Figure 2. Knocking down the expression of p16^INK4A reverses the senescent phenotype of hCPCs.

Real time qPCR data (A) and quantitative analysis of Western blot (B) confirmed that p16^INK4A protein expression was knocked down in one line (AMC20) of the hCPCs-A group at passage 8 (P8) infected with lentivirus expressing shRNA against p16^INK4A compared to the control which was infected with scramble shRNA. Senescence-associated β-galactosidase staining showed a decrease in the number of senescent cells in the group of hCPCs that were infected with lentivirus expressing shRNA against p16^INK4A (D) compared to the group of hCPCs expressing the scramble shRNA (C). E. Quantitative analysis of β-galactosidase positive staining in panel C and D. In hCPCs with 48-hour knockdown of p16^INK4A, senescence-associated genes (F) and quiescence-associated genes (G) exhibited decreased expression compared to hCPCs with 48-hour infection by a scramble control. Data labels shown are for the gene expression levels in p16^INK4A shRNA-infected hCPCs, relative to those in scramble shRNA-infected hCPCs. H. Quantitative analysis of Western blot confirmed a decrease in p21^CIP1 protein expression after knocking down p16^INK4A in hCPCs, consistent with its decreased gene expression (CDKN1A) shown in panel F. I. Representative FACS analysis showed the increased proliferation ability of hCPCs after knocking down p16^INK4A. J. Quantitative data analysis for panel I. Data were presented as means ± SD from 3 independent experiments (n=3); * indicates p<0.05 vs. scramble shRNA.