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. 2018 Nov 21;11(6):1479–1492. doi: 10.1016/j.stemcr.2018.10.018

Figure 1.

Figure 1

Role of c-MYC in Cell Reprogramming-Induced Mitochondrial Fission

(A) Representative bright-field images after alkaline phosphatase (AP) staining of plates containing MEFs after 25 days of either OSK (right panels) or OSKM (left panels) retroviral delivery in the presence of DMSO (control) or the MYC inhibitor 10058-F4 (ic-MYC, 10 μM). Inset shows a magnification of a selected area from the AP-stained plates. Data on the bottom left-hand side of the pictures represent the mean ± SEM of three independent experiments.

(B) MEFs were mock-infected (control) or transduced with the indicated factors. At day 4 post transduction, cells were fixed and mitochondrial morphology assessed by immunofluorescence. Left panels: representative confocal images of MEFs stained with anti-TOM20 antibodies (red) before (control) or after expressing the indicated factors. Inset shows a black-and-white magnification of the pictures. DAPI (blue) was used as a nuclear counterstaining. Graph on the right shows the quantification of the different mitochondrial morphologies.

(C) Representative confocal images of MEFs before (Control) or 4 days after OSKM, OSK, or c-MYC expression stained with anti-DRP1 (green) or anti-TOM20 (red) antibodies. DAPI (blue) was used as a nuclear counterstaining. Middle panels show a magnification of the pictures displayed in the upper panels. Bottom images are color map representations of the pictures in the middle panels to display co-localized pixels between both fluorophores according to the color bar shown on the upper-right corner of the pictures. Warm colors depict pixels with highly correlated intensity and spatial overlap while cold colors are indicative of random or anti-correlation. Graph on the right shows the quantification of the Pearson's correlation coefficient (PCC) to display the degree of co-localization between DRP1 and TOM20 in cells transduced with the indicated factors. Red dashed line indicates the levels of DRP1 and TOM20 co-localization found in ESCs.

(D) Lysates of MEFs control or expressing OSKM, OSK, or c-MYC for 4 days were analyzed by immunoblotting using the indicated antibodies. Graphs on the right show the quantification of the data.

Data represent mean ± SEM, one-tailed unpaired t test (n = 3): ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001. Scale bars, 24 μm in (B) and upper panels of (C); 12 μm in middle and bottom panels of (C). See also Figure S1.