Knockdown of <i>Utx</i> in 3T3-L1 cells resulted in an enhancement of adipocyte differentiation.

<p>(A) Oil-red O staining of adipocytes. Representative results from three independent experiments are shown. mRNA levels of (B) <i>Utx</i>, (C) <i>C/EBP alpha</i>, <i>Pparg2</i>, (D) <i>Pparg2</i> normalized to <i>β-actin</i>. The experiments were performed independently three times in triplicates and the representative results are shown and expressed as mean ± SE. (n = 3) *p<0.05. (E) Schematic model of Utx during differentiation of mESCs to adipocytes.</p

<i>Utx</i>-deficient mESCs showed diminished differentiation to adipocytes.

<p>(A) Oil-red O staining of adipocytes. Representative results from three independent mESC clones are shown. (B-F), mRNA levels of <i>aP2</i>, <i>Pparg2</i>, <i>C/EBP alpha</i>, <i>C/EBP beta</i>, <i>C/EBP delta</i> (B)<i>; Nanog</i>, <i>Sox2</i> (C); <i>Pou5f1</i>, <i>Brachyury</i> (D); <i>Prdm16</i>, <i>Ucp1</i>, <i>Pgc1 alpha</i> (E); <i>Utx</i> (F) normalized to <i>β-actin</i> in mESCs and adipocytes. The experiments were performed with three independent clones, and the results are expressed as mean ± SE (n = 3); *p<0.05.</p

Construction and characterization of <i>Utx</i>-deficient mESCs.

<p>(A) Conditional gene targeting of the murine <i>Utx</i> locus. (B) Immunoblotting for Utx and β-actin in <i>Utx</i>-deficient and control mESCs. A representative result from three independent mESC clones is shown. Band densities of Utx and β-actin were quantified by ImageQuant TL ver. 8.1 and normalized to β-actin. The data are presented relative to the signal of control mESCs. (C) Immunoblotting for H3, H3K4me3, H3K9me3, H3K27me3, and H3K36me3 in mESCs. A representative result from three independent mESC clones is shown. Band density of histones was quantified by ImageQuant TL ver. 8.1 (GE healthcare life sciences, Pittsburgh, PA, USA), and data are shown by the relative value of histone methylations normalized to H3.</p

Effects of Utx deficiency on gene expression in mESCs and adipocytes.

<p>(A) Heat map visualizations of the 79 genes that are differentially expressed in <i>Utx</i>-deficient and control mESCs and adipocytes. Two clones were used for each group. Data for the heat maps are normalized using average linkage clustering on entities and represent median-centered log-transformed values. Red and blue correspond to high and low expression, respectively, compared with the experiment-wide median. (B) mRNA levels of <i>Mgp</i> and <i>Dcn</i> normalized to <i>β-actin</i>. The experiments were performed independently with three clones, and the results are expressed as mean ± SE (n = 3); *p<0.05. (C) GSEA enrichment score curves described with two sets of adipocytes from the Molecular Signatures Database (MSigDB). The graph at the bottom of each panel represents the ranked, ordered, non-redundant list of genes. Vertical black lines indicate the position of genes from the studied gene set in the ordered, non-redundant data set. The green curve corresponds to the enrichment score (ES) curve, which is the running sum of the weighted enrichment score obtained with the GSEA software. NES, normalized enrichment score: FDR, false discovery rate; NOM, nominal. (D, E) mRNA levels of <i>c-Myc</i> normalized to <i>β-actin</i> in (D) mESC and adipocytes, (E) mESCs, EBs, and RA-induced differentiation. The experiments were performed independently with three clones, and the results are expressed as mean ± SE. (n = 3) *p<0.05. (F) mRNA levels of <i>aP2</i> normalized to <i>Gapdh</i> in <i>Utx</i>-deficient or control mESCs and adipocytes in the presence of 10058-F4. The representative result is shown as mean ± SE. (n = 3) *p<0.05.</p

<i>Utx</i>-deficient mESCs showed some potential to differentiate into mesoderm.

<p>(A) Scheme of the differentiation assay of mESCs to adipocytes. (B, C) mRNA levels of <i>Brachyury</i> (B), <i>Utx</i> (C) normalized to <i>β-actin</i> in mESCs and adipocytes. The experiments were performed independently with three clones, and the results are expressed as mean ± SE (n = 3); *p<0.05.</p

Expression of proteins which were reported to be associated with sensitivity to anti-microtubule agents.

<p>(A) Protein expression was evaluated by western blot analysis. Expression values of class Ⅲ beta-tubulin (TUBB3) relative to beta-actin were determined using Just TLC software. (B) Representative images of HCC4006 and HCC4006ER cells immunohistochemically stained with antibodies to ATP-binding cassette subfamily B, member 1 (ABCB1).</p

Additional effects of entinostat in HCC4006 and HCC4006ER cells.

<p>(A-C) Tumor cells were incubated with various concentrations of docetaxel (D), paclitaxel (P) and vinorelbine (V) and with or without 1μM entinostat (E) for 72 hours. Percent growth relative to DMSO-treated controls was evaluated by Cell Counting Kit-8 assay. (D) Total cell lysates were harvested after addition of 1μM entinostat.</p

Effect of siRNA-mediated knockdown for ABCB1 in HCC4006ER cells.

<p>(A) Total cell lysates were harvested 72 hours after reverse-transfection of negative control siRNA (siNC) or validated two siRNAs for ATP-binding cassette subfamily B, member 1 (ABCB1) mixed with Lipofectamine RNAiMAX. (B-E) Tumor cells were reverse-transfected at the same time plating into 96-wells and then incubated for 24 hours. They were incubated with various concentrations of docetaxel, paclitaxel, vinorelbine and erlotinib for additional 72 hours. Percent growth relative to DMSO-treated controls was evaluated by Cell Counting Kit-8 assay.</p

Relative expression levels of ABC transporter family in HCC4006ER cells compared with HCC406.

<p>Relative expression levels of ABC transporter family in HCC4006ER cells compared with HCC406.</p

Summary of eMLST, monoclonal antibody typing and antibiotic susceptibility testing results for antibiotic-resistant <i>P. acnes</i> strains.

a<p>Antibody typing with type IA and type II monoclonal antibodies QUBPa1 and QUBPa2, respectively <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041480#pone.0041480-McDowell1" target="_blank">[12]</a>.</p>b<p>Tetracycline resistance MIC≥1.0 mg/L; erythromycin resistance MIC≥0.5 mg/L; clindamycin resistance MIC ≥0.25 mg/L.</p