Cell cycle distribution of the FPCs and the ASCs.

<p>Cell cycle distribution of the FPCs and the ASCs.</p

Expression levels of p21 mRNA in the ASCs and the FPCs.

<p>Total RNA was isolated from the ASCs or the FPCs at the third passage, RT-qPCR was performed for each cell type. Note that mRNA level for p21 in the FPCs accounted for only half of that in the ASCs. Expression level of the p21 mRNA in the ASCs was significantly higher than that in the FPCs (P<0.05).</p

Effects of p21 knockdown on DNA damage in the ASCs.

<p>Observation of the cells containing DNA breaks (with long tails) under a fluorescent microscopy. A, Untreated ASCs. No obvious comet tails were observed. B, S1-iRNA ASCs. Almost every cell in the field had a long and obvious comet tail (arrow). C, Empty-vector-iRNA ASCs. Weak comet tails were observed.</p

Effects of RNAi on the p21 expression in the ASCs.

<p>GFP expression was monitored under a fluorescent microscope 48h after lentiviral infection. A and B, the p21-shRNA-infected ASCs. C and D, the empty-vector-infected ASCs. Fluorescence was clearly observable in both the p21-shRNA-infected ASCs (3A) and the empty-vector-infected ASCs (3C). Cells from both groups were healthy. E: RT-qPCR of the p21 expression. Note that both of the target sequences (S1 and S2) significantly down-regulated p21 mRNA expression in the ASCs (P<0.05). No significant difference was found between the empty-vector-iRNA ASCs and the untreated ASCs (P>0.05).</p

Primers used for qPCR.

<p>Primers used for qPCR.</p

Effects of p21 knockdown on ASC cell-cycle distribution.

<p>Cells were infected at the third passage and harvested at the sixth, tenth and fifteenth passages, and analyzed by flow cytometry. The data were analyzed with ModFit LT Software to estimate the percentages of cells at G1, S, and G2/M. A and B: Cell cycle distribution of the ASCs. A, S1-iRNA ASCs. B, Untreated ASCs. Note that there was no distinct G2 accumulation in either of the groups. C: Bar graph depicts percentage of the cells at G1, S and G2 phases. Percentage of the S1-iRNA ASCs at G2 phase was similar to that of the untreated ASCs. There was no significant difference in cell-cycle distribution between the two groups (P>0. 05).</p

Effects of p21 knockdown on senescence in the ASCs.

<p>A and B: β-Galactosidase staining. A, S1-iRNA ASCs. Note these cells were barely stained, changes in cell volume were not detectable. B, Empty-vector-iRNA ASCs. Note most of these cells were stained in deep blue (senescent cells), with an increase in cell volume. C, Comparisons between the S1-iRNA ASCs, the empty-vector-iRNA ASCs and the untreated ASCs in the percentage of the senescence positive ratio of the cells. Note that senescent rate of the cells infected by S1-siRNA was significantly lower than those of the other two groups (P<0.05).</p

Effects of p21 knockdown on ASC growth.

<p>MTT assay of the S1-iRNA ASCs, the empty-vector-iRNA ASCs and the untreated ASCs. A, Growth curve of the ASCs at passage six. No significant difference was observed between these three groups. B, Growth curve of the ASCs at passage fifteen. Note that the proliferation rate of the S1-iRNA ASCs was significantly higher than those of the other two groups (P<0.05).</p

Interfering sequences of siRNA targeting for p21 gene.

<p>Interfering sequences of siRNA targeting for p21 gene.</p

Effects of p21 knockdown on apoptosis of the ASCs.

<p>Detection of cell apoptosis by Annexin V-PE/7-AAD staining. A-C: Annexin V-PE was read in FL2 and plotted on the x-axis. 7-AAD was read in FL3 and plotted on the y-axis. Living cells were located in the lower left quarter (both annexin and 7AAD negative), apoptotic cells were in the lower right quarter (annexin positive, 7AAD negative) and the dead cells (and late apoptosis) were in the top right quarter (both annexin and 7AAD positive). A, Untreated ASCs. B, S1-iRNA ASCs. C, Empty-vector-iRNA ASCs. D, Comparisons of the apoptotic rate between the S1-iRNA ASCs, the empty-vector-iRNA ASCs and the untreated ASCs. Note that the rate of apoptosis in the S1-iRNA ASCs was significantly increased compared to the untreated ASCs and the empty-vector-iRNA ASCs (P<0.05).</p