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. 2021 Nov 14;297(6):101410. doi: 10.1016/j.jbc.2021.101410

Figure 4.

Figure 4

The mitochondrial phosphatase PGAM5 activates the Wnt/β-catenin pathway during differentiation.A, iPSCs were treated with 10 μM oligomycin and 5 μM antimycin (or DMSO vehicle) for 3 h to induce mitophagy. Cells were then lysed and run on a SDS-PAGE gel. Membranes were immunoblotted for PGAM5. Full PGAM5 is the upper band and cleaved PGAM5 is the lower band. β-actin was used for loading control. B, quantification of (A), which shows a higher level of cleaved PGAM5 after mitophagy induction. Mean ± SEM, n = 4 with ∗p < 0.05. C, cells were lysed at different stages of differentiation. Western blots are shown of full and cleaved PGAM5. D, quantification of (C), which shows a higher level of cleaved PGAM5 on day 4 of differentiation. Mean ± SEM, n = 5 with ∗p < 0.05. E, cells were lysed at different stages of differentiation and PGAM5 was immunoprecipitated with 1 μg β-catenin specific antibody followed by Western blotting for PGAM5 and β-catenin. F, quantification of (E), which shows a higher level of interaction between cleaved PGAM5 from IP and β-catenin on Day 4 of differentiation. Mean ± SEM, n = 3 with ∗p < 0.05. G, cells were collected at different stages of differentiation and then subjected to a subcellular fractionation. An equal amount of nuclear and cytosolic extracts was loaded onto an SDS-PAGE gel and then probed for β-catenin. p84 and GAPDH were used as nuclear and cytosolic loading controls, respectively. H, quantification of (G), which shows an increased amount of nuclear β-catenin on day 4. Mean ± SEM, n = 3 with ∗p < 0.05. I, immunohistochemistry of cells fixed at days 0 and 4 of differentiation on coverslips and imaged by Z-sectioning with confocal microscopy. β-catenin is shown in green and the nuclei are in blue. Scale bar = 20 μm. A zoomed image of the white box is shown on the right. Scale bar = 10 μm. J, quantification of (I) as the colocalization of nuclei to β-catenin. There is more nuclear β-catenin on day 4 of differentiation. Mean ± SEM, n = 7 fields of view, three independent experiments with ∗p < 0.05.