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. 2015 Nov 13;10(11):e0142554. doi: 10.1371/journal.pone.0142554

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This is an open-access article distributed under the terms of the Creative Commons Public Domain declaration, which stipulates that, once placed in the public domain, this work may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose.

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Fig 5 — (A) Schema of 2-NBDG accumulation and retention (glycogen labeling) experiments. (B) 2-hour 2-NBDG accumulation in the presence of 10 mM D-glucose. Upper panel: green fluorescence intensity (Fluor) images that include signals from both Oct4-EGFP and 2-NBDG. These images were obtained (immediately after replacing with fresh mTeSR1 medium) by non-saturated time-exposure guided by an autoexposure software (Zeiss Inc.). No 2-NBDG indicates fluorescence background images produced from Oct4-EGFP signal without the use of 2-NBDG. The fluorescence intensity was determined by the signal/noise ratio between cellular fluorescence (signal) and background (noise). The fluorescent background in the 2-NBDG control was due to non-saturated auto exposure using the Zeiss Axiovert imaging system. Lower panel: the corresponding phase images of the upper panel. Only brightness was adjusted in phase images (presented in both B and C) to enhance the image presentation in this figure. (C) 2-NBDG retention and glycogen labeling carried out in the presence of 10 mM D-glucose and absence of 2-NBDG. Upper panel: unique fluorescence loci (dots) were derived from 2-NBDG signals as indicated by arrows in the inset of Fig 5C4. Lower panel: the corresponding phase images of the upper panel. (D) Quantitative analysis of Oct4-EGFP signals without the addition of 2-NBDG. (E) Quantitative analysis of 2-NBDG accumulation in Fig 5B. (F) Quantitative analysis of 2-NBDG retention and glycogen labeling by counting 2-NBDG loci as presented in Fig 5C. Columns represent mean fluorescence intensity measured from at least 4 random colonies and bar standard deviations. Abbreviations (depicted sequentially): BIO, 2 μM GSK3i (BIO); 2i, 2 μM BIO + 1 μM MEKi; 3i, 2i + 1 μM BMP4i; 2iL, 2i + 10 ng/mL LIF; 3iL, 3i + 10 ng/mL LIF; 2-NBDG, (2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose), a fluorescent glucose derivative, overlapping with EGFP signals. Scale bars represent 100 μm.