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. 2016 Mar 1;30(5):535–552. doi: 10.1101/gad.274142.115

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© 2016 Young et al.; Published by Cold Spring Harbor Laboratory Press

This article is distributed exclusively by Cold Spring Harbor Laboratory Press for the first six months after the full-issue publication date (see http://genesdev.cshlp.org/site/misc/terms.xhtml). After six months, it is available under a Creative Commons License (Attribution-NonCommercial 4.0 International), as described at http://creativecommons.org/licenses/by-nc/4.0/.

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Figure 1. — Dynamic AMPK signaling during EB differentiation and generation and characterization of AMPK double-knockout ESCs. (A) RT-qPCR analysis of AMPK subunits during EB differentiation. γ3 was not detected at any time point. Data are from two independent experiments. Bar graphs depict mean ± SEM. (B) ESCs and differentiating EBs were lysed on the indicated days 1 h after a medium change and subjected to Western blotting with the antibodies listed. (C) Western blot analysis of AMPKα1 and AMPKα2 in wild-type (WT) parental and two independent AMPK double-knockout (DKO) CRISPR clones. (D) Immunoblot on lysates from wild-type and AMPK double-knockout ESCs following vehicle or 1 h of 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) treatment (0.5 mM). (E) Levels of pluripotency markers Oct4 and Nanog in wild-type and AMPK double-knockout ESCs as determined by Western blot. (F) Bright-field images of wild-type and two AMPK double-knockout ESC lines grown on feeders indicating normal ESC-like morphology (panel i) and equivalent amounts of alkaline phosphatase activity between the different genotypes (panel ii). Bar, 100 µm. (G) Proliferation curves of wild-type and AMPK double-knockout ESCs grown in the absence of feeders in both high glucose (HG; 25 mM) and low glucose (LG; 2.5 mM). n = 2 samples per condition.