Figure 1.

There is an initial burst of mitophagy during differentiation. A, schematic of differentiation protocol. hiPSCs were differentiated for 7 days in serum-free media toward an endothelial lineage. Cells were collected at days 0 (iPSC), 2, 4, and 7 after sorting for VE-Cadherin positive cells (iPSC-EC). B, representative confocal image of VE-Cadherin after VE-Cadherin (CD144) sorting of day 7 cells. iPSC-ECs express VE-Cadherin at the junctions between cells. DAPI depicts nuclei staining. Scale bar = 10 μm. C, live cells were stained with MitoTracker (mitochondria, green) and Lysotracker (lysosomes, red) and imaged at specified timepoints with confocal microscopy. iPSC indicates cells at day 0, 2D = day 2, 4D = day 4, and iPSC-EC indicates 7 days differentiated cells, sorted for VE-Cadherin. Scale bar = 10 μm. D, quantification of (C) indicating the percent of overall mitochondria within lysosomes. Mitophagy is increased on days 2 and 4. Mean ± SEM; n = 8 fields of view, image shown is representative of three independent experiments, with ∗∗p < 0.01. E, cells were lysed at different stages of differentiation. Western blots show Ser443 Mitofusin 2 phosphorylation, overall Mfn2 levels, PINK1 levels, and GAPDH as loading control. F, quantification of (E), which shows a higher level of phosphorylated Mfn2 on days 2 and 4 of differentiation. Mean ± SEM; n = 5 with ∗p < 0.05. G, quantification of (E), which shows a higher level of PINK1 on days 2 and 4 of differentiation. Mean ± SEM; n = 5 with ∗p < 0.05.