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. 2015 Apr 17;22(12):1957–1969. doi: 10.1038/cdd.2015.43

Figure 5.

Figure 5

Reciprocal inhibition of p53/p21 and Mfn1/2 activates the Ras-Raf-HIF1α pathway. (a) WT, Mfn1−/−, and Mfn2−/− MEFs were reprogrammed via retroviral transduction of OSKM in the absence and presence of 25 μM Nutlin3a, an MDM2 inhibitor that stabilizes p53. Western blot analysis of p53 and p21 on D11 of reprogramming. (b) WT, p53−/−, and p21−/− MEFs were reprogrammed via retroviral transduction of OSKM with or without retroviral Mfn1 overexpression. Western blot analysis of Mfn1/2 on D11 of reprogramming. (c and d) Representative images of AP+ colonies (top and middle) and the total numbers of AP+ colonies (bottom) were obtained in each indicated group. (e) Western blot analysis of the indicated Ras-Raf signaling proteins in MEFs transduced with OSKM and Mfn1/2 shRNAs on D7 of reprogramming. (f) Cellular ROS levels in MEFs transduced with OSKM and Mfn1/2 shRNAs on D7 of reprogramming. (g) Western blot analysis of HIF1α in Mfn1/2 knockdown reprogramming cultures absence and presence of indicated concentration of N-acetylcysteine. (h) Model showing the activation of Ras-Raf-HIF1α signaling and ROS-mediated HIF1α stabilization in Mfn1/2-depleted cells. β-actin was used as an internal control. The data are presented as the mean±S.E. (n=3). *P<0.05; **P<0.01 (Student's t-test)