Journal: Brain : a journal of neurology

Article Title: Integrin control of the transforming growth factor-β pathway in glioblastoma.

doi: 10.1093/brain/aws351

Figure Lengend Snippet: Figure 3 Integrin inhibition decreases Smad2 phosphorylation and TGF-b-induced reporter gene activity. (A) Whole cell protein lysates of LN-308 glioma cells, cultured with specific antibodies to different integrins as indicated (top), or transiently transfected with small interfering RNA oligonucleotides specific for one integrin or a control small interfering RNA (middle), were assessed for pSmad2 and total Smad2/3 levels by immunoblot, using GAPDH as a loading control. In the bottom panel, LN-308, LN-18, U87MG, T98G or LNT-229 cells were exposed to control peptide () or cilengitide (10 mM) as indicated and assessed for pSmad2 and total Smad2/3 levels. (B) LN-308 or LNT-229 cells were exposed to blocking antibodies (top) or small interfering RNA oligonucleotides (middle) as in (A). In the bottom panel, LN-308 or LNT-229 cells were exposed to the control peptide RAD (black bars), 2.5 mM (light grey) or 10 mM (dark grey) cilengitide for 24 h. Intracellular TGF-b signalling was assessed by reporter assays using the pGL3-SBE4-Luc and pGl2-3TP-Luc constructs (*P 5 0.05 and **P 5 0.01 compared with isotype control and scrambled small interfering RNA or RAD, respectively).

Article Snippet: TGF-b1, phospho-Smad2 (Ser465/467) (clone 138D4) and Smad2 (clone 86F7) antibodies were purchased from Cell Signaling Technology.

Techniques: Inhibition, Phospho-proteomics, Activity Assay, Cell Culture, Transfection, Small Interfering RNA, Control, Western Blot, Blocking Assay, Construct

Journal: Brain : a journal of neurology

Article Title: Integrin control of the transforming growth factor-β pathway in glioblastoma.

doi: 10.1093/brain/aws351

Figure Lengend Snippet: Figure 6 Effects of cilengitide on Smad2 phosphorylation in LN-308 gliomas in vivo. (A) LN-308 glioma cells were exposed to RAD or cilengitide (10 mM), TGF-b2 (10 ng/ml) or SD-208 (1 mM) and assessed for pSmad2 expression after 48 h by immunofluorescence microscopy (original magnification 40). (B) LN-308 cells were inoculated intracerebrally in athymic CD1 nude mice. The animals were treated intraperitoneally with cilengitide (90 mg/g body weight) or PBS for 5 days starting 3 weeks after tumour cell inoculation. Subsequently, the animals’ brains were removed, shock-frozen and integrin expression was assessed by immunohistochemistry as indicated (original magnification 10 for haematoxylin and eosin and 40 for pSmad2). (C) pSmad2 levels were assessed by immu- nohistochemistry (C) and quantified (D).

Article Snippet: TGF-b1, phospho-Smad2 (Ser465/467) (clone 138D4) and Smad2 (clone 86F7) antibodies were purchased from Cell Signaling Technology.

Techniques: Phospho-proteomics, In Vivo, Expressing, Microscopy, Immunohistochemistry

Journal: Journal of cell science

Article Title: FGF2-induced Ras-MAPK signalling maintains lymphatic endothelial cell identity by upregulating endothelial-cell-specific gene expression and suppressing TGFβ signalling through Smad2.

doi: 10.1242/jcs.137836

Figure Lengend Snippet: Fig. 7. H-Ras overexpression inhibits TGFb1-induced Smad2 phosphorylation. (A) Western blots of C-terminally phosphorylated, linker phosphorylated and total Smad2 in control and H-Ras-overexpressing mLECs stimulated by TGFb1, and quantitative analysis of western blots. Error bars s.d.; n53. ***P,0.005 (versus TGFb1-stimulated control mLECs), **P,0.01 (versus starved control mLECs). (B) Western blots of phosphorylated and total Smad1/3/5 in control and H-Ras-overexpressing mLECs stimulated with TGFb1, and quantitative analysis of western blots. (C) Real-time RT-PCR assay for mRNA expression in control and H-Ras-overexpressing mLECs cultured in EGM2-MV CM and FGF2-depleted EGM2-MV CM supplemented with TGFb1 (CM-FGF2+TGFb). Error bars represent s.d.; n53. **P,0.01, ****P,0.001 (versus control mLECs cultured in CM).

Article Snippet: Western blot analysis was performed using the following primary antibodies: mouse anti-Pan-Ras (Calbiochem/Merck, Darmstadt, Germany), rabbit anti-phosphorylated p42/44 MAPK, anti-p42/44 MAPK, anti-phosphorylated Smad2 (Ser465/ 467; 138D4), anti-phosphorylated Smad2 (Ser245/250/255), antiphosphorylated Smad3 (Ser423/425; C25A9), anti-phosphorylated Smad1/5 (Ser463/465; 41D10), anti-Smad2 XP (D43B4), anti-Smad3 (C67H9), anti-Smad1 XP (D59D7) and anti-Smad5 (Cell Signaling Technology).

Techniques: Over Expression, Phospho-proteomics, Western Blot, Control, Quantitative RT-PCR, Expressing, Cell Culture

Journal: Journal of cell science

Article Title: FGF2-induced Ras-MAPK signalling maintains lymphatic endothelial cell identity by upregulating endothelial-cell-specific gene expression and suppressing TGFβ signalling through Smad2.

doi: 10.1242/jcs.137836

Figure Lengend Snippet: Fig. 8. MAPKs repress Smad2 activation by phosphorylating the Smad2 linker region in the absence of TGFb1 and suppressing the TGFb1-driven phosphorylation of Smad2 C-terminal region. (A) Western blots of Smad2 and MAPK phosphorylation in mLECs stimulated with TGFb1 in the presence or absence of a MEK inhibitor U0126, and quantitative analysis of western blots. Error bars represent the s.d.; n53. *P,0.05, ****P,0.001. (B) Real-time RT-PCR assay for expression of Snai1 and Snai2 mRNA in mLECs cultured in EGM2-MV CM and TGFb1-supplemented EGM2-MV (CM+TGFb) in the presence or absence of U0126. Error bars represent the s.d.; n53. ****P,0.001. (C) Schematic representation of the role of FGF2 in EndMT of LECs. In LECs, the FGF2– FGFR–Ras–MAPK pathway not only maintains endothelial marker gene expression, but also induces phosphorylation of the Smad2 linker, leading to suppression of TGFb-induced Smad2 activation. FGF2 represses EndMT by maintaining endothelial marker gene expression and suppressing TGFb–Smad2- induced activation of mesenchymal genes.

Article Snippet: Western blot analysis was performed using the following primary antibodies: mouse anti-Pan-Ras (Calbiochem/Merck, Darmstadt, Germany), rabbit anti-phosphorylated p42/44 MAPK, anti-p42/44 MAPK, anti-phosphorylated Smad2 (Ser465/ 467; 138D4), anti-phosphorylated Smad2 (Ser245/250/255), antiphosphorylated Smad3 (Ser423/425; C25A9), anti-phosphorylated Smad1/5 (Ser463/465; 41D10), anti-Smad2 XP (D43B4), anti-Smad3 (C67H9), anti-Smad1 XP (D59D7) and anti-Smad5 (Cell Signaling Technology).

Techniques: Activation Assay, Phospho-proteomics, Western Blot, Quantitative RT-PCR, Expressing, Cell Culture, Marker, Gene Expression

Journal: JCI Insight

Article Title: TGF- β promotes fibrosis after severe acute kidney injury by enhancing renal macrophage infiltration

doi: 10.1172/jci.insight.123563

Figure Lengend Snippet: Peritoneal macrophages from Tgfbr2fl/fl (WT) and LysM-Cre Tgfbr2fl/fl (macrophage TGF-βRII–/–; MφTGF-βRII–/–) mice 3 days after peritoneal thioglycollate administration were used. (A) TGF-βRII levels were markedly lower in TGF-βRII–/– macrophages than in WT macrophages. ***P < 0.001, n = 3 in each group. Data were expressed as mean ± SEM. (B) Macrophages were stimulated with 2 ng/ml recombinant TGF-β for 30 minutes. TGF-βRII deletion led to decreases in TGF-β–stimulated phosphorylation of Smad2 and Smad3, an indication of TGF-βRII deficiency. p-Smad3, phospho-Smad3; p-Smad2, phospho-Smad2; FAK, focal adhesion kinase.

Article Snippet: Rat anti-mouse F4/80 (MCA497R, a marker of macrophages) and CD3 (MCA1477, a marker of T lymphocytes) were purchased from AbD Serotec (now Bio-Rad); mouse anti–mannose receptor (CD206, MAB25341) and mouse anti–4-HNE (a marker of oxidative stress, 198960) were from R&D Systems; rabbit anti–TGF-βRII (SC-400) and goat anti-human CTGF (SC-14939) were from Santa Cruz Biotechnology; rabbit anti-human Smad2 (600-401-A59), phospho-Smad2 (600-401-K09S), and phospho-Smad3 (600-401-919) as well as rabbit anti-murine collagen type I (600-401-103-01) were from Rockland Immunochemicals; mouse anti–α-SMA (A5228, a marker of myofibroblasts) was from Sigma-Aldrich; and rabbit anti–TGF-β1 (NBP1-80289) and mouse anti–TGF-β2 (Mab612-SP) were from Novus Biologicals.

Techniques: Recombinant

Journal: Cell Cycle

Article Title: Circ_0008450 downregulates Runx3 to promote the proliferation and epithelial-mesenchymal transition of human keratinized epithelial cells

doi: 10.1080/15384101.2020.1842665

Figure Lengend Snippet: Circ_0008450 silence repressed the TGF-β/Smad signal pathway via the up-regulation of Runx3. (a and b) Keratinized epithelial cells treated with LY2109761 were applied for the detection of phosphorylation of Smads (Smad2 and Smad3) by Western blot. *** p < 0.001. (c and d) Keratinized epithelial cells treated with LY2109761, sh-circ_0008450 (sh-circ_0008450), sh-Runx3 (sh-Runx3) or TGF-β1 were applied for the detection of phosphorylation of Smads (Smad2 and Smad3) by western blot. * p < 0.05, ** p < 0.01 or *** p < 0.001

Article Snippet: Primary antibodies included CyclinD1 (orb77046, Biorbyt), CDK4 (orb225521), cleaved caspase-3 (orb106556), cleaved caspase-9 (orb129033), E-cadherin (orb378741), Vimentin (orb379309), Fibronectin (orb95640), ZO-1 (orb35080), Runx3 (orb153370), p-Smad2 (phospho-S467, orb304593), t-Smad2 (orb99462), p-Smad3 (phospho-S204, orb214215), t-Smad3 (orb304596), and β-actin (orb181785).

Techniques: Western Blot