Increased TCTP secretion by over-expression of proton pump is inhibited by pantoprazole in HEK293.

<p>(A) Comparison of protein expressions in control and Hαβ samples. Control sample was transfected with two empty vectors (pEGFP-N1 and pcDNAI-neo) and TCTP-3Xflag construct. Hαβ sample was transfected with rat H⁺/K⁺-ATPase α1-GFP, HA-rat Na⁺/K⁺-ATPase β1, and TCTP-3Xflag constructs. WB: anti-GFP Ab (purified rabbit polyclonal antibody, InVitrogen), anti-HA Ab (mouse 12CA5 monoclonal antibody, Santa Cruz), and anti-flag Ab, (B) TCTP secretion is increased by over-expression of H⁺/K⁺-ATPase α1 and Na⁺/K⁺-ATPase β1. Both groups of cells were incubated for 3 h in conditioned media. WB: anti-flag Ab, (C) The secretion assay data for the cells transfected with H⁺/K⁺-ATPase α1, Na⁺/K⁺-ATPase β1, and TCTP. Pantoprazole (1 mM) or omeprazole (1 mM) was treated during 3 h secretion assay. ‘O’ in the graph means omeprazole and ‘P’ does pantoprazole. WB: anti-flag Ab. Data of panel B and panel C represent means±S.D. from three independent experiments. #: inhibition, p<0.05, vs. non-treated cells, *: increase, p<0.05, vs. the cells transfected with two empty vectors (pEGFP-N1 and pcDNAI-neo) and TCTP-3Xflag construct.</p

Restoration of BMD in TH mice by testosterone and alendronate.

<p>B6 and TH mice (n = 6 each group) were orchiectomized at 8 weeks of age and subsequently injected with vehicle (sesame oil) or testosterone (50 mg/kg) for 6 weeks. (A) Micro-CT images of trabecular bone were reconstructed, and one representative image is presented in each group. (B) BMD was calculated using a micro-CT scanner and micoView. All results are expressed as the mean ± SEM for 6 mice. (C) Serum testosterone levels of 8 week-old WT and TH mice (n =  5) were determined using a high-sensitivity ELISA kit. The testosterone/estradiol ratio was determined after determination of serum estradiol level. (D) Relative expression level of aromatase was quantitated in testis by real time PCR and normalization to the level of β-actin (n = 4). (E) TH mice at 4 weeks of age (n = 7) were orally administered alendronate (ALD, 5 mg/kg/day) for 4 weeks. BMD and BMC were determined in B6 and TH mice at 8 weeks of age using a micro-CT scanner.</p

Altered CD4+ T cell functions in TH mice.

<p>All B6 and TH mice were analyzed at 8 weeks of age (n = 8). (A) Bone marrow cells were stained with fluorescence-conjugated anti-Ly6C Ab (BD Pharmingen) and analyzed using FACS Calibur and CellQuest program (BD Biosciences). (B) Single cell suspensions of thymus and lymph node were harvested and stained with Abs against CD4 and CD8, followed by flow cytometry analysis. (C) CD4+ T cells were isolated from the lymph node and stimulated with anti-CD3 and anti-CD28 for 24 h. Cells were then collected for real time-PCR to determine the relative expression levels of IL-4 and IFN-γ. (D-F) CD4+ T cells were stimulated for 48 h. IFN-γ production was analyzed in the cell supernatant by ELISA (D) and intracellular cytokine staining (E). (F) T cells were stained with anti-IFN-γ and anti-IL-4 Abs (BD PHarmingen), followed by flow cytometry. (G) T cells were stimulated and subsequently treated with TGF-β and IL-6 for 48 h. IL-17 was measured in the cell supernatant by ELISA. (H) T cells were stimulated with anti-CD3/28 for 48 h, followed by incubation with anti-RANKL Ab and flow cytometry analysis.</p

Increased serum levels of leptin and inflammatory cytokines in TH mice.

<p>Whole blood was collected from mice (males, n = 6) at 8 weeks and used to measure the serum levels of leptin (A), OCN (B), OPG (C), IL-6 (D), IFN-γ (E), and TGF-β (F). All data are expressed as the mean ± SEM. P value was calculated by student t-test.</p

Knockdown of Sox10 leads to inhibition of migration of B16F10 melanoma cells.

<p>B16F10 murine melanoma cells were transfected with control siRNAs, MT1-Sox10 (A, B; MT, mutant) and MT2-Sox10 (E, F) or siRNAs specific for Sox10, WT1-Sox10 (C, D; WT, wild type) and WT2-Sox10 (G, H). Nuclei were stained with DAPI (A, C, E, G) and anti-Sox10 antibody (B, D, F, H). Nucleotide sequences of MT1-Sox10 and MT2-Sox10 differ from those of WT1-Sox10 and WT2-Sox10 by 5 nucleotides respectively (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031477#pone.0031477.s007" target="_blank">Table S1</a>). Sox10 was down-regulated only with WT siRNAs (D, H) but not with MT siRNAs (B, F). B16F10 cells treated with siRNAs were put to transwell migration assay (I–M). Transfection of WT1-Sox10 (K) and WT2-siRNA (L) led to significant reduction in migration of the cells compared to transfection of MT1-Sox10 (I) or MT2-Sox10 (J). (M) Quantitation of transwell migration assay. The effect of Sox10 knockdown on the number of cells that migrated through the filter pores is shown in percentile relative to the matching control case. Values represent the average of 5 independent trials, and error bars represent standard deviations. The asterisk (*) represents a significant difference with the <i>p</i> value of <0.05.</p

Confirmation of the role of Mc1r on cell migration using an in vivo metastasis model.

<p>(A, B) Effect of Mc1r knockdown on the development of pulmonary metastatic colony was determined. B16F10 melanoma cells were treated with MT1-Mc1r (A) or WT1-Mc1r (B) and injected into tail vein of C57BL/6 mice. Representative lungs harvested after 18 days are shown. (C) B16F10 colonies visible on the lung surface were counted and plotted. N = 9 for MT1-Mc1r and N = 8 for WT1-Mc1r. The significance of difference (p<0.0001) was determined by <i>t</i>-test.</p

Mc1r promotes migration of melanoma cells.

<p>(A) Quantitative Real time RTPCR assays were carried out using B16F10 murine melanoma cells transfected with the universal control siRNA, MT1-Mc1r, or one of the three siRNAs specific for Mc1r, WT1-Mc1r, WT2-Mc1r, and WT3-Mc1r. The nucleotide sequence of MT1-Mc1r differs from that of WT1-Mc1r by 5 nucleotides. The expression levels of Mc1r, Ald1a, and Ctbp1 were examined. The effect of Mc1r knockdown is expressed relative to that of the universal control siRNA after normalization with GAPDH expression level. Values represent the average of three independent real-time PCR experiments each carried out in duplicates, and error bars represent standard deviations. (B–G) B16F10 cells were treated with the universal control siRNA (B), MT1-Mc1r (C), WT1-Mc1r (D), WT2-Mc1r (E), or WT3-Mc1r (F) and put to transwell migration assay. (G) Quantitation of transwell migration assay. The effect of Mc1r knockdown on the number of cells that migrated through the filter pores is shown in percentile relative to the universal control case. Values represent the average of 5 independent trials, and error bars represent standard deviations. The asterisk (*) represents a significant difference with the <i>p</i> value of <0.05.</p

Confirmation of microarray expression profiling.

<p>(A) Quantitative real time RTPCR assays were carried out using B16F10 cells transfected with the MT1-Sox10 or WT1-Sox10. A subset of genes that showed down-regulation by WT1-Sox10 in the microarray assay by 2.5 fold or higher in all triplicates (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031477#pone-0031477-t001" target="_blank">Table 1</a>) and two non-target genes whose expression levels were unchanged (Ald1a and Ctbp1) were used to validate the results from the microarray assay. The effect of Sox10 knockdown by the specific siRNA on the expression level of each target gene is expressed relative to that of the control siRNA after normalization with GAPDH expression level. Values represent the average of three independent real-time PCR experiments each carried out in duplicates, and error bars represent standard deviations. (B) Real time RTPCR carried out with MT2-Sox10 and WT2-Sox10. The asterisk (*) represents a significant difference with the <i>p</i> value of <0.05.</p