Down-regulation of Notch1 signaling promoted BMP2-induced chondrogenic differentiation and inhibited BMP2-induced osteogenic differentiation of MSCs in vitro. (IMAGE)

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Down-regulation of Notch1 signaling promoted BMP2-induced chondrogenic differentiation and inhibited BMP2-induced osteogenic differentiation of MSCs in vitro.

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(A) C3H10T1/2 cells were transfected with AdGFP, AdBMP2, AdDnNotch1, and AdBMP2+DnNotch1, respectively. On day 7, quantitative reverse transcription PCR was used to detect the expression of chondrogenic differentiation markers (Sox9 and Col2a1) and osteoblastic differentiation markers (Runx2, Col1a1, and OPN) of MSCs. (B) To detect the expression of sulfated glycosaminoglycan during C3H10T1/2 cell differentiation, alcian blue staining was performed on day 7 after cells were transfected with recombinant adenovirus. (C) Alkaline phosphatase (ALP) staining experiments were used to determine ALP activity on day 3 (a) and day 7 (b) respectively. (D) For matrix mineralization, alizarin red S staining was performed on day 14 (a); microscopic (b) observations showed that down-regulation of Notch1 signaling inhibited BMP2-induced calcium deposition. (E) Quantitative analysis of ALP activities and calcium deposition. The ALP activity was quantified at OD 405 nm and normalized by protein concentration per well (unit/mg protein) on day 3 (a) and day 7 (b). Alizarin red staining was quantified at OD 405 nm and normalized to total DNA per well (OD405 nm/μg DNA) (c). (F) Western blot analysis for the chondrogenic differentiation markers Sox9 and Col2a1 and the osteogenic markers Runx2, Col1a1, and OPN. Protein bands (a) and quantitative analysis (b). The relative expression of Sox9, Col2a1, Runx2, Col1a1, and OPN proteins were analyzed using GAPDH as control (b). One-way analysis of variance; ∗∗∗∗p < 0.0001, ∗∗∗p < 0.001, ∗∗p < 0.01, and ∗p < 0.05 versus the AdGFP group; ####p < 0.0001, ###p < 0.001, ##p < 0.01, and #p < 0.05 versus the indicated group; ns, p > 0.05. BMP2, bone morphogenetic protein 2; Col1a1, collagen type I alpha 1 chain; Col2a1, collagen type II alpha 1 chain; MSC, mesenchymal stem cell; Notch1, Notch receptor 1; OPN, osteopontin; Runx2, RUNX family transcription factor 2; Sox9; SRY-box transcription factor 9.

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Genes & Diseases

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