Journal: Experimental and Therapeutic Medicine

Article Title: miR-877-3p promotes TGF-β1-induced osteoblast differentiation of MC3T3-E1 cells by targeting Smad7

doi: 10.3892/etm.2019.7570

Figure Lengend Snippet: TGF-β1 promotes the osteoblastic differentiation of MC3T3-E1 cells. (A) RT-qPCR analysis of the osteogenic-related genes RUNX2, OSX and COL1A1 in MC3T3-E1 cells after treatment with TGF-β1 (4 ng/ml) for 0, 7 and 14 days. (B) Western blotting of RUNX2, COL1A1-and OSX protein in MC3T3-E1 cells cultured with TGF-β1 (4 ng/ml) for 0, 7 and 14 days. (C) ALP activity in MC3T3-E1 cells after treatment with TGF-β1 (4 ng/ml) for 0, 7 and 14 days. NS, no significance change; *P<0.05, **P<0.01 as indicated. TGF, transforming growth factor; RUNX2, runt-related transcription factor 2; OSX, osterix; COL1A1 collagen type I α1 chain; ALP, alkaline phosphatase.

Article Snippet: Mineralization assay The MC3T3-E1 cells were cultured with TGF-β1 (4 ng/ml) and osteoblastic differentiation medium for 7 days in 24-well plates at a density of 2×10 4 cells per well, and the mineralization of the MC3T3-E1 cells was observed via Alizarin Red S (ARS) and ALP staining with an optical light microscope (ARS, Solarbio, China; ALP staining kit, Beyotime Institute of Biotechnology, Haimen, China).

Techniques: Quantitative RT-PCR, Western Blot, Cell Culture, Activity Assay

Journal: Experimental and Therapeutic Medicine

Article Title: miR-877-3p promotes TGF-β1-induced osteoblast differentiation of MC3T3-E1 cells by targeting Smad7

doi: 10.3892/etm.2019.7570

Figure Lengend Snippet: miR-877-3p promotes the osteoblastic differentiation of MC3T3-E1 cells. (A) miR-877-3p expression in MC3T3-E1 cells after treatment with TGF-β1 (4 ng/ml) for 0, 7 and 14 days. (B) Expression of miR-877-3p was validated in MC3T3-E1 cells transfected with miR-877-3p mimics or inhibitor and their corresponding controls by RT-qPCR. (C) RUNX2, OSX and COL1A1 mRNA expression in MC3T3-E1 cells. (D) MC3T3-E1 cells transfected with miR-877-3p mimics or inhibitor and their corresponding controls were cultured in osteogenic medium. At day 14, the mineralization of differentiated MC3T3-E1 cells was detected with ARS and ALP staining. (E) Transfected MC3T3-E1 cells were cultured in osteogenic medium for 14 days and then mixed with tricalcium phosphate/hydroxyapatite and transplanted into the dorsal region of nude mice for 4-weeks. The results were then evaluated by H&E and Masson's trichrome staining. (F) (F) Transfected MC3T3-E1 cells were cultured in osteogenic medium for 14 days and then mixed with tricalcium phosphate/hydroxyapatite and transplanted into the dorsal region of nude mice for 4-weeks. The Expression expression of miR-877-3p in the xenografts from each group was detected by RT-qPCR analysis. *P<0.05, **P<0.01 and ***P<0.001, as indicated. miR, microRNA; TGF, transforming growth factor; RUNX2, runt-related transcription factor 2; OSX, osterix; COL1A1 collagen type I α1 chain; NC, negative control; ARS, Alizarin Red S; ALP, alkaline phosphatase; H&E, hematoxylin and eosin.

Article Snippet: Mineralization assay The MC3T3-E1 cells were cultured with TGF-β1 (4 ng/ml) and osteoblastic differentiation medium for 7 days in 24-well plates at a density of 2×10 4 cells per well, and the mineralization of the MC3T3-E1 cells was observed via Alizarin Red S (ARS) and ALP staining with an optical light microscope (ARS, Solarbio, China; ALP staining kit, Beyotime Institute of Biotechnology, Haimen, China).

Techniques: Expressing, Transfection, Quantitative RT-PCR, Cell Culture, Staining, Negative Control

Journal: Experimental and Therapeutic Medicine

Article Title: miR-877-3p promotes TGF-β1-induced osteoblast differentiation of MC3T3-E1 cells by targeting Smad7

doi: 10.3892/etm.2019.7570

Figure Lengend Snippet: Smad7 is identified as a downstream target of miR-877-3p during the osteoblastic differentiation of MC3T3-E1 cells. mRNA expression of (A) Smad7 and (B) osteoblast differentiation-associated genes in MC3T3-E1 cells transfected with inhibitor-NC, miR-877-3p inhibitor or miR-877-3p inhibitor plus TGF-β1. (C) Expression of RUNX2, Smad7, p-Smad2, t-Smad2, p-Smad3 and t-Smad3 protein in MC3T3-E1 cells transfected with inhibitor-NC, miR-877-3p inhibitor or miR-877-3p inhibitor plus TGF-β1. (D) The mRNA expression of Smad7 was validated in MC3T3-E1 cells transfected with pcDNA-NC or pcDNA-Smad7. The mRNA expression of (E) Smad7 and (F) osteoblast differentiation-associated genes in MC3T3-E1 cells transfected with miR-877-3p mimics and/or pcDNA-Smad7. (G) Protein levels of Smad7, RUNX2, p-Smad2, t-Smad2, p-Smad3 and t-Smad3 in MC3T3-E1 cells transfected with miR-877-3p mimics and/or pcDNA-Smad7. (H) ALP activity of MC3T3-E1 cells transfected with miR-877-3p mimics and/or pcDNA-Smad7 after culture with osteogenic medium for 7 days. (I) MC3T3-E1 cells transfected with miR-877-3p mimics and/or pcDNA-Smad7 were induced with osteogenic medium. At day 14, the mineralization of differentiated MC3T3-E1 cells was detected with ARS and ALP staining. *P<0.05, **P<0.01 as indicated. miR, microRNA; NC, negative control; TGF, transforming growth factor; RUNX2, runt-related transcription factor 2; p, phospho; t, total; ALP, alkaline phosphatase; ARS, Alizarin Red S.

Article Snippet: Mineralization assay The MC3T3-E1 cells were cultured with TGF-β1 (4 ng/ml) and osteoblastic differentiation medium for 7 days in 24-well plates at a density of 2×10 4 cells per well, and the mineralization of the MC3T3-E1 cells was observed via Alizarin Red S (ARS) and ALP staining with an optical light microscope (ARS, Solarbio, China; ALP staining kit, Beyotime Institute of Biotechnology, Haimen, China).

Techniques: Expressing, Transfection, Activity Assay, Staining, Negative Control

Journal: Open Veterinary Journal

Article Title: Generation and characterization of mesenchymal stem cells from the affected femoral heads of dogs with Legg Calvé Perthes disease

doi: 10.5455/OVJ.2024.v14.i5.12

Figure Lengend Snippet: Trilineage differentiation potency. Canine LCPD-affected femoral head-derived adherent cells (A-C) and BMMSCs (D-F) differentiated into adipocytes (A and D), osteoblasts (B and E), and chondrocytes (C and F). (A and D): Sudan III staining, (B and E): Alizarin Red staining, and (C and F): Alcian blue staining. Scale bars = 100 μm or 1 mm.

Article Snippet: Canine osteoblast differentiation medium (Cell Applications, Inc.) was used for osteoblast differentiation, and the same static culture was performed for 2 weeks.

Techniques: Derivative Assay, Staining