MEST Regulates the Stemness of Human Periodontal Ligament Stem Cells

Figure 3

The effects of MEST knockdown on the expression of stem cell markers and the proliferative potential in 2-23 cells. (a, b) The expression of MEST mRNA (a) and protein (b) in 2-23 cells transfected with negative control siRNA (siCont) or MEST siRNA (siMEST_#1 and siMEST_#2) was assessed by quantitative RT-PCR and western blot analyses, respectively. (c) The expression intensities of CD105 and CD146 in 2-23 cells transfected with siCont. (black line) or siMEST_#1 (red line) were demonstrated by flow cytometric analysis. In the gated region, positive cells. (d) The gene expression of p75NTR, N-cadherin, and NANOG in 2-23 cells transfected with siCont. or siMEST was assessed by quantitative RT-PCR. (e) The proliferation of 2-23 cells transfected with negative control siRNA (siCont) or MEST siRNA (siMEST) was examined by WST-1 proliferation assay. The graph shows the time-course of the increase in cell numbers of 2-23 cells transfected with siCont. (grey line) or siMEST (black line). All values are (error bars) of quadruplicate assays. , . (f) The gene expression of Cyclin D1 (CCND1) and Cyclin E1 (CCNE1) in 2-23 cells transfected with siCont. or siMEST was assessed by quantitative RT-PCR. (f) The expression of Ki-67 in 2-23 cells transfected with siCont. (upper) or siMEST (lower) was observed by immunocytofluorescence staining (anti-Ki-67: green, DAPI: blue; bars: 500 μm). (a, 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. , .

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