ICI182,780 dose response and tumor cell survival.

<p><b>Panel A:</b> Evaluation of cell viability (ViaCount) of exponentially growing Daoy cells (FBS) treated with cisplatain (Cis; 1 µg/ml for 48 hrs) in the presence or absence of ERβ antagonist, ICI182,780 at indicated concentrations. In one instance the cells were preincubated for 48 hrs with siRNA against ERβ mRNA (siRNA ERβ; 200 nM). <b>Inset:</b> Western blot showing effectiveness of ERβ siRNA (200 nM for 48 hrs) tested in exponentially growing Daoy cells. Data represent average values from 3 experiments in triplicate (n = 9) with standard deviation. *indicate values significantly different from Cis (paired student t-test P≤0.05). <b>Panel B:</b> Evaluation of cell viability (ViaCount) in three medulloblastoma (Daoy, D283Med and D384Med) and two breast cancer (MCF7 and BT-20) cell lines. The cells were cultured in 10%FBS (FBS); 10%FBS+ICI182,780 (10 µM) (ICI); 10%FBS+Cisplatin (1 µg/ml) (Cis); and 10%FBS+ICI182,780 (10 µM) + Cisplatin (1 µg/ml) (Cis+ICI) for 48 hrs. Data represent average values from 2 experiments in triplicate (n = 6) with standard deviation. *indicate values significantly different from Cis (paired student t-test P≤0.05). <b>Panel C:</b> Evaluation of cell viability (ViaCount) in exponentially growing Daoy cells (10%FBS) treated with different doses of ICI182,780 ranging from 10 nM to 100 µM.</p

Inhibition of ERβ improves homologous replication directed DNA repair (HRR) and increases clonogenic growth of Daoy cells treated with cisplatin.

<p><b>Panel A:</b> HRR was evaluated by the assay based on the reconstruction of the wild type green fluorescent protein (GFP) from two non-functional heteroallelic fragments of GFP cDNA delivered into cells by the pDRGFP expression vector <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033867#pone.0033867-Pierce1" target="_blank">[37]</a>. HRR was evaluated in Daoy/DRGFP cells following transient transfection with the expression vector coding for I-<i>Sce-I</i> (rare cutting endonuclease), to inflict DNA double strand break in GFP cDNA, and with mito-red containing expression vector (control for the efficiency of transfection). The results were collected from three separate experiments in duplicate (n = 6) in which about 10,000 transfected cells <i>per</i> experiment were counted in at least ten randomly selected microscopic fields. * indicates value statistically different from the sample labeled Cis (paired student t-test; P≤0.05). The histogram labeled “DRGFP control” illustrates baseline HRR in exponentially growing Daoy cells in 10%FBS and in 10%FBS+10 µM ICI182,780. <b>Panel B:</b> Clonogenic assay. The monolayer cultures of Daoy cells were exposed to cisplatin (0.25 µg/ml) in the presence and in the absence of 10 µM ICI182,780 for 24 hours. Next, the medium containing cisplatin was replaced with the fresh medium and the cells were plated at the clonal-density (ranging from 1×10³ to 1×10⁴ cells per 35 mm dish) in the presence of 10%FBS. Clonogenic growth was evaluated after 14 days of a continuous cell growth as described in our previous work <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033867#pone.0033867-Urbanska2" target="_blank">[50]</a>. In control conditions (Panel C), the cisplatin treatment was omitted. The data represent average number of clones with standard deviation calculated from three independent experiments in duplicate (n = 6) *indicates values statistically different (paired student t-test; P≤0.05).</p

Inhibition of ERβ activates recruitment of Rad51 during S phase DNA repair.

<p><b>Panel A:</b> Fluorescent images of Daoy cells immunolabeled with anti-histone γH2AX (upper panel) and anti-IRS-1 (lower panel) antibodies. The nuclei are visualized by DAPI staining (blue fluorescence). The histograms represent quantification of the co-localization between γH2AX and DAPI; IRS-1 and DAPI. The data represent average percentage of nuclear voxels (3-D pixels) of γH2AX (red fluorescence) and IRS-1 (green fluorescence) calculated from three independent experiments (n = 3) in which ten randomly selected cells have been evaluated by the Mask analysis included in SlideBook 5 deconvolution software. * indicates value statistically different from the sample labeled Cis (paired student t-test; P≤0.05). <b>Panel B:</b> Fluorescent images of the cells labeled with anti-Rad51 (green fluorescence) and with anti-BrdU (red fluorescence) antibodies. Exponentially growing cultures of Daoy cells (10%FBS) were exposed for one hour to bromodeoxyuridine (BrdU) during the 6 hours treatment with cisplatin (1 µg/ml) in the absence (Cis) or in the presence of 10 µM ICI182,780 (Cis+ICI). The histogram represents quantification of Rad51 positive cells in which Rad51 nuclear foci co-localize with BrdU-labeled DNA. Note, almost 40% increase in the number of cells utilizing Rad51 to repair cisplatin-induced DNA damage (during DNA replication) when the cisplatin treatment is accompanied by ICI182,780. * indicates value statistically different from the sample labeled Cis (paired student t-test; P≤0.05).</p

Inhibition of ERβ modulates cisplatin-induced phosphorylation of cell cycle checkpoint proteins.

<p><b>Panel A:</b> Western blot analyses showing levels of the phosphorylated ATM, ATR, Chk1 and Chk2 in constitutively growing Daoy cells (10%FBS) treated with cisplatin (1 µg/ml) in the presence (Cis+ICI) or absence (Cis) of ICI182,780 (10 µM). The cells without treatment (FBS), or cells treated with ICI182,780 only (ICI) were used as controls. <b>Panel B:</b> Densitometry of Western blots depicted in Panel A evaluated by EZQuant-Gel 2.17 software. Levels of pATM, pATR, pChk1 and pChk2, were normalized with the corresponding levels of Grb-2. Data represent averages obtained from densitometric measurements of 3 blots with standard deviation and each band was normalized with corresponding loading control, Grb-2.</p

Inhibition of ERβ improves cell survival in the presence of cisplatin.

<p><b>Panel A:</b> Fluorescent images showing nuclear morphology following labeling of DNA by fluorescent dye 4′,6-diamidino-2-phenylindole (DAPI). Exponentially growing monolayer cultures of Daoy cells (10%FBS) were treated with cisplatin (1 µg/ml) or with cisplatin + ICI182,780 (10 µM) for 48 hours. The images were taken with Nikon Eclipse 400 upright fluorescent microscope equipped with the motorized Z-axis, EXI-Aqua camera and deconvolution software (SlideBook5). Rectangles indicate magnified area containing cells in mitosis (asterix); and cells with damaged nuclei (arrowhead). Note that abundant presence of micronuclei (arrow) and nuclear fragmentation in cisplatin, and much less of the nuclear damage in cells treated by cisplatin+ICI182,780. Original magnification ×20. <b>Panel B:</b> Daoy cell viability evaluated by ViaCount and TUNEL assays. Both assays were adopted for the use with the GUAVA easyCyte 8HT flowcytometer (Millipore). The Guava/Express Plus and Guava/ViaCount software were used for data analysis and quantification according to the manufacturer recommendations (Millipore).</p

Induced pluripotent stem cells derived from human amnion in chemically defined conditions

<p>Fetal stem cells are a unique type of adult stem cells that have been suggested to be broadly multipotent with some features of pluripotency. Their clinical potential has been documented but their upgrade to full pluripotency could open up a wide range of cell-based therapies particularly suited for pediatric tissue engineering, longitudinal studies or disease modeling. Here we describe episomal reprogramming of mesenchymal stem cells from the human amnion to pluripotency (AM-iPSC) in chemically defined conditions. The AM-iPSC expressed markers of embryonic stem cells, readily formed teratomas with tissues of all three germ layers present and had a normal karyotype after around 40 passages in culture. We employed novel computational methods to determine the degree of pluripotency from microarray and RNA sequencing data in these novel lines alongside an iPSC and ESC control and found that all lines were deemed pluripotent, however, with variable scores. Differential expression analysis then identified several groups of genes that potentially regulate this variability in lines within the boundaries of pluripotency, including metallothionein proteins. By further studying this variability, characteristics relevant to cell-based therapies, like differentiation propensity, could be uncovered and predicted in the pluripotent stage.</p

Quantitative Reverse Transcriptase Primers.

<p>Quantitative Reverse Transcriptase Primers.</p

Activation of the IL-2 receptor is associated with induction of JAK/STAT protein expression.

<p>Expression of JAK 3, pJAK 3, STAT 5a, and pSTAT 5 in non-stimulated podocytes were compared to podocytes stimulated with IL-2. (A)There is increased expression of JAK 3 after both 30 and 60 minutes of IL-2 stimulation (P<0.05, One-way ANOVA with Tukey’s multiple comparisons test). N = 3 in individual groups. (B) Phosphorylated JAK 3 is significantly increased after 30 minutes of stimulation with IL-2 (*, P<0.01); however, after 60 minutes the expression has returned to within unstimulated levels (**, P<0.01; One-way ANOVA with Tukey’s multiple comparisons test). N = 3 in individual groups. (C) Cytoplasmic STAT 5a protein expression is significantly increased in podocytes after 60 minutes of IL-2 stimulation (P<0.0001, One-way ANOVA with Tukey’s multiple comparisons test) compared to unstimulated podocytes. N = 3 in individual groups. (D) Nuclear STAT 5a protein expression in podocytes was significantly decreased at both time points with IL-2 stimulation (*, P<0.0001) compared to unstimulated podocytes. However, podocytes stimulated for 60 minutes showed a significantly higher STAT 5a protein expression when compared to 30 min (**, P<0.0001; One-way ANOVA with Tukey’s multiple comparisons test). N = 3 in individual groups. (E) Cytoplasmic phosphorylated STAT 5 protein expression was significantly decreased following 30 minutes of stimulation with IL-2, but this effect was not sustained after 60 minutes of stimulation (P<0.05, One-way ANOVA with Tukey’s multiple comparisons test). N = 3 in individual groups. (F) Similar to phosphorylated JAK3, phosphorylated nuclear STAT 5 protein expression was significantly increased after 30 minutes of IL-2 stimulation, but was significantly decreased after 60min of IL-2 stimulation compared to both unstimulated podocytes and podocytes stimulated with IL-2 for 30 minutes (Fig 4F). (P<0.001, One-way ANOVA with Tukey’s multiple comparisons test). N = 3 for individual groups. Bar columns and error bars represent mean and standard deviation respectively.</p

Antibody list for western blot.

<p>Antibody list for western blot.</p

Stimulation with IL-2 increases mRNA expression of STAT 5 subunits.

<p>Expression of STAT 5a-b subunits in non-stimulated podocytes were compared to podocytes stimulated with IL-2. The mRNA expression of STAT 5a-b was significantly increased after stimulation with IL-2 (P< 0.01, P<0.05 respectively; One-way ANOVA). N = 3 in individual groups. Bar columns and error bars represent mean and standard deviation respectively.</p