tgf β receptor inhibitor ly2109761  (MedChemExpress)

Journal: Biomaterials Research

Article Title: Targeting Smad3 Phosphorylation Attenuates Anastomotic Intimal Hyperplasia and Perigraft Fibrosis in Decellularized Tissue-Engineered Vascular Grafts

doi: 10.34133/bmr.0241

Figure Lengend Snippet: In vitro co-culturing of M2 macrophages with fibroblasts reveals that M2 macrophages promote myofibroblast activation via the TGF-β1/Smad3 signaling pathway. (A) Schematic of the in vitro co-culture experiment with M2 macrophages and fibroblasts. (B) M2 macrophage co-culture enhances the expression of collagen I and α-SMA proteins in NIH 3T3 fibroblast cells, while the TGFβR1 inhibitor LY2109761 (1 and 10 μM) inhibits this effect. (C and D) Relative quantification of collagen I (C) and α-SMA (D) protein levels in NIH 3T3 cells after intervention. (E) M2 macrophage co-culture enhances the phosphorylation of Smad3 in NIH 3T3 fibroblast cells, while the TGFβR1 inhibitor LY2109761 (1 and 10 μM) inhibits this effect. This suggests activation of the TGF-β1/Smad3 signaling pathway. (F and G) Relative quantification of p-Smad3 (F) and Smad3 (G) protein levels in NIH 3T3 cells after intervention. (H) M2 macrophage co-culture enhances the phosphorylation of Smad3 in NIH 3T3 fibroblast cells, while the Smad3 phosphorylation inhibitor SIS3 (1 and 10 μM) inhibits this effect. (I and J) Relative quantification of p-Smad3 (I) and Smad3 (J) protein levels in NIH 3T3 cells after intervention. (K) IF characterized cell morphology and coexpression of α-SMA and collagen I. NIH 3T3 cells were treated with SIS3 (10 μM), M2 macrophage co-culture, or M2 macrophage co-culture + SIS3 (10 μM), while the control group used ordinary cell culture medium. Scale bar, 50 μm. * indicates comparison with the “no M2 macrophage co-culture, no LY2109761 group”, and # indicates comparison with the “only M2 macrophage co-culture group”. DMEM, Dulbecco’s modified Eagle’s medium.

Article Snippet: In inhibition experiments, NIH 3T3 cells were co-treated with LY2109761 (1 and 10 μM) or SIS3 (1 and 10 μM) and then co-cultured with M2 macrophages for 12 h. Cells were washed twice with PBS and lysed with RIPA buffer for protein extraction; for IF, cells were fixed with 4% PFA for 15 min. SIS3 and LY2109761 were purchased from MedChemExpress (Shanghai, China).

Techniques: In Vitro, Activation Assay, Co-Culture Assay, Expressing, Quantitative Proteomics, Phospho-proteomics, Control, Cell Culture, Comparison, Modification

Journal: Biomaterials Research

Article Title: Targeting Smad3 Phosphorylation Attenuates Anastomotic Intimal Hyperplasia and Perigraft Fibrosis in Decellularized Tissue-Engineered Vascular Grafts

doi: 10.34133/bmr.0241

Figure Lengend Snippet: In vitro co-culturing of M2 macrophages with fibroblasts reveals that M2 macrophages promote myofibroblast activation via the TGF-β1/Smad3 signaling pathway. (A) Schematic of the in vitro co-culture experiment with M2 macrophages and fibroblasts. (B) M2 macrophage co-culture enhances the expression of collagen I and α-SMA proteins in NIH 3T3 fibroblast cells, while the TGFβR1 inhibitor LY2109761 (1 and 10 μM) inhibits this effect. (C and D) Relative quantification of collagen I (C) and α-SMA (D) protein levels in NIH 3T3 cells after intervention. (E) M2 macrophage co-culture enhances the phosphorylation of Smad3 in NIH 3T3 fibroblast cells, while the TGFβR1 inhibitor LY2109761 (1 and 10 μM) inhibits this effect. This suggests activation of the TGF-β1/Smad3 signaling pathway. (F and G) Relative quantification of p-Smad3 (F) and Smad3 (G) protein levels in NIH 3T3 cells after intervention. (H) M2 macrophage co-culture enhances the phosphorylation of Smad3 in NIH 3T3 fibroblast cells, while the Smad3 phosphorylation inhibitor SIS3 (1 and 10 μM) inhibits this effect. (I and J) Relative quantification of p-Smad3 (I) and Smad3 (J) protein levels in NIH 3T3 cells after intervention. (K) IF characterized cell morphology and coexpression of α-SMA and collagen I. NIH 3T3 cells were treated with SIS3 (10 μM), M2 macrophage co-culture, or M2 macrophage co-culture + SIS3 (10 μM), while the control group used ordinary cell culture medium. Scale bar, 50 μm. * indicates comparison with the “no M2 macrophage co-culture, no LY2109761 group”, and # indicates comparison with the “only M2 macrophage co-culture group”. DMEM, Dulbecco’s modified Eagle’s medium.

Article Snippet: In inhibition experiments, NIH 3T3 cells were co-treated with LY2109761 (1 and 10 μM) or SIS3 (1 and 10 μM) and then co-cultured with M2 macrophages for 12 h. Cells were washed twice with PBS and lysed with RIPA buffer for protein extraction; for IF, cells were fixed with 4% PFA for 15 min. SIS3 and LY2109761 were purchased from MedChemExpress (Shanghai, China).

Techniques: In Vitro, Activation Assay, Co-Culture Assay, Expressing, Quantitative Proteomics, Phospho-proteomics, Control, Cell Culture, Comparison, Modification