Gene expression primers.

<p>Gene expression primers.</p

hESC-inducible EZH2 (hPSC- <i>i</i>EZH2) cell line and definitive endoderm formation.

<p><b>A.</b> The original master cell line flanked by heterotypic FRT sequences and resulting RMCE line (Recombinase-Mediated Cassette Exchange, hPSC-<i>i</i>EZH2) are depicted. RMCE donor vector <i>pZ</i>:<i>F3-P TetOn hEZH2</i> for inducible expression was introduced by flippase (additional details in the Supplemental Information). <b>B.</b> Expression of pluripotency markers (TRA-1-60 and OCT4) of a representative hPSC-<i>i</i>EZH2 clone and wild-type hPSCs by immunocytochemistry. Right: isotype control of TRA-1-60 and OCT4. <b>C.</b> Upper part, directed differentiation of hPCS-<i>i</i>EZH2 toward endoderm (endo_d4) with addition of 5μg/ml doxycycline (doxy) in the first 4 days of differentiation (red arrow). Bottom part, protein analysis of untreated and doxy treated EZH2 induced cells (doxy -/+) using EZH2, GATA4 and GAPDH antibodies. GAPDH was used as loading control. Signals were quantified and indicated as % to loading control. <b>D.</b> Double extracellular staining for CXCR4 (PE channel) and cKIT (APC channel) measured by FACS in untreated and doxy treated EZH2 induced cells (doxy-treated) at definitive endoderm stage on day 4. Right: the percentage of cells co-expressing CXCR4 and cKIT. Data as mean ± SEM of n ≥ 3 IEs. ** p < 0.01 by Student’s t test. <b>E.</b> Representative immunofluorescence images for SOX17 (red signal) in untreated and EZH2 induced cells (doxy-treated) at day 4 of differentiation. EZH2 induced cells showed significantly high levels of SOX17 expression. Around 58% of positive cells were counted in EZH2 doxy induced cells, right part. Nuclei are staining with DAPI (blue). Data as mean ± SEM of n ≥ 3 IEs. *** p < 0.001 by Student’s t test. <b>F.</b> mRNA expression profile of the endoderm marker <i>SOX17</i> in untreated (-) and EZH2 doxy induced cells (+) at endo_d4. <b>G.</b> mRNA expression profile of untreated (-) and EZH2 doxy induced cells (+) at endo_d4 and HP_d8 of endoderm markers <i>FOXA2</i>, <i>EOMES</i>, <i>MIXL1</i>, <i>GOOSECOID</i> and <i>CXCR4</i>. Relative gene expression to GAPDH. Data as mean ± SEM of n ≥ 3 IEs.</p

EZH2 regulation and fetal hepatocytes differentiation.

<p><b>A.</b> Upper part, directed differentiation of hPCS-<i>i</i>EZH2 toward fetal hepatocytes (FH_d16) with addition of 5μg/ml doxycycline (doxy) in the first 8 days of differentiation (red arrow). Bottom part, relative gene expression of the <i>EZH2</i> transgene (Exogenous <i>EZH2</i>) and both endogenous and exogenous EZH2 (Total <i>EZH2</i>) at endo_d4, HP_d8 and FH_d16 in untreated (-) and EZH2 doxy induced cells (+). Relative gene expression to GAPDH and same scale bar. Data as mean ± SEM of n ≥ 3 IEs. <b>B.</b> Western blot for EZH2 and GAPDH in untreated (-) and EZH2 doxy induced cells (+) at hPSC, endo_d4, HP_d8 and FH_d16. Positive control: HEK293T cells transiently transfected with pLVX-IRES-Hygro-hEZH2. The molecular weight size marker was cropped from the gel. Signals were quantified and indicated as % to loading control. <b>C.</b> Relative expression (to <i>GAPDH</i>) of hepatic markers <i>HNF1A</i>, <i>HNF3B</i>, <i>HNF6</i>, <i>GSTp</i> and transcription factors <i>CAR</i> and <i>CEBPA</i> in HP_d8 and FH_d16 untreated (-) and EZH2 doxy induced cells (+). Data as mean ± SEM of n ≥ 3 IEs. * p < 0.05 and ** p < 0.01. <b>D.</b> Relative gene expression (to <i>GAPDH</i>) of hepatic marker genes <i>AFP</i>, <i>ALBUMIN and AAT</i> in HP_d8 and FH_d16 untreated (-) and EZH2 doxy induced cells (+). Data as mean ± SEM of n ≥ 3 IEs. * p < 0.05, ** p < 0.01. <b>E.</b> Left part, relative gene expression (to <i>GAPDH</i>) of hepatic transcription factor HNF4A in HP_d8 and FH_d16 untreated (-) and EZH2 doxy induced cells (+). Right part, representative immunofluorescence images for HNF4A (red signal) on day 8 for cells treated without (untreated) or with doxy (doxy-treated). Nuclei are staining with DAPI (blue). On the right, more than 80% of HNF4A positive cells on HP_d8 EZH2 doxy induced cells were counted. Data as mean ± SEM of n ≥ 3 IEs. * p < 0.05. <b>F.</b> Immunofluorescence staining for ALBUMIN (red signal) at HP (day8) and FH (d16) in untreated (-) and EZH2 induced cells (+) (left part). Nuclei are staining with DAPI (blue). Data as representative images of n = 2 IES. <b>G.</b> ELISA for ALBUMIN secretion on FH_d16 of the hepatocyte differentiation protocol. EZH2 doxy induced cells (+) secrete significant amounts of albumin compared to untreated (-) cells. Data as mean ± SEM of n ≥ 3 IEs. ** p < 0.01. <b>H.</b> Immunofluorescence staining for AAT (red signal) at HP (day 8) and FH (day 16) showed abundant expression of the hepatocyte protein in doxy-treated cells compared to the untreated (left part). Nuclei are stained with DAPI (blue). Data as representative images of n = 2 IES. <b>I.</b> Intracellular flow cytometry analysis for AAT demonstrated that more then around 60% of EZH2 doxy induced cells (+) progeny were positive for AAT. Results represent the mean of three independent experiments ± SEM. ** p < 0.01.</p

miRNA and EZH2 mRNA degradation.

<p><b>A.</b> Relative gene expression (to <i>GAPDH</i>) of endogenous and exogenous <i>EZH2</i> (Total <i>EZH2</i>) daily during the transition from endoderm stage (day 4, endo_d4) to hepatoblast progenitor (day 8, HP_d8) in untreated (-) and EZH2 doxy induced cells (+). Data as mean ± SEM of n = 3 IEs. <b>B.</b> Relative expression of miR-101, miR-138, miR-214 and miR-124 during hepatocytes differentiation from hPSC-<i>i</i>EZH2 cell line doxy induced the first 8 days of differentiation. Relative gene expression to U6. Data as mean ± SEM of n = 3 IEs. <b>C.</b> Name, functions and reference papers of the miRNAs analyzed.</p

EZH2 expression during hepatocyte differentiation.

<p><b>A.</b> Upper part, differentiation protocol of hPSCs into hepatocytes-like cells. hESC were harvested and plated at ±7 x 10⁴ cell/cm² and differentiated with a combination of growth factors: 50ng/ml of Activin-A and Wnt3a for the first 2 days and 50ng/ml Activin-A until day 4, followed by BMP4 50 ng/ml from day 4 until day 8, FGF1 50 ng/ml from day 8 until day 12, and HGF 20 ng/ml from day 12 until day 16. Differentiated progeny of the hESC line were harvested on day 4 (endo_d4, definitive endoderm cells), on day 8 (HP_d8, hepatoblast progenitor) and on day 16 (FH_d16, fetal hepatocyte). Bottom part, transcript expression levels of <i>EZH2</i> and <i>RING1B</i> in hPSCs, endo, HP and FH stages. <i>GAPDH</i> was used as a control. Data as mean ± SEM of n ≥ 3 IEs. *** p < 0.001 by Student’s t test. <b>B.</b> Western blot analysis of EZH2, RING1B and GAPDH on hPSCs, endo, HP and FH stages. GAPDH was used as loading control. HEK293T transiently transfected with pLVX-IRES-Hygro-hEZH2 cells were used as positive control. Signals were quantified and indicated as % to loading control. <b>C.</b> Schematic representation of the <i>EZH2</i> regulatory region. Approximately 270bp (arrow in front of the TTS) in a CpG island were bisulfite sequenced in undifferentiated hPSC, HP and FH. Percentages of the results of bisulfite sequencing of the regions are at right side of the figure for each stage.</p

miRNAs expression primers.

<p>miRNAs expression primers.</p

Total H3 levels and H3K27me3 levels on endodermal and hepatocyte genes.

<p><b>A.</b> Left, representative immunofluorescence image for H3K27me3 (green signal) of three different differentiations (n = 1, 2 and 3) at endo_d4, HP_d8 and FH_d16 stages in untreated and doxy-treated cells. Right, the fluorescence of H3K27me3 signal was digitally quantified (additional details in the Supplemental Information). Results represent the mean of three independent experiments ± SEM and displayed as arbitrary units (A.U.). ** p < 0.01. <b>B.</b> Histone modification levels of H3K27me3 at SOX17, CXCR4 and FOXA2 (left part) and GAPDH, MYOD1 and HOXD11 promoter (right part) at endo_d4 in untreated and doxy-treated cells. Results represent the mean of three independent experiments ± SEM. * p < 0.05. <b>C.</b> Histone modification levels of H3K27me3 at ALBUMIN and AAT (left part) and GAPDH, MYOD1 and HOXD11 promoter (right part) at FH_d16 in untreated and doxy-treated cells. Results represent the mean of three independent experiments ± SEM. * p < 0.05, ** p < 0.01.</p

Functional links between MK3 and PRC in proliferative life span.

<p>TIG3 cells were sequentially transduced with either <i>Bmi1</i>.<i>ires</i>.<i>GFP</i> (<i>Bmi1OE</i>) or <i>GFP</i> (con) virus, and <i>MK3</i>/<i>puromycin</i> (<i>MK3OE</i>) or control <i>puromycin</i> virus (con) at 48 hrs intervals. Retroviral vectors expressing murine <i>Bmi1/</i>GFP reporter were transduced first (or empty vector control), followed by a MK3/<i>puromycin</i> resistance marker (or empty vector control). Transduction of <i>Bmi1OE</i> and control transduced cells was simultaneously carried out with the same <i>MK3</i>^<i>WT</i><i>OE</i> viral preparation (or control virus) to minimize inter-experimental variation. Cells were grown on selection medium and proliferation capacity was tested ± 2–3 weeks post-transduction. (A) Proliferation curves of normal human TIG3 fibroblasts transduced with a retroviral <i>MK3</i>^<i>WT</i><i>overexpression</i> vector (<i>MK3</i>^<i>WT</i><i>OE</i>; black symbols) or <i>shcon</i> vector (white symbols), in conjunction with either an empty vector control (con; circles) or a murine <i>Bmi1</i> expression vector (<i>Bmi1OE</i>; triangles). (B) Proliferation curves of normal human TIG3 fibroblasts transduced with a retroviral <i>MK3</i> knock-down vector (<i>shMK3</i>; black symbols) or <i>shcon</i> vector (white symbols), in conjunction with either an empty vector control (con; circles) or a murine <i>Bmi1</i> expression vector (<i>Bmi1OE</i>; squares). Cell counts at t = 2 through t = 8 (A, B) were normalized to cell counts at t = 0 for each transduced cell culture individually (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118840#sec013" target="_blank">Methods</a> section for details); statistical significance was determined by two-tailed Student’s t-test and is presented relative to the empty vector control (* p < 0.05). (C) Comparative morphology of TIG3 cells expression <i>Bmi1</i> and/or <i>MK3</i> versus control cells as recorded by GFP fluorescent imaging ± 3 weeks after transduction (D) Immunoblot analysis of EZH2, CBX4, RNF2 and H3K27me3 in <i>MK3</i>^<i>WT</i><i>OE</i>, <i>Bmi1OE</i>, <i>Bmi1OE/MK3</i>^<i>WT</i><i>OE</i> and <i>control</i> TIG3 cell lysates. (E) Expression analysis of BMI1, MK3, and TP53 at the indicated time points in (corresponding to experiment in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118840#pone.0118840.g004" target="_blank">Fig 4C</a>). Cells were grown on selection medium and analysed at 1 or 4 weeks after serial transduction; expression vectors and antibodies are indicated in the figure. Early and late samples were loaded on the same gel for BMI1 analysis; corresponding sections are shown separately.</p

Proliferative regulation by MK3 in cancer cell lines.

<p>(A) Proliferation curves (left) of U-2OS cells expressing a retroviral <i>MK3</i> vector (<i>MK3</i>^<i>WT</i><i>OE</i>; filled circles) or an empty vector (con; open circles); overexpression of GST-MK3 (<i>MK3</i>^<i>WT</i><i>OE</i>) in U-2OS cells detected with and GST or a MK3-antiserum (right panel). Cell counts at t = 2 through t = 8 were normalized to cell counts at t = 0 for each transduced cell culture individually (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118840#sec013" target="_blank">Methods</a> section for details); statistical significance was determined by two-tailed Student’s t-test and is presented relative to the empty vector control (* p < 0.05). (B) Phase contrast images showing cell morphology in U-2OS/<i>MK3</i>^<i>WT</i><i>OE</i> cells and control cells. (C) Protein expression levels of the check-point regulator proteins TP53 and p21^CIP1/WAF1 (P21) in U-2OS/<i>MK3</i>^<i>WT</i><i>OE</i> cells; loading control b-Actin (bAct). (D) DNA profile analysis of U-2OS/<i>MK3OE</i> versus control cells (4–6 days post-transduction; representative experiment). <i>MK3</i>^<i>WT</i>overexpression elicits an intra S-phase arrest: table shows a substantially increased S-phase occupancy. (E) Immunohistochemical staining for phosphorylated H2A.X (γH2A.X) and phosphorylated KAP1pSer824 (pKAP1; arrows) to visualize DNA damage in U-2OS/<i>MK3</i>^<i>WT</i><i>OE</i> cultures; control (top panels) or <i>MK3</i>^<i>WT</i><i>OE</i> (bottom panels).</p

<i>MK3</i>^<i>WT</i> overexpression induces a proliferative arrest in normal human fibroblasts.

<p>(A) Retroviral expression systems were applied to enhance (<i>MK3</i>^<i>WT</i><i>OE</i>) or remove (<i>shMK3</i>) MK3 expression. Western blot shows MK3 proteins: endogenous (MK3^endo) and overexpressed (GST:MK3). (B) Proliferation curves of TIG3 cells transduced with: a retroviral <i>MK3</i> expression vector (<i>MK3</i>^<i>WT</i><i>OE</i>; filled circles), an empty vector (con; open circles) or a murine <i>Bmi1</i> expression vector (open triangles); proliferation was determined at 1 week (top panel) or ±4 weeks (bottom panel) after retroviral transduction of TIG3 cells. Cell counts at t = 2 through t = 8 were normalized to cell counts at t = 0 for each transduced cell culture individually (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118840#sec013" target="_blank">Methods</a> section for details); statistical significance was determined by two-tailed Student’s t-test and is presented relative to the empty vector control (* p < 0.05). (C) Quantification of DNA profiles (BrdU pulse-labeling and S-phase quantification by FACS) in TIG3/<i>MK3</i>^<i>WT</i><i>OE</i> cells at approximately 1 week post-transduction. <i>RasV12</i>-transduced cells were used as a positive control. (D) <i>MK3</i>^<i>WT</i><i>OE</i> reduces <i>de novo</i> DNA synthesis in TIG3/<i>MK3</i>^<i>WT</i><i>OE</i> cells; cell counts: control (con) 699 BrdU-positive cells/10 fields; <i>MK3</i>^<i>WT</i><i>OE</i>: 442 BrdU-positive cells/9 fields.</p