The consequence of p53 accumulation in hESCs.

<p>(A) Cell cycle analysis. hESCs transfected with non-target (siControl) or siRNA specific to p53 (<i>siTP53</i>) or p21 (<i>siCDKN1A</i>) and treated with RA were stained with PI and subjected to flow cytometry analysis to determine DNA content. <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001268#s2" target="_blank">Results</a> quantitated as fold change in cell cycle are shown. (B) qRT-PCR. RNA from hESCs treated with RA for 4 d or Adr for 6 h were subjected to qRT-PCR assay using primers specific for human <i>CDKN1A</i>. (C) ChIP. p53-bound chromatin was immunoprecipitated from hESCs, and p53 enrichment on <i>CDKN1A</i> was analyzed by qRT-PCR using primers encompassing p53REs (*, <i>p</i><0.05). Scheme representing location of p53RE and primers used for ChIP-qRT-PCR are shown on the top (asterisk indicates the 3′ end of the gene). (D) hESCs treated with RA or Adr were lysed, and cell lysates were blotted for γ-H2AX. (E) Apoptosis assay. hESCs treated as in (D) were stained with Annexin V and PI, and percent apoptotic cells was determined by flow cytometry (mean ± SEM). (Also see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001268#pbio.1001268.s003" target="_blank">Figures S3</a> and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001268#pbio.1001268.s004" target="_blank">S4</a>.).</p

p53 drives differentiation of hESCs.

<p>(A) AP staining. hESCs transfected with non-target (siControl) or siRNA specific to p53 (<i>siTP53</i>) or p21 (<i>siCDKN1A</i>) were treated with RA and stained for AP (blue colonies). (B and C) hESCs transfected and treated as in (A) were used in Western blotting (B) and qRT-PCR (C) analyses. The blots in (B) were quantitated, and average density of three different blots is plotted (bottom panel) (*, <i>p</i><0.05) (mean ± SEM). (D) OCT4 + SSEA4 staining. hESCs transfected with siRNA followed by RA treatment were stained for SSEA4 and OCT4 and subjected to dual flow cytometry analysis. Triplicate samples were analyzed in each experiment, and data analyzed with FACSDiva software. Decreases in fraction of OCT4-positive cells, as compared to siControl untreated, are indicated in red. (E) AP staining. hESCs transfected with <i>HDM2</i> or <i>TRIM24</i> siRNA were treated with RA and stained for AP. (F) Quantified AP-stained colonies. Date shown are for 50 colonies per treatment in three separate experiments (in [A] and [E]), scored as undifferentiated, partially differentiated, or fully differentiated colonies, mean ± SEM. (G) OCT4 + SSEA4 staining and flow cytometry analysis as in (D) after transfection with siRNA targeting <i>HDM2</i> or <i>TRIM24</i>. (H) Cell cycle analysis. hESCs transfected with <i>HDM2</i> or <i>TRIM24</i> siRNA were stained with PI and subjected to cell cycle analysis. (Also see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001268#pbio.1001268.s003" target="_blank">Figures S3</a> and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001268#pbio.1001268.s005" target="_blank">S5</a>.).</p

DNA binding activity of p53 is required to induce differentiation of hESCs.

<p>(A) hESCs stably expressing p53WT and mutant p53 (p53R175H and p53R175P) under control of tet-inducible promoter cultured in CM + FGF were treated with 100 ng/ml Dox for 2 d. p53 and OCT4 protein levels were analyzed by blotting. (B) AP staining. hESCs in (A) treated with Dox for 2 d (2D) or 4 d (4D) and AP stained. (Arrows indicate differentiated cells.) (C) qRT-PCR. hESCs treated with Dox for 1 d (1D) or 2 d (2D). RNA analyzed by qRT-PCR assay for expression of exogenous <i>TP53, CDKN1A, OCT4</i>, and <i>AFP</i> (*, <i>p</i><0.05) (mean ± SEM). (D) Cell cycle analysis. hESCs treated with RA for 1 d or Dox for 1 d or 2 d, stained with PI, and subjected to cell cycle analysis (mean ± SEM). (E) hESCs stably expressing p53WT were transfected with siRNA and treated with Dox for 2 d. p53, p21, and OCT4 protein levels were analyzed. (F and G) hESCs expressing p53WT were treated with Dox for 2 d or 4 d, and lysed to analyze protein (F) or RNA (G) for various differentiation markers; AFP and GATA4 (endoderm), Brachyury (mesoderm), and PAX6 (ectoderm). (H) p53 acetylation. Lysates from hESCs treated with RA and Dox-inducible p53WT treated with Dox were blotted for p53K373ac and p53. (Also see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001268#pbio.1001268.s006" target="_blank">Figure S6</a>.).</p

p53 protein is induced during differentiation of hESCs.

<p>(A) qRT-PCR. RNA from hESCs treated with RA in medium without FGF for 5 d (R0–R5), were subjected to qRT-PCR assay (data are presented as mean ± SEM). (B and C) Western blot. Lysates (total cell lysate [TCL]) prepared from hESCs cultured as in (A) were analyzed by Western blotting, the blots in (B) were quantitated (C): the average density of three different blots is plotted (*, <i>p</i><0.05). (D) <i>TP53</i> qRT-PCR. RNA samples prepared as in (A) were subjected to qRT-PCR assay (mean ± SEM). (E) Immunofluorescence. hESCs in complete CM or treated with RA for 3 d were stained with antibodies against p53 and OCT4, and nuclei were stained with DAPI. Scale bar is 50 µm. (F) p53 nuclear localization. Nuclear extracts prepared from hESCs cultured as in (A) were analyzed by Western blotting. (Also see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001268#pbio.1001268.s001" target="_blank">Figure S1</a>.).</p

Acetylation of Lys373 leads to stabilization of p53.

<p>(A) p53 acetylation. Equal amounts of p53 were immunoprecipitated by adjusting the amounts of total cell lysates prepared from hESCs and probed with p53K373ac antibody. (B) Immunofluorescence. hESCs treated with RA for 3 d were stained with antibodies against p53K373ac and OCT4; nuclei were stained with DAPI. (C) Co-immunoprecipitation. Cell lysates from RA-treated hESCs were immunoprecipitated with p53 followed by Western blotting. (D) p53 acetylation. p53 immunoprecipitated from hESCs cultured under differentiating conditions and treated with either circumin on day 2 or nicotinamide on day 4 and probed with p53K373ac antibody. (E) Co-immunoprecipitation. Cell lysates from differentiating hESCs were immunoprecipitated with HDM2 or TRIM24 antibodies and analyzed by Western blotting. (F) Endogenous p53 ubiquitination. hESCs cultured under differentiating conditions were treated with MG132 + RA or MG132 + Adr; endogenous p53 was immunoprecipitated and probed for ubiquitin (top panel). Same blot was reprobed with p53 antibody to show the equal p53 pull down (bottom panel). (Also see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001268#pbio.1001268.s002" target="_blank">Figure S2</a>.) IP, mmunoprecipitation; Ub, ubiquitin; WB, Western blot.</p

Model depicting role of p53 in inducing differentiation of hESCs.

<p>In pluripotent hESCs, p53 is negatively regulated by HDM2 and TRIM24. Differentiation induces acetylation at Lys373 of p53 via CBP/p300, p53K373ac then activates transcription by binding to p53REs on <i>CDKN1A</i> (p21), <i>miR-34a</i>, and <i>miR-145</i>. Induction of p21 leads to p53-dependent elongation of G₁ phase, whereas induction of <i>miR-34a</i> supports G1 elongation, blocks deactivation of p53 by targeting the deacetylase SIRT1, and counteracts pluripotency by targeting <i>LIN28A</i>. On the other hand, <i>miR-145</i> targets OCT4, KLF4, and SOX2 and antagonize pluripotency. Thus, p53 exerts a cumulative pro-differentiation effect by elongating hESC G₁ phase via p21 and synergistically up-regulating <i>miR-34a</i> and <i>miR-145</i> to counteract pluripotency. Ub, ubiquitin.</p

Forced down-regulation of Jag1 in endothelial cells removes Notch pathway cis inhibition by HoxA3 and initiates EHT.

<p><b>A)</b> Jag1 RNA expression level in Bend3 cells infected with empty vector (Con) or Jag1 shRNA (Jag1 KD). <b>B)</b> Western blot using specific antibody against Jag1 and GAPDH in Bend3 cells infected with empty vector (pGIPZ) or Jag1 shRNA (Plko.1 Jag1). <b>C)</b> Frequency of Flk1+/VE-cadherin+ cells obtained from day 6 EBs, transduced with empty vector (CON) or with shRNA-Jag1-GFP (JKD) and co-cultured on OP9 for 5 days in Control (Con) or HoxA3 overexpression. <b>D)</b> Gene expression levels of Notch pathway components Hes1, Hey1, Hey2 from purified control (white bar) or HoxA3-overexpressing cells (black bars), transduced with empty vector (CON) or with shRNA-Jag1-GFP (JKD) and co-cultured on OP9-DLL1 for 5 days. Graphs in panel D show one representative experiment with triplicate measurements. *: p<0.05; **: p<0.01; ***: p<0.001. Statistical analysis is reported on <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186818#pone.0186818.s011" target="_blank">S6 Table</a></b>.</p

Proposed model.

<p><b>A)</b> Illustration of ligand <i>cis</i> inhibition in the hemogenic endothelium. HoxA3 dependent Jag1 overexpression in <i>cis</i> interacts with Notch receptors, inhibiting Notch ligand in <i>trans</i> to interact with the receptor. When HoxA3 is withdrawal the pathway is activated in <i>trans</i> and Runx1 can be induced in the hemogenic endothelium. <b>B)</b> Effect of HoxA3 on Notch pathway. HoxA3 upregulates Jag1 to inhibit the Notch pathway. The pathway in turn will promote downregulation of endothelial specific transcripts, and initiate the EHT.</p

Notch signaling in <i>trans</i> does not rescue HoxA3 mediated inhibition of Notch.

<p><b>A)</b> Experimental procedure <b>B)</b> Representative flow cytometric profile of endothelial surface markers Flk-1/Ve-Cadherin and hematopoietic surface markers c-Kit/CD41, and c-Kit/CD45 obtained from 200,000 EB-derived Flk1⁺/VE-cadherin⁺ cells and co-cultured on OP9 control (CON) or OP9 overexpressing Dll1 (OP9-Dll1) for 5 days in Control or HoxA3-overexpressing HE cells. <b>C)</b> Quantification of frequencies of hematopoietic surface markers (CD41, CD45) of the same cell as in <b>B</b>. <b>D)</b> Gene expression levels of the Notch pathway target genes (Hes1, Hey1, Hey2) and hematopoietic gene markers (PU.1, Runx1, Gata1) on control (white bar) or HoxA3-overexpressing (black bars) HE cells co-cultured on OP9 controls (CON) or OP9-DLL1 for 5 days (Flk1⁺/VE-cadherin⁺ and CD41⁺/c-Kit⁺ cells were pulled together). <b>E)</b> Iimmunofluorescence staining for activated Notch1 (NICD-red), VE-Cadherin (VECad-green) and Hoechst (blue) showing adherent endothelial clusters growing in Control (Con) or HoxA3 overexpression (HoxA3), derived from endothelial cells (Flk1⁺/VE-cadherin⁺) co-cultured on OP9-DLL1 cells *: p<0.05. Statistical analysis is reported on <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186818#pone.0186818.s010" target="_blank">S5 Table</a></b>.</p

Repression of EHT by HoxA3 is not affected by inhibition Notch pathway.

<p><b>A)</b> Experimental procedure, Endothelial Derived Cells (ENDO). <b>B)</b> Representative flow cytometric profile of endothelial surface markers Flk-1/Ve-Cadherin and hematopoietic surface markers c-Kit/CD41, and c-Kit/CD45 on 200,000 EB-derived Flk1⁺/VE-cadherin⁺ cells without or with HoxA3 overexpression and co-cultured on OP9 for 5 days in the presence or absence of the Notch inhibitor DAPT (20 μM). <b>C)</b> Frequency of endothelial surface markers Flk-1⁺/Ve-Cadherin⁺ in EB-derived cells. *: p<0.05; **: p<0.01. Two way ANOVA analyses of Flk-1⁺/Ve-Cadherin⁺, CD41⁺ and CD45⁺ frequencies are reported on <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186818#pone.0186818.s008" target="_blank">S3 Table</a></b>.</p