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

Jak inhibitor and siRNA targeted to Stat2 prevent IFN-λ antiviral mechanisms against HCV in FFA treated cell culture.

<p>(<b>A and B</b>). FFA treated HCV culture is treated with Jak1 inhibitor for seven days. Antiviral activity of IFN-αand IFN-λwas determined each day by Renilla Luciferase. (<b>C</b>). FFA treated HCV cell culture was treated with siRNA targeting Stat1, Stat2 and Stat3 and after 24 hours culture was treated with IFN-λ. After 72 hours antiviral effect of was determined by Luciferase assay. Silencing Stat2 only prevent IFN-λantiviral activity against HCV in FFA treated culture.</p

Quantitative Reverse Transcriptase Primers.

<p>Quantitative Reverse Transcriptase Primers.</p

Antibody list for western blot.

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

FFA treatment for 48 hours induced decrease of IFNAR1 in Huh-7.5 cells is mediated by a lysosomal degradation pathway.

<p>Huh-7.5 cells were treated with FFA for 24 hours in the presence or absence of lysosome inhibitors such as: NH4Cl (30mM) or BafilomycinA1. The expression levels IFNAR1, IL10Rβ and β-actin were examined using the cell lysates by Western blotting.</p

FFA treatment for 48 hours induced autophagy response in Huh-7.5 cells.

<p>Huh-7.5 cells were treated with increasing concentrations of FFA and after 72 hours and examined for autophagy induction by immunostaining and Western blot analysis. (<b>A)</b> Immunostaining showing expression of p62 in FFA treated Huh-7.5 cells. (B). Western blot show that p62 levels are decreased in FFA treated cells. ATG5 level are induced in FFA treated Huh-7.5 cells. LAMP2A and HSC70 levels and b-actin levels were not altered significantly as compared to untreated Huh-7.5 cells. (C). The expression of p62 was decreased by both HCV and FFA treated culture. The expression of ATG5 and LAMP2A was induced by HCV infected and FFA treated Huh-7.5 cells.</p