Figure 2.

p53- and p21-KO cells and pluripotent reprogramming intermediates express low levels of mitochondrial fusion proteins. (a) OSKM-transduced WT, p53−/−, and p21−/− MEFs on D7 of reprogramming were stained with Tom20 (mitochondria, green) and DAPI (nuclei, blue) (top). High-magnification images (bottom). (b) Mitochondrial morphology of WT, p53−/−, and p21−/− MEFs stained with Mitotracker (red) and enlarged images (inset in top right corner). (c) Cell proliferation of WT, p53−/−, and p21−/− MEFs were determined by cell number counting. (d) Western blot analysis of Cyclin B1 and mitochondrial fission (Drp)-fusion (Mfn) components. β-actin was used as an internal control. (e) Reprogramming intermediates were sorted based on Thy1 and SSEA1 expression using MACS on D11. Representative images of each subgroup (top), the mitochondrial morphology stained with Mitotracker (red) (middle), and enlarged images (inset in middle right corner) on D14. The percentages of cells with fragmented/intermediate/fused mitochondria were determined in each subgroup (bottom). (f) Western blot analysis of mitochondrial fission (Drp)-fusion (Mfn) components in the mitochondrial fraction and of the pluripotency marker Nanog in the whole-cell lysates of MEFs and each subpopulation. HSP60 and β-actin were used as internal controls. (g) Schematic presentation of the p53- and p21-KO cells. Scale bar=50 μm