Figure 5.

CD146 knockdown in human umbilical cord blood-derived mesenchymal stem cells accelerates the senescence process. (A): Cell growth was monitored by measuring cumulative PD. (B): Cells were assessed for their expression of stemness genes by quantitative real-time polymerase chain reaction (qPCR) at passage (P) 9 (mean ± SD; n = 3; ∗∗, p < .01). (C): Telomerase activities was measured at P9 using the telomerase PCR enzyme-linked immunosorbent assay kit (mean ± SD; n = 4; ∗∗, p < .01). (D): Expression of the cell cycle inhibitors was measured by immunoblotting at P9, with β-actin serving as a loading control fold (right panel; mean ± SD; n = 4; ∗, p < .05; ∗∗, p < .01). (E): qPCR data showing the mRNA expression levels (p16 and Bmi-1) at P9 (mean ± SD; n = 3; ∗∗, p < .01). (B, D, E): Expression levels were normalized to β-actin, with the expression levels in naïve defined as 1. (F): SA β-gal-positive cells were measured at P9 and P12. Results are shown as mean ± SD (n = 4; ∗∗, p < .01). (G): Cell areas compared at P9, which was normalized to the mean area in naïve cells, defined as 1 (mean ± SD; n = 25; ∗∗, p < .01). The black lines indicate the cell margins. (H): Multilineage differentiation was assessed by quantifying the percentage of positively stained cells at P9 (mean ± SD; n = 3; ∗, p < .05). (F–H): Scale bar = 50 μm. Abbreviations: OD, optical density; PD, population doubling; pho-p53, phospho-p53; SA β-gal, senescence-associated β-galactosidase; siCD146, CD146 small interfering RNA; siCon, small interfering scrambled RNA; siRNA, small interfering RNA; TRAP, telomeric repeat amplification protocol.