Figure 3.

Mitophagy is required for metabolic reprogramming in differentiation.A, cells were transduced with the lentivirus PercevalHR, which has an excitation wavelength at 405 nm for ADP, an excitation wavelength at 488 nm for ATP, and one emission wavelength of 529 nm. Cells were also transduced with a doxycycline-inducible Mfn2 shRNA lentivirus. Cells were first treated with 200 ng/ml doxycycline (or DMSO) for 3 days to knockdown Mfn2. They were differentiated for 4 days and then imaged live with confocal microscopy. Ratiometric images created with ImageJ are shown. The calibration bar shows that red has the highest ATP-to-ADP ratio followed by green and then blue. Scale bar = 20 μm. B, quantification of (A) where control is DMSO vehicle treated and Mfn2 KD is doxycycline treated. Day 4 cells have a significantly higher ATP-to-ADP ratio, whereas Mfn2 KD cells have a blunted increase in ATP generation. Mean ± SEM with n = 36 fields of view, representative of three independent experiments with ∗∗∗p < 0.001. C, Western blot image of knockdown of Mitofusin 2 on Day 3 after doxycycline induction in iPSCs. D–G, mRNA levels of (D) K-Glutaminase, (E) L-Glutaminase, (F) Fatty Acid Binding Protein 4 (FABP4), and (G) CPT1A were evaluated in iPSCs and VE-Cadherin sorted cells (iPSC-ECs) by RT-qPCR. B2M (β-2 microglobulin) was used as housekeeping control. Mean ± SEM, n = 3 with ∗∗p < 0.01. H, Seahorse Analyzer assessment depicting mitochondrial OCR (oxygen consumption rate) of iPSC and iPSC-ECs with previous addition of either vehicle (0.1% BSA, basal) or 0.5 μM palmitate. I and J, quantification of the basal respiration and maximal respiration in (H) showing a significant increase of OCR in iPSCs as compared with iPSC-ECs at basal levels and after the addition of palmitate. n = 22 ∗∗∗∗p < 0.0001.