Figure 4
The effects of P18 overexpression from the early stage on hematopoietic differentiation can be counteracted by inhibition of TGF-β signaling. When P18/hESCs co-cultured with AGM-S3 cells were treated with DOX from D0, flow cytometry with 7-AAD and combination of anti-CD34/CD43 antibodies at (a) D8 or (b) D14 revealed that production of CD34highCD43−, CD34−CD43+, and CD34+CD43+ populations was reduced. (c) Cell cycle analysis at D4 indicated that the proportion of KDR+ cells in G2/M phase decreased significantly, whereas the proportions of cells in G0/G1 and S phases increased significantly. (d) P18/hESC cocultured with AGM-S3 cells were treated with or without DOX, or with both DOX and 0.33 μM RepSox started from D0. At D4 these cocultures were performed apoptosis analysis by corresponding kit using 7-AAD and anti-KDR antibody. The results indicated that P18 overexpression from D0 increased the apoptosis of co-cultures at D4, which can be counteracted by the inhibition of TGF-β signaling. (e) Co-cultured P18/hESCs were treated without or with DOX or with both DOX and 0.33 μM RepSox from D0, and analyzed by qRT-PCR at D4. The expression of KDR, which is related to mesoderm induction, was stable (i), while important hematopoiesis related genes were downregulated (ii). All of these effects can be counteracted by addition of 0.33 μM RepSox from D0. All results were expressed as means ± SD of three repeated experiments, and p < 0.05 was considered significant (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).
