Figure 2.

There is a subsequent burst of mitochondrial biogenesis after mitophagy.A, schematic of MitoTimer doxycycline-inducible construct. Mitochondria fluoresce green as they are newly synthesized and then gradually fluoresce red as they age over 72 h. Doxycycline pulses depicted for experiment in (B). Three days before endpoint, cells were pulsed for 24 h with 2 μg/ml of doxycycline. After doxycycline is removed, mitochondria age and the network emit a red fluorescence. On day 4 of differentiation, a second 2 μg/ml doxycycline pulse was added for 24 h and then removed. Eight hours is sufficient time for these mitochondria to fluoresce green. Afterward, the cells were fixed with 4% PFA. B, MitoTimer confocal images of fixed iPSCs and 4-day differentiated cells. 2 μg/ml doxycycline pulses are outlined in (A). Red mitochondria represent old mitochondria and green mitochondria represent new mitochondria. Merged image includes both channels as well as DAPI nuclear staining. Scale bar= 10 μm. C, quantification of (B) indicating the intensity of green fluorescence (488 nm) over the intensity of red fluorescence (561 nm). Mean ± SEM, with n = 12 fields of view from three independent experiments, ∗p < 0.05. D, mRNA was extracted from cells at different stages of differentiation and mRNA levels of PGC-1α, which is increased on day 4 as compared with iPSCs. Mean ± SEM, n = 3 with ∗∗∗p < 0.001. E, immunohistochemistry of cells fixed at different days of differentiation on coverslips and imaged with confocal microscopy. TOMM20 is shown in green and the nuclei are in blue. Top right inset is a zoom image of one cell. Scale bar = 10 μm. F, quantification of (E) as the amount of total pixels of TOMM20 over the amount of nuclei per frame. There are more mitochondria on day 4 of differentiation followed by a larger increase of mitochondria in iPSC-ECs. Mean ± SEM with n = 12 fields of view from three independent experiments, ∗∗p < 0.01, ∗∗∗p < 0.001.