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. 2014 Nov 11;111(47):E5039–E5048. doi: 10.1073/pnas.1419272111

Fig. 1.

Fig. 1.

Constitutive c-Myc expression across cellular differentiation prevents the development of circadian clock oscillation. (A) mBmal1:luc ESCs or PER2Luc ESCs carrying Dox-inducible c-Myc (tetO:c-myc mBmal1:luc ESCs or tetO:c-myc PER2Luc ESCs) were established using PiggyBac transposon vectors with a Dox-inducible c-Myc. (B) Immunofluorescent staining of tetO:c-myc ESCs using anti-c-MYC antibody. c-Myc was induced by addition of 40 ng/mL Dox (Left, tetO:c-myc mBmal1:luc ESCs) or 500 ng/mL Dox (Right, tetO:c-myc PER2Luc ESCs) for 1 d. (C) Schematic of method to express c-Myc constitutively. During in vitro differentiation, culture medium was changed with EFM containing 0, 40 (for tetO:c-myc mBmal1:luc ESCs), or 500 ng/mL Dox (for tetO:c-myc PER2Luc ESCs). (D) Morphological observation of in vitro 2-, 7-, 14-, and 21-d differentiated tetO:c-myc mBmal1:luc ESCs (Upper) or tetO:c-myc PER2Luc ESCs (Lower) in the absence or presence of Dox. (Scale bars, 100 µm.) (E) Quantitative RT-PCR analysis of differentiation markers, Nanog, Oct3/4 (Pou5f1), and Sox2, in ESCs or in vitro-differentiated ESCs. Data are mean ± SD (n = 3). (F) Bioluminescence rhythms of tetO:c-myc mBmal1:luc ESCs (Left) and tetO:c-myc PER2Luc ESCs (Right) after 28-d differentiation culture with or without Dox. (mean with SD, n = 12). (G) Representative single-cell bioluminescence traces of in vitro 28-d-differentiated tetO:c-myc mBmal1:luc ESCs in the absence or presence of Dox. The red line is the LOWESS curve of the fitted values. (H) FFT (Fast Fourier Transform) spectral power analysis of bioluminescences of in vitro 28-d-differentiated tetO:c-myc mBmal1:luc ESCs in the absence or presence of Dox. Bars are mean (n = 12). Student t test, ***P < 0.001. (I) Representative single-cell bioluminescence traces of in vitro 28-d-differentiated tetO:c-myc PER2Luc ESCs in the absence or presence of Dox. (J) FFT spectral power analysis of bioluminescence of in vitro 28-d-differentiated tetO:c-myc PER2Luc ESCs in the absence or presence of Dox. Bars are mean (n = 15–19). Student t test, ****P < 0.0001.