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. 2014 Dec 1;9(12):e113775. doi: 10.1371/journal.pone.0113775

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© 2014 Doppler et al

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.

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Scale bars: 200 µm for all panels. A. Lentiviral constructs for the production of the doxycyclin-inducible tetOMzf1-Nkx2.5 CE eGFP ES line. LTR: long terminal repeats. TRE: tetracyclin responding element. CMV: cytomegalovirus promoter. IRES: internal ribosomal entry site. rtTA: reverse tetracyclin transactivator. B. In vitro differentiation protocols with time-schedules of dox-treatment. C. Morphology of differentiating EBs on day eight. Permanent and day 0–5 dox-treatment led to closely packed globular clusters. In contrast dox-treatment from day 5 showed a normal differentiation pattern comparable to the control w/o dox. D/E. FACS analysis revealed a significant increase in eGFP⁺ CPCs for dox-treatment from day 5 of differentiation whereas a continuous and day 0-5 dox-treatment resulted in a significant decrease of eGFP⁺ CPCs compared to control w/o dox; ** = p <0.01.