Mechanism of Akt1 in promoting reprogramming.

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Mechanism of Akt1 in promoting reprogramming. Mechanism of Akt1 in promoting reprogramming. (A) Western blotting of total protein extracts from B6/129-MEFs transduced with either vector control or CA-Akt1, and with (+) or without (−) a 20 minute exposure to LIF. The specific proteins probed were Akt, Stat3, Erk1/2 and their respective phosphorylated forms. Tubulin was used as the loading control. Stimulation with LIF and CA-Akt1 co-stimulated Stat3 phosphorylation. (B) Western blotting of total protein extracts from B6/129-MEFs transduced with either vector control or CA-Akt1 and treated with DMSO, 5 µM rapamycin (Rap) or 1 µM MK2206 (MK), with (+) or without (−) LIF stimulation. The specific proteins probed were Akt, Stat3 and their respective phosphorylated forms. GAPDH was used as the loading control. Both mTOR and Akt inhibitors suppressed Stat3 phosphorylation. (C) Relative signal density of pStat3 and pAkt from western blots shown in B. The values were normalized with GAPDH and calculated relative to the levels in DMSO/Vector/LIF-treated cells. (D) Relative levels of mRNA for LIFR, Akt1, Stat3 and Socs3 in B6/129 MEFs transfected with retroviral GFP control or CA-Akt1, with (+) or without (–) 20 minutes of stimulation with LIF; c denotes combined endogenous and retroviral Akt1 expression. Values were normalized to GAPDH and expressed relative to the GFP control without stimulation with LIF. Compared to the GFP control without stimulation with LIF, the level of LIFR was significantly increased in CA-Akt1-transduced cells and highest in CA-Akt1 cells treated with LIF. As an indicator of Stat3 activation, the level of Socs3 was significantly increased when cells had been treated with LIF and was highest in CA-Akt1 cells treated with LIF, demonstrating a positive feedback effect of Akt1 on the LIF pathway. (E) Effect of Stat3 inhibition on formation of GFP-positive colonies induced by OKSM plus vector control or CA-Akt1. Transduced OG-MEFs were cultured in LIF-containing medium with either DMSO or 1 µM Jaki and observed 2 weeks after OKSM transduction. Inhibiting Stat3 completely blocked GFP-positive colony formation even in the presence of CA-Akt1. (F) Representative ESC-like colonies induced in OG-MEFs and treated with either DMSO or Jaki as described in E. (Scale bar: 610 µm). Inhibiting Stat3 completely blocked GFP-positive expression but not colony formation. Yong Tang et al. J Cell Sci 2014;127:3998-4008 © 2014. Published by The Company of Biologists Ltd