD. Right panel shows a representative Western blot. For statistical analysis the Mann-Whitney test was used. (D, E) BMP-4 and BMP-7 immunoreactivity (red) in the anterior chamber angle of 2-month-old βB1-CTGF1 mice and wildtype littermates. Immunoreactivity of BMP-4 and BMP-7 was dramatically reduced in the TM of transgenic animals, compared to control animals. Nuclei were stained with Dapi; n = 5 each. (F) Real-time RT-PCR analysis of Bmp-4 and Bmp-7 in the anterior eye segment of 2-month-old βB1-CTGF1 mice and wildtype littermates. mRNA expression of Bmp-4 and Bmp-7 was significantly reduced in βB1-CTGF1 mice compared to wildtype mice ( Bmp-4 : WT: n = 8, TG: n = 8; Bmp-7 : WT: n = 9, TG: n = 6). mRNA expression was normalized to Gnb2l, and mean value of wildtype mice was set to 1. For statistical analysis the Mann-Whitney test was used. (G) Western blot analyses of BMP-4 and BMP-7 in the anterior eye segment of 2-month-old βB1-CTGF1 mice and wildtype littermates. Protein synthesis of BMP-4 and BMP-7 was significantly reduced in transgenic animals compared to wildtype controls (BMP-4: n = 4 each; BMP-7: n = 5 each). Integrated Blots in the graph show a representative Western blot for both proteins. For statistical analysis the Mann-Whitney test was used. (H) Real-time RT-PCR analyses of Gremlin in the anterior eye segment of 2-month-old βB1-CTGF1 mice and wildtype littermates. mRNA expression of Gremlin was dramatically increased in βB1-CTGF1 mice, compared to wildtype littermates (WT: n = 5, TG: n = 7). mRNA expression was normalized to Gnb2l and mean value of wildtype mice was set to 1. For statistical analysis the Mann-Whitney test was used. (I) Western blot analysis of Gremlin in the anterior eye segment of 2-month-old βB1-CTGF1 mice and wildtype littermates. Protein synthesis was significantly increased in βB1-CTGF1 mice, compared to wildtype (n = 6). For statistical analysis the Mann-Whitney test was used. (J) Immunoreactivity of Gremlin (green) was increased in the TM of 2-month-old βB1-CTGF mice, compared to wildtype control littermates. Nuclei were stained with Dapi (blue). CB: ciliary body; I, iris; TM: trabecular meshwork; c, cornea; n = 5. * p ≤ 0.05; ** p < 0.01.

Article Snippet: Afterwards sections were incubated with the primary antibody as follows: goat anti-BMP-4 (1:50, Santa Cruz Biotechnology; RRID:AB_2243391), goat anti- BMP-7 (1:50, Santa Cruz Biotechnology; RRID:AB_2227926), rabbit anti-Gremlin (1:50, Santa Cruz Biotechnology; RRID:AB_2279266), rabbit anti-pSmad1/5/8 (1:100, Cell Signaling Technology, Danvers, MA, United States; RRID:AB_331671), rabbit anti-pSmad2 (1:50, Cell Signaling Technology; RRID:AB_390732) and rabbit anti-TGF-β2 (1:50, Cell Signaling Technology) at 4°C overnight.

Techniques: Transgenic Assay, Staining, Western Blot, MANN-WHITNEY, Quantitative RT-PCR, Expressing

Journal: Frontiers in Molecular Biosciences

Article Title: CCN2/CTGF tip the balance of growth factors towards TGF-β2 in primary open-angle glaucoma

doi: 10.3389/fmolb.2023.1045411

Figure Lengend Snippet: BMP signaling in CCN2/CTGF treated HTM-N cells in vitro . (A, B) Verification of BMP signaling activity in HMT-N cells in vitro . Immunoreactivity of pSmad1/5/8 (green) in HMT-N cells was increased after the treatment with 10 ng/mL BMP-4 (A) and 10 ng/mL BMP-7 (B) . Nuclei were stained with Dapi (blue). n = 3 (C) Western blot analysis of pSmad1/5/8 in the cytoplasmic fraction of HTM-N cells after the treatment with 10 ng/mL BMP-4 or BMP-7 for 1 h. Protein synthesis of pSmad1/5/8 was significantly increased after the treatment with BMP-4 for. (n = 5). GAPDH was used to normalize protein synthesis. Data represented as mean ± SD. Right panel shows a representative Western blot. For statistical analysis unpaired two-tailed t -test was used. (D) Western blot analysis of pSmad1/5/8 in the nuclear fraction of HTM-N cells after the treatment with 10 ng/mL BMP-4 or BMP-7 for 1 h. Protein synthesis of pSmad1/5/8 was significantly increased after 1 h with both treatments (n = 5). LaminB1 was used to normalize protein synthesis. Data represented as mean ± SD. Right panel shows a representative Western blot. For statistical analysis unpaired two-tailed t -test was used. (E) Real-time RT-PCR analysis of Bmp-4 and Bmp-7 after the treatment with 50 ng/mL and 100 ng/mL CCN2/CTGF for 24 h in HMT-N cells. mRNA expression of Bmp-4 and Bmp-7 was significantly reduced after the treatment with CCN2/CTGF ( Bmp-4 : control n = 4, 50 ng/mL CCN2/CTGF n = 3, 100 ng/mL CCN2/CTGF n = 3; Bmp-7 : control n = 5, 50 ng/mL CCN2/CTGF n = 4, 100 ng/mL CCN2/CTGF n = 3). mRNA expression was normalized to Gnb2l, and mean value of untreated control cells was set to 1. For statistical analysis the Kruskal–Wallis test was used. (F) Real-time RT-PCR analyses of Smad6 , Smad7 , and Id2 after the treatment with 50 ng/mL and 100 ng/mL CCN2/CTGF for 24 h in HMT-N cells. mRNA expression of Smad6 was significantly increased after the treatment with 100 ng/mL CCN2/CTGF (n = 6). mRNA expression of Smad7 was significantly increased after the treatment with 100 ng/mL CCN2/CTGF (control: n = 5, 50 ng/mL CCN2/CTGF: n = 5, 100 ng/mL CCN2/CTGF: n = 4). mRNA expression of Id2 was significantly reduced after the treatment with 50 ng/mL and 100 ng/mL CCN2/CTGF (control: n = 5, 50 ng/mL CCN2/CTGF: n = 5, 100 ng/mL CCN2/CTGF: n = 4). mRNA expression was normalized to Gnb2l, and mean value of untreated control cells was set to 1. For statistical analysis the Kruskal–Wallis test was used. (G) Western blot analyses of BMP-7 after the treatment with 5 ng/mL, 25 ng/mL, 50 ng/mL and 100 ng/mL CCN2/CTGF for 24 h in HMT-N cells. Protein synthesis of BMP-7 was significantly reduced after the treatment with 50 ng/mL and 100 ng/mL CCN2/CTGF (control: n = 4, 5 ng/mL CCN2/CTGF: n = 3, 25 ng/mL CCN2/CTGF: n = 3, 50 ng/mL CCN2/CTGF: n = 3, 100 ng/mL CCN2/CTGF: n = 3). Mean value of wildtype animals (control) was set to 1. α -Tubulin was used to normalize protein synthesis. For statistical analysis the Kruskal–Wallis test was used. (H) Western blot analysis of pSmad1/5/8 after the treatment with 10 ng/mL BMP-4, 60 ng/mL Noggin and 10 ng/mL BMP-4, and 50 ng/mL CCN2/CTGF and 10 ng/mL BMP-4 in HTM-N cells. pSmad1/5/8 protein synthesis was increased after the treatment with BMP-4 (n = 5), compared to untreated control cells and significantly reduced after the treatment with the combination of Noggin and BMP-4 (n = 5) and the combination of CCN2/CTGF and BMP-4 (n = 5), compared to the treatment with BMP-4 only. Mean value of wildtype animals (control) was set to 1. GAPDH was used to normalize protein synthesis. Data represented as mean ± SD. For statistical analysis the One-way ANOVA test was used. * p ≤ 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Afterwards sections were incubated with the primary antibody as follows: goat anti-BMP-4 (1:50, Santa Cruz Biotechnology; RRID:AB_2243391), goat anti- BMP-7 (1:50, Santa Cruz Biotechnology; RRID:AB_2227926), rabbit anti-Gremlin (1:50, Santa Cruz Biotechnology; RRID:AB_2279266), rabbit anti-pSmad1/5/8 (1:100, Cell Signaling Technology, Danvers, MA, United States; RRID:AB_331671), rabbit anti-pSmad2 (1:50, Cell Signaling Technology; RRID:AB_390732) and rabbit anti-TGF-β2 (1:50, Cell Signaling Technology) at 4°C overnight.

Techniques: In Vitro, Activity Assay, Staining, Western Blot, Two Tailed Test, Quantitative RT-PCR, Expressing

Journal: bioRxiv

Article Title: Selective disruption of synaptic BMP signaling by a Smad mutation adjacent to the highly conserved H2 helix

doi: 10.1101/811109

Figure Lengend Snippet: (A) Western blot analysis of whole extracts from S2 cells expressing Flag-Mad and Flag-Mad-8 and treated with Dpp as indicated. Compared to control Mad, Mad-8 variant has significantly reduced pMad levels upon Dpp exposure. The pMad signals were normalized as the relative ratio pMad/Flag (A’). (B) Confocal images of S2 cells transfected with Flag-Mad variants and Tac-Tkv chimeras as indicated, spread on anti-Tac coated surfaces and labeled for pMad (red), Flag (green), actin (phalloidin-magenta), and DNA (DAPI-blue). The activated Tac-TkvA chimera induces nuclear pMad accumulation when co-transfected with Flag-Mad and, to a lesser extent, with Flag-Mad-8, as quantified in (B’). The pMad signals localize to cell surfaces only in Tac-TkvA/ Flag-Mad co-transfected cells. (C) Structure of the Type I receptor (PDM code 3TZM). The L45 loop (magenta) interacts specifically with R-Smads. The N-lobe of the receptor, including the GS box and L45, forms a docking surface for MH2 and positions the S-V-S C-tail of Mad in the catalytic pocket. (D-E) Structure of the MH2 domain of Drosophila Mad (PDM code 3DIT) shown as monomer (D) and trimer (E). The L3 loop (yellow) is engaged in exclusive interactions with either the L45 loop of the receptor or with the phosphorylated C-tail of another MH2 domain. (F) Map of MH2 Mad residues mutated in various Mad alleles. The two views of the structure are related by a 90° rotation around a vertical axis. (G) S359L in silica mutagenesis. S359 and adjacent peptide backbone form hydrogen bonds with H357 and residues on the H2 helix (purple); the S359L substitution breaks the hydrogen bonds with H357 (red asterisk) and introduces a bulky moiety, shifting the H2. (H) Lateral view of the Mad MH2 trimer showing the close proximity of the H2 helix (purple) to the charged L3 surface (colored by atoms). (I) Alignment of R-Smad sequences indicating class specific residues in the H2 region, including the S359 (yellow). Scale bars: 10 μm (B). Error bars indicate SEM. ***, p <0.0001; **, p <0.001; *, p <0.01

Article Snippet: The surface attached cells were fixed in 4% formaldehyde (Polysciences) for 15 min, then stained for pMad (anti-phospho-Smad 1/5, 41D10, 1:200, Cell Signaling), and Flag (anti-Flag, M2, 1:500, Sigma).

Techniques: Western Blot, Expressing, Control, Variant Assay, Transfection, Labeling, Mutagenesis