Zscan4 protein expression is highly restricted in mouse ESC and iPSC lines.

<p><b>A.</b> The ZE3-MC1 Zscan4 reporter line <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089821#pone.0089821-Zalzman1" target="_blank">[25]</a> was cultured in serum replacement plus LIF, cells were fixed and immunostaining was carried out with pan-Zscan4 antibodies (Red). Expression of the Emerald reporter is shown (Green) and cells were counterstained with DAPI (Blue). (<b>i</b>) Scale bars = 100 µm. (<b>ii</b>) Scale bars = 5 µm. <b>B.</b> The mouse ESC and iPSC lines indicated were cultured in the presence of serum replacement plus LIF and subsequently fixed and immunostained with the pan-Zscan4 antibody, while nuclei were counter-stained with DAPI. The proportion of Zscan4 positive cells present were determined by counting several thousand cells for each cell line sample. Data represent average of three independent replicates ± SEM, unless otherwise indicated. <b>C.</b> A colony of Nanog-GFP reporter cells stained with the anti-Zscan4 antibody. (<b>i</b>) The GFP reporter (Green), Zscan4 (Red) and nuclei (DAPI, Blue) are shown, along with a merged image. (<b>ii</b>) Nanog-GFP cells were stained for Zscan4 protein. The levels of Nanog-GFP and Zscan4 protein expression were calculated using Cell-P (Olympus) using fluorescent images captured using an Olympus IX51 epifluorescence microscope. Mean fluorescence intensity for GFP (Nanog) was compared between all cells, Zscan4 expressing (+ve) and non-expressing (−ve) cells.</p

Zscan4 gene expression is regulated by the p110α isoform of PI3Ks.

<p><b>A.</b> (<b>i</b>) and (<b>ii</b>) E14 ESCs were cultured in the presence of LIF, with the addition of DMSO (control) or the inhibitors indicated: 5 µM LY294002 (broad spectrum PI3K inhibitor), 10 µM TGX121 (p110β), 5 µM IC87114 (p110δ), 25 nM PIK75 (p110α), 100 nM TGX221 (p110β), for 48 h. RNA was extracted and levels of Zscan4 expression analysed by qRT-PCR and normalised to levels of β-actin. Mean values are shown with standard deviations (n = 4). **, p<0.005, ***, p<0.0005 in a Student's t-test. <b>B.</b> ESCs were cultured in LIF and treated with either DMSO as a control or with 5 µM LY294002 (LY), 10 nM PIK75, 50 nM TGX221 (TGX), or 5 µM IC87114 for 48 h prior to immunostaining for Zscan4. The mean percentage of Zscan4 positive cells with SEM are shown. *, p<0.05, **, p<0.005 following an ANOVA and Tukey's post-hoc test. <b>C.</b> ESC clones over-expressing myristoylated p110α catalytic subunit of PI3Ks were cultured in the presence or absence of LIF. As a control parental OCRG9 ESCs were grown in presence and absence of LIF for 4 days. Expression of Zscan4 was analysed by qRT-PCR and Zscan4 expression normalised to levels of GAPDH. The averages and SEM of triplicate samples from each of three independent biological replicates are shown: ***, p<0.0005, in a Student's t-test.</p

Zscan4 expression is enhanced in late S/early G2 phase of the cell cycle.

<p><b>A.</b> (<b>i</b>) The proportion of Zscan4 positive cells appearing as individual single cells or in proximity with one, two or three other Zscan4 positive cells were determined. Mean and SEM are shown. Average frequency of Zscan4 positive cells was 0.4%. (<b>ii</b>) ZE3-MC1 ESCs were grown in the presence of LIF for 48 h, trypsinised and Nuclear-ID Red (Enzo) used to stain cellular DNA. Cells were analysed by flow cytometry and G1, S and G2/M gates were assigned to the population based on DNA content and using a Nocodazole treated sample as a reference. The cell cycle distribution of GFP negative and positive populations are presented, the mean and SEM are shown (n = 3). *, p<0.05, **, p<0.005, ***, p<0.0005, in a Student's t-test. (<b>iii</b>) The mean fluorescence intensity of the GFP positive (Zscan4c expressing) cells distributed in the different cell cycle phases. In each case the mean and SEM are shown. *, p<0.05, **, p<0.005, ***, p<0.0005 following an ANOVA and Tukey's post-hoc test. <b>B.</b> E14 ESCs were cultured in LIF and treated with 100 ng/ml Nocodazole for 12 h to induce mitotic arrest. Following release from the block, cell cycle analyses ((<b>i</b>), (<b>ii</b>) and (<b>iii</b>)) and immunoblotting, to detect Zscan4 protein expression (<b>iv</b>) were performed at the times indicated (n = 3). Mean and SEM are shown on the graphs. Zscan4 protein expression was normalised to GAPDH ((<b>iv</b>), lower panel).</p

The ability of Gsk-3 inhibition to promote ESC proliferation is dependent upon the culture environment.

<p>ESCs were grown in (<b>A</b>) GMEM supplemented with Serum and LIF; (<b>B</b>) N2B27 supplemented with BMP4 and LIF or <b>(C</b>) N2B27 without extrinsic stimuli, in the presence of Gsk3 inhibitor CHIR (CHIR99201) or MEK inhibitor PD (PD0325901) alone or in combination (2i) for 3 days and their growth measured. Control ESCs had DMSO vehicle added instead of inhibitors (CON). The data are the average and S.E.M of triplicate experiments. **, p<0.01, ***, p<i><</i>0.005 (2-way ANOVA with Bonferroni post-hoc test).</p

Expression patterns of Zscan4 gene paralogues in mouse ESC lines.

<p><b>A.</b> The mouse ESC lines E14 or CCE were cultured in the presence of KO serum replacement and LIF, RNA was extracted, limited amplification by RT-PCR was performed and fragments cloned. 96 independent clones were sequenced and categorised as representing one of the 9 mouse Zscan4 paralogues present in the NCBIm37 database based on SNPs. The relative expression of each Zscan4 paralogue, as a percentage of the total number of Zscan4 gene sequences obtained for E14 and CCE ESCs, are shown in comparison with the 129.3 ESC line, data derived from that presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089821#pone.0089821-Falco1" target="_blank">[26]</a>. <b>B.</b> The suggested evolution of Zscan4 genes in the mouse based on sequence variation. Dendrogram (Unweighted Pair-Groups Method using Averages) showing genetic relationship between Zscan4 paralogs based on Nei's [1972] original distance with 1000 bootstrap replications (confidence values given at each node). The dendrogram was produced using Tools for population genetic analysis (<a href="http://www.marksgeneticsoftware.net" target="_blank">www.marksgeneticsoftware.net</a>).</p

Zscan4 acts as a transcriptional regulator and interacts with LSD-1 and CtBP2.

<p><b>A.</b> Schematic showing the structure of GAL4-DNA binding domain fusions with Zscan4c. <b>B.</b> Plasmids expressing the GAL-4-DNA binding domain fusions indicated were co-transfected into HEK293 cells along with pGL4-GAL4-UAS plasmid and the pRenilla plasmid (to assess transfection efficiency). 48 h after transfection luciferase activity was determined using Dual-Glo system, according to the manufacturer's recommendations (Promega). Mean and SEM of 3 independent experiments are shown. *, p<0.05, **, p<0.005 following an ANOVA and Tukey's post-hoc test. <b>C.</b> ESCs engineered to express an N-Terminal eGFP-Zscan4c fusion protein under the control of the pTRE tight Dox-inducible expression system were cultured in the absence (−) or presence (+) of Dox for 72 h. Nuclear extracts were prepared (Nuc) and immunoprecipitates prepared from 80 µg of protein per sample using either GFP-Trap-A beads or anti-LSD-1 antibodies. Precipitates were divided into two aliquots and separated through 10% polyacrylamide on duplicate gels, prior to immunoblotting. Immunoblotting was performed with anti-GFP, anti-CtBP2 or anti-Zscan4 antibodies, sequentially with one immunoblot. The duplicate immunoblot was probed with anti-LSD-1 antibodies. Positions of precipitated proteins are indicated. <b>D.</b> ESCs engineered to express a C-Terminally V5 epitope-tagged version of Zscan4c under the control of the Tet-off expression system were cultured in the presence (+) or absence (−) of Tet for 72 h. Nuclear extracts were made (Nuc) and immunoprecipitates prepared from 80 µg of protein per sample using either anti-LSD-1 or anti-CtBP2 antibodies or protein-A sepharose (PAS) alone as a control. Precipitates were separated through 7.5% polyacrylamide gels prior to immunoblotting. Immunoblotting was first performed with anti-Zscan4 antibodies. The blots were then stripped and reprobed with anti-V5 epitope antibodies. Positions of precipitated proteins are indicated. <b>E.</b> Nuclear extracts (Nuc) were prepared from E14 ESCs and precipitates from 200 µg of protein per sample generated using anti-Zscan4, anti-LSD-1 or anti-CtBP2 antibodies. Precipitates were separated through a 6.5% polyacrylamide gel prior to immunoblotting and the lanes between the samples were left blank to avoid the potential of any bleed-through. Immunoblotting was performed with anti-Zscan4 antibodies. Positions of precipitated proteins are indicated. <b>F.</b> ESCs engineered to express a C-Terminally V5 epitope-tagged version of Zscan4c under the control of the Tet-off expression system were cultured in the presence (+) or absence (−) of Tet for 72 h. Nuclear extracts were prepared (Nuc) and immunoprecipitates prepared from 80 µg of protein per sample using anti-V5 epitope antibodies. Precipitates were separated through a 7.5% polyacrylamide gel prior to immunoblotting. Immunoblotting was performed with anti-Zscan4 antibodies. Positions of precipitated proteins are indicated.</p

Zscan4 expression is enhanced in response to DNA damage induction.

<p>E14 ESCs were plated in the presence of LIF for 4<b>A.</b> Zeocin or <b>B.</b> Cisplatin for 12 h. After this cells were washed extensively and fresh media added. Following a further 8 or 24 h samples were taken and cells fixed for cell cycle analysis or protein extracted into RIPA buffer. (i) Levels of H2AX phosphorylation at S139 (γH2AX) and Zscan4 protein expression were examined by immunoblotting. (ii) Zscan4 protein expression normalized to GAPDH is shown. (iii) The percentage of cells in G2/M phase of the cell cycle are shown for different samples. <b>C.</b> (<b>i</b>) The percentage of Zscan4 positive cells were determined after 24 h treatment with the indicated doses of Zeocin. (<b>ii</b>) The effect of inhibition of PI3Ks by LY294002 (LY, 5 µM) on the ability of Zeocin to induce Zscan4 expression was examined following 24 h treatment (12.5 µg/ml). In each case the mean and SEM are shown. *, p<0.05, **, p<0.005, ***, p<0.0005 following an ANOVA and Tukey's post-hoc test. <b>D.</b> (<b>i</b>) E14 ESCs were co-stained for γH2AX and Zscan4 and the mean fluorescence intensity of γH2AX staining determined for Zscan4 positive and negative populations using Cell-P (Olympus). ***, p<0.0005, in a Student's t-test. Representative images are shown in (<b>ii</b>).</p

Gsk-3 inhibition or ablation increases Nanog protein synthesis in the absence of increased transcription.

<p>WT and Gsk-3 DKO ESCs were incubated in (<b>A</b>) GMEM supplemented with LIF and serum or (<b>B</b>) N2B27 medium supplemented with LIF and BMP4 prior to addition of 10 mg/ml CHX for 4 hours to halt protein synthesis. CHX was then washed out and fresh media (<b>A</b>, GMEM plus LIF plus serum and <b>B</b>, N2B27 plus LIF and BMP4) added back. (i) & (ii) Protein and (iii) RNA were extracted between 1 and 4 hours after CHX washout. (i) Immunoblotting was performed with the antibodies indicated. (ii) Antibody signals were quantified and normalised to Gapdh. A value of 1 was given to CHX-treated samples. The data are the average relative protein expression levels and SD of duplicate experiments. (iii) Quantitative RT-PCR was carried out and Nanog expression normalized relative to ß-actin levels. A value of 1 was given to CHX-treated samples in (iii). The data are the average gene expression levels and S.E.M of quadruplicate samples. (<b>C</b>) Nanog reporter Nd ESCs were sorted by flow cytometry and cells with low to no VNP expression collected and grown in GMEM supplemented with LIF and serum in the presence or absence of DMSO (controls) or in the presence of 3 µM CHIR99201. The data shown are the average Nanog:VNP expression levels (relative to DMSO control) and standard deviation of at least 3 biological replicates. All <i>p</i>-values were calculated using a two-tailed distribution, two-sample equal variance t-test (** p-value <0.01; *** p-value <0.0001).</p

Gsk-3 inhibition does not alter Nanog, Tbx3, Oct4 or c-Myc protein stability.

<p>(<b>A</b>) E14tg2a WT ESCs and (<b>B</b>) WT and Gsk-3 DKO ESCs were grown in the presence of N2B27 plus LIF and BMP4 for 24 h. In (<b>A</b>) 1 m (2 µM) or vehicle alone control (CTL) were added to the cells for this 24 h period. All samples were then incubated with cycloheximide (CHX) to halt protein synthesis for the times indicated. (i) Protein samples were extracted after 0, 1, 3 and 6 hours of CHX treatment and immunoblotting performed with the indicated antibodies. (ii) Antibody signals were quantified and protein levels normalised to Gapdh. In each case a value of 100% was given to the t = 0 samples to enable direct comparisons to be made. The data are the average relative protein expression levels and S.E.M of triplicate experiments.</p

Inhibition or ablation of Gsk-3 enhances expression of Nanog, Tbx3 and c-Myc proteins in ESCs.

<p>E14tg2a wild-type (WT) and Gsk-3α/ß double knockout (DKO) mESCs were cultured in the presence of serum plus LIF (<b>A</b>) or chemically defined medium (N2B27) plus LIF and BMP4 (<b>B</b>). Gsk-3 inhibitors 1 m or CHIR99201 (CHIR) were added to WT ESCs at 2 and 3 µM respectively, as indicated in both <b>A</b> and <b>B</b>. (i) Images show colonies formed from WT ESCs that were untreated (WT), or cultured in the presence of 2 µM 1 m or 3 µM CHIR99201 for 48 h and Gsk-3 DKO ESCs. Protein and RNA were extracted at the times indicated. (ii) 12 µg of nuclear protein extracts were immunoblotted with the antibodies specified. (iii) Antibody signals were quantified and normalised to Gapdh. A value of one was given to normalised protein levels in WT ESCs at 8 hours and values for other samples related to these. The data show relative protein levels and are the average and SD of duplicate experiments for <b>A</b> and a single representative experiment in <b>B</b>. (iv) Quantitative RT-PCR was carried out and gene expression normalized relative to ß-actin levels. A value of one was given to normalised RNA levels in WT ESCs at 8 hr and other samples related to these. The data show relative gene expression levels and are the average and S.E.M of quadruplicate samples. *, p<0.05; **, p<0.01, ***, p<0.005. * for Nanog, #for Tbx3 and+for c-Myc (2-way ANOVA with Bonferroni post-hoc test). (<b>C</b>) Nanog-TNG reporter ESCs were cultured in serum and LIF in the presence of 3 µM CHIR99201 (CHIR) or DMSO (CON) and imaged over a period of 24 h using confocal microscopy, with images taken every 10 mins. Representative still images of ESC colonies are shown for the times specified. 10 fields of view chosen at random and within each field at least 15 colonies were imaged. White arrows indicate cells where Nanog reporter expression increases at early time points. Complete time-lapse series for control and CHIR-2 are available Videos S1 and S2.</p