PI3 Kinase activation regulates Zscan4 metastate induction.

<p>(A) Western Blot analysis on ESCs treated with either RA or RA with 5.0 μM LY294002 for 12h. NANOG was considered the positive control for treatment evaluation. (B) The mRNA expression levels were assessed by qRT-PCR and normalized to RM condition. The average and SD of duplicate samples from each of three independent biological replicates are shown: **, p < .01; ***, p < .001, in a Student’s <i>t</i> test. (C) Western Blot analysis on ESCs were treated with either RA or RA with 20 μM PD0325901 for 12h. ESCs treatment with RA increased pERK1/ERK2, whereas PD treatment hampered pERK1/ERK2. (D) The mRNA expression levels are assessed by qRT-PCR and normalized to RM condition. The average and SD of duplicate samples from each of three independent biological replicates are shown: *, p < .05, in a Student’s <i>t</i> test.</p

RA-Zscan4⁺ cells molecular and cellular characterization.

<p>(A) The global gene expression profiles between RA-Zscan4⁺ and RA-Zscan4^- cell populations by DNA chip microarray hybridization. List of RA-Zscan4⁺ probes upregulated more than 20-fold compared to RA-Zscan4^-. (B) RA-Zscan4⁺ cells expression versus RM-Zscan4⁺. (C) RA-Zscan4⁺ and RA-Zscan4^- cells were collected, separated by FACS and plated in RM. The mean % RA-Zscan4⁺ or RA-Zscan4^- cells ± SD of three independent experiments is presented with statistical analysis performed using Student’s <i>t</i> test (**, p < .01; ***, p < .001). (D) RA-Zscan4⁺ and RA-Zscan4^- cells were collected and separated by FACS, plated in RM and were characterized based on AP-positive colonies after 5 days in RM (<i>n</i> = 3).</p

RA induces Zscan4 metastate transition.

<p>(A) <i>Zscan4</i> and <i>Gm12794</i> expressions were visualized by Emerald and Strawberry reporter respectively. (B) Percentage analyses of ES^{Gm12794_St/Zscan4_Em} cells upon 3 days of treatment in RM or RA by flow cytometry. The mean % ES^{Gm12794_St/Zscan4_Em} cells ± SD of three independent experiments is presented with statistical analysis performed using Student’s <i>t</i> test: **, p < .01. (C) Cell live imaging reported in 4 time frames of 2 hour each. (D) ES^{Gm12794_HSVTK} cell line and control E14Tg2a.4 cultured in media supplemented with RA in presence or absence of GCV (2.0 μM, Sigma). The mRNA expression levels were assessed by qRT-PCR and normalized to RA/GCV- condition. The average and SD of duplicate samples from four independent biological replicates are shown: ***, p < .001, in a Student’s <i>t</i> test.</p