Fig 1.
RA induces Zscan4 ESC metastate.
(A) ESCs were cultured for 36 and 72 h in differentiating conditions: Lif-, 1% DMSO and 1.5 μM RA. 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 t test. (B) Zscan4 expression pattern in ESCs cultures by RNA in situ hybridization upon 5 days of treatment in RM or RA. RNA ISH showed “spotted” patterns on ESC colonies (40x). (C) Percentage of RM-Zscan4+ and RA-Zscan4+ cells was evaluated by flow cytometry analyses. The mean % ESZscan4_Em cells ± SD of three independent experiments is presented with statistical analysis performed using Student’s t test (**, p < .01). (D) Distribution of Zscan4 related gene signature in ESC cultures by RNA in Situ hybridization showed “spotted” patterns on ESC colonies. Most representative colonies were magnified (20x) to show the detailed staining patterns.
Fig 2.
Zscan4 metastate induction requires canonical RAR signaling.
(A) (I) The percentage of ESZscan4_Em cells was assessed on ESCs cultured for 3 days in: RM+DMSO; RM supplemented with RA; RA plus BMS493; BMS753, and UVI2060. The mean % ESZscan4_Em cells ± SD of two independent experiments is shown. (II) The mRNA expression levels were assessed on ESCs treated with RA and RA plus BMS493 by qRT-PCR and normalized to RM+DMSO condition. The average and SD of duplicate samples from each of three independent biological replicates are shown: *, p < .05; **, p < .01; ***, p < .001, in a Student’s t test. B (I) The mRNA expression levels were assessed on ESCs treated with BMS753 by qRT-PCR and normalized to RM+DMSO 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 t test. (II) The mRNA expression levels were assessed on ESCs treated with UVI2060 by qRT-PCR and normalized to RM+DMSO condition. The average and SD of duplicate samples from each of three independent biological replicates are shown: *, p < .05; ***, p < .001, in a Student’s t test.
Fig 3.
PI3 Kinase activation regulates Zscan4 metastate induction.
(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 t 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 t test.
Fig 4.
RA-Zscan4+ cells molecular and cellular characterization.
(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 t 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 (n = 3).
Fig 5.
RA induces Zscan4 metastate transition.
(A) Zscan4 and Gm12794 expressions were visualized by Emerald and Strawberry reporter respectively. (B) Percentage analyses of ESGm12794_St/Zscan4_Em cells upon 3 days of treatment in RM or RA by flow cytometry. The mean % ESGm12794_St/Zscan4_Em cells ± SD of three independent experiments is presented with statistical analysis performed using Student’s t test: **, p < .01. (C) Cell live imaging reported in 4 time frames of 2 hour each. (D) ESGm12794_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 t test.
Fig 6.
Model the activation of RA-Zscan4+ metastate during ESC differentiation through RA treatment.