Figure 1

Establishment of p53 stable knockdown in rabbit mesenchymal stem cells. Passage 4 of mesenchymal stem cells (MSCs) generated from femur bone marrow was transduced with lentiviral particles containing p53 shRNA or control (Ctrl) shRNA. (A) Stable clones were harvested after puromycin selection, and cells of passages 3 to 9 were used for the following tests. Scale bar, 500 μm. (B) Representative western blot image of p53 in MSCs. (C) Quantification of p53 protein level in western blot by densitometry. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) used as internal control (n = 3, *P <0.01 vs. control group). (D) 3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethonyphenol)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay showed no significant difference in cell viability between the two groups. P >0.05. (E) Representative images of cell colony formation. Viable cells (2,000 cells/well in six-well plates) were allowed to grow for 12 days before being fixed with methanol and stained with crystal violet. Scale bar, 1 cm. (F) Quantification of the number of colony formation in p53 shRNA and control shRNA groups. P >0.05. (G) Flow cytometry analysis of cell surface markers. Blue line, negative control. (H) Differentiation capacity of the cells. Both control shRNA-transduced and p53 shRNA-transduced MSCs differentiated into mineralizing cells stained with alizarin red, or adipocytes stained with oil red. Scale bar, 200 μm. Data presented as mean ± standard error of the mean, n = 3 individual experiments.