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. 2015 Apr 8;5:9604. doi: 10.1038/srep09604

Figure 5. mTORC1 Supports the De-differentiate Potential of Early SSEA-4− Progeny.

Figure 5

(a) SSEA-4− cells were sorted at different time points during MG63 cells' differentiation to osteocytes and individually inoculated into fresh medium for measuring their clonal efficiency or into special medium for measuring their tumorsphere-forming potential (*P < 0.05, ***P < 0.001). (b) Enforced activation of AKT-mTOR pathway increases SSEA-4+ cell frequency in MG63 cells undergoing mesenchymal differentiation. Transduced cells were plated at 60% confluence and the expression of Myr-AKT was induced by Dox; SSEA-4+ cell frequency was measured 48 hours later. Results are shown as means ± SDs. The cropped blots were run under the same experimental conditions. The full-length blots can be seen in Supplementary Figure 8. (c) mTOR inhibitor relieves the AKT activation-caused differentiation arrest of MG63 cells. Data for ALP activity and OCN mRNA levels are presented as means ± SDs (*P < 0.05, **P < 0.01, ***P < 0.001). The cropped blots were run under the same experimental conditions. The full-length blots can be seen in the Supplementary Figure 8. (d) Tumorsphere-forming rates of single SSEA-4− MG63 cells expressing vector or Myr-AKT (as in b or c) are shown on the upper panel (***P < 0.001). The enhancing effect of Myr-AKT induction on the tumorsphere-forming potential of SSEA-4− cells was abolished by RAD001 (bottom panel).