MEST Regulates the Stemness of Human Periodontal Ligament Stem Cells

Figure 4

The effects of MEST knockdown on the multidifferentiation capacity in 2-23 cells. (a) The formation of mineralized nodules in 2-23 cells transfected with control siRNA (siCont.) or MEST siRNA (siMEST) was examined by Alizarin red S staining after culture in osteoblastic differentiation medium (DM) for 3 weeks (bars: 5 mm). (b) The gene expression of ALP and Osterix in 2-23 cells transfected with siCont. or siMEST, which were cultured with or without DM for 3 days, was assessed by quantitative RT-PCR. (c) The lipid vacuoles in 2-23 cells transfected with siCont. or siMEST were examined by Oil red O staining after culture in adipogenic DM for 3 weeks (bars: 20 μm). (d) The gene expression of PPARγ and C/EBPα in 2-23 cells transfected with siCont. or siMEST, which were cultured with or without DM for 3 days, was assessed by quantitative RT-PCR. (e) The cartilaginous matrix in 2-23 cells transfected with siCont. or siMEST was examined by Alcian blue staining after culture in chondrogenic DM for 4 weeks (bars: 200 μm). (f) The gene expression of Type-2 collagen (COL-2) and Aggrecan in 2-23 cells transfected with siCont. or siMEST, which were cultured with or without DM for 3 days, was assessed by quantitative RT-PCR. (b, d, f) In quantitative RT-PCR, the expression levels of these genes were normalized against β-actin expression. All values are (error bars) of quadruplicate assays. . n.s.: not significant.

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