Figure 5.

Increased Mitochondria Polarization during Cell Reprogramming

(A) Representative flow-cytometry histograms of MEFs, induced pluripotent stem cells (iPSCs), and ESCs stained with TMRM to assess mitochondrial membrane potential. Graph underneath shows the quantification of the mean fluorescence intensity of the histograms shown above.

(B) Representative confocal images of live MEFs before (Control) or 12 days after OSKM expression (Day 12), iPSCs, and ESCs stained with anti-THY1 (green) or anti-SSEA1 (yellow) antibodies, combined with the cell-permeable TMRM dye (red). Hoechst 33342 (blue) was used as a nuclear counterstaining. Lower pictures are color map representations of the pictures in the middle panels showing TMRM intensity according to the displayed color bar. Scale bars, 24 μm (upper images) and 12 μm (middle and bottom images).

(C) Right: histograms of TMRM staining in MEFs expressing the indicated factors for the days shown. Left graph represents the TMRM/MitoTracker green ratio dynamics along the indicated days.

(D) Lysates of MEF control, expressing OSKM for the specified days or ESCs (upper panels, black and red bars in the right graph), or the indicated factors for 4 days (lower panels, colored bars in the right graph) were analyzed by immunoblotting using the shown antibodies. Graphs on the right show the quantification of the data.

(E) MEFs transduced with OSKM factors were transfected with esiRNAs targeting either eGFP (Control) or Atpif1 at day 1 post transduction. Graph shows the number of AP-positive colonies obtained after 25 days of retroviral delivery. Panels on the right show representative bright-field images from the plates of the indicated cultures after AP staining; inset shows magnification of a selected area from the AP-stained plates.

Data represent mean ± SEM, one-tailed unpaired t test (n = 3): ^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001; ^∗∗∗∗p < 0.0001. See also Figure S6.