Analysis of gene expression using exon expression array and qRT-PCR.

<p>A) Gene expression profiling using exon expression arrays. RNA samples of mock-transfected HeLa cells respectively HeLa cells expressing the WT SLCO5A1 both treated with 1 µg/ml tetracycline for 24 h were collected and analyzed on Affymetrix Exon Arrays. Results of the WT SLCO5A1 sample were compared to the mock sample and expression values of genes with a fold-change of at least 2.0 were analyzed using the GeneSpring® GX 12.0 software. Selected genes were clustered according to their biological function using the GeneSpring Gene Ontology (GO) analysis tool (for complete results see supplemental information – <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083257#pone.0083257.s003" target="_blank">Table S1</a>). A fold-change expression of 30.2-fold was observed for SLCO5A1 (control) (not shown). B) Analysis of GeneChip Human Exon 1.0 ST microarray data by quantitative <i>real-time</i> PCR. The expression of the indicated genes was analyzed after application of mock-transfected HeLa cells and HeLa cells expressing the WT SLCO5A1 with tetracycline for 24 h. The relative expression levels of the WT SLCO5A1 sample were compared to the mock sample ( = 1) and normalized to GUSB (glucuronidase, beta) expression. Mean values with standard deviation of 3 biological replicates are displayed.</p

Inducible expression of hSLCO5A1 in HeLa cells.

<p>A) RNA and protein expression of the YFP-tagged WT SLCO5A1 and its L³³F mutant. mRNA expression was measured after 24 h and 48 h treatment with 1 µg/ml tetracycline. As a basal mRNA expression control cells were left untreated. SLCO5A1 mRNA, measured by TaqMan qRT-PCR, was normalized to GAPDH mRNA expression. The relative RNA levels are presented as fold-change compared to SLCO5A1 expression in untransfected HeLa cells ( = 1). SLCO5A1 protein expression after treatment with 1 µg/ml tetracycline for 48 h was determined by western blot analysis. Tubulin served as loading control. B) Western blot analysis of the deglycosylated HA-tagged WT and mutant (L³³F) SLCO5A1 protein. Protein expression was induced with tetracycline for 48 h. The proteins were deglycosylated with either endoglycosidase H (EndoH) (E) or PNGase F (P). Tubulin served as loading control. C) Protein expression of the YFP-tagged WT SLCO5A1 or its L³³F mutant after induction with 1 µg/ml tetracycline for 24 h was analyzed by confocal fluorescence microscopy (blue: DAPI; yellow: YFP). The diagrams represent YFP fluorescence intensities along the length of the red arrows (x-axis: distance [μm]; y-axis: relative signal intensity).</p

Proliferation assay of hSLCO5A1-expressing HeLa cells.

<p>The proliferation rate of stably transfected HeLa cells was measured by counting cells incubated in the presence or absence of tetracycline (1 µg/ml). At the indicated time points the total cell number was determined by using the Casy Counter System. Mean values with standard deviation of 3 biological replicates (*p = 0.023) are displayed.</p

Transport assay of the hSLCO5A1 protein with Tritium-labelled substrates in <i>X. laevis</i> oocytes.

<p><i>X. laevis</i> oocytes (8–12 oocytes) were injected with the cRNA of the WT SLCO5A1 or its L³³F mutant, or with the control (Tris-HCl). Oocytes were incubated with 1 µCi/ml Tritium-labelled substrate and 0.04 µCi/ml [¹⁴C]sucrose at room temperature for 30 minutes. [¹⁴C]sucrose served as internal leakage control. Radioactivity was measured using a Beckman scintillation counter. Mean CPM (counts per minute) values with standard deviation are displayed.</p

IFNα-activated signalling pathways in THP1 and A375 cells.

<p>THP1 (A) and A375 (B) cells were stimulated with the indicated concentrations of IFNα. As controls, THP-1 cells were additionally stimulated with IL-6/sIL-6R and LPS; A375 cells were stimulated with OSM or IL-1β. The phosphorylation levels of STAT1, STAT3, ERK1/2 and Akt were detected via Western blot analysis. Displayed are mean values of at least three independent experiments.</p

IFNα stimulates TAP1 expression in mouse blood and tumor tissue and suppresses subcutaneous melanoma metastasis in a murine model for malignant melanoma.

<p>C57BL/6 mice were inoculated subcutaneously with B16F1 malignant melanoma cells (day 0). The mice received either no treatment (control group, n = 3) or recombinant murine IFNα (n = 3). IFNα treatment (10.000 IU) started on day +3 after application of B16F1 cells for 5 consecutive days. (A) Measurement of tumor growth in control mice and IFNα treated mice. (B) On day +7.5, three IFNα treated and three control mice were sacrificed and blood was collected for qRT-PCR analysis of TAP1 expression. (C) Three days after the last injection of IFNα (day +11), three mice of the IFNα treated and three mice of the control group were sacrificed and tumors were excised for qRT-PCR analysis of TAP1 expression. Statistical analysis was performed using unpaired Student’s t-test (* p<0.05; not significant (n.s.)).</p

The inhibition of Janus kinases abrogates the IFNα-upregulated TAP1 expression in THP1 and A375 cells.

<p>(A) THP1 (left) or A375 (right) cells were pre-incubated with the indicated amounts of JAK inhibitor I (JI-1), followed by stimulation with 100 U/ml or 1000 U/ml IFNα, respectively. Western blot analysis was performed using a specific antibody against phosphorylated STAT1 (STAT1-pY701), a STAT1 antibody recognizing the protein irrespective of its phosphorylation status and with an α-tubulin antibody. (B) Phosphorylation intensities were quantified by chemiluminescence analysis and normalization to loading controls. Shown are the means (n = 3) with standard error of mean (SEM, two-tailed, paired Student’s t-test, * p<0.05, ** p<0.01, *** p<0.001). (C) THP1 (left) or A375 (right) cells were pretreated with JI-1, and subsequently exposed to 100 U/ml or 1000 U/ml IFNα. Relative expression levels of TAP1 were analyzed by qRT-PCR and normalized to 18SrRNA as internal reference. Shown are -fold changes relative to unstimulated control = SEM (n = 3). Statistical significance was evaluated by performing a two-tailed, paired Student’s t-test (** p<0.01, *** p<0.001).</p

IFNα-2b (IntronA) stimulates TAP1 expression in peripheral blood mononuclear cells (PBMC) of patients (n = 18) with malignant melanoma receiving adjuvant high-dose immunotherapy.

<p>(A) The actual administered dose of IFNα-2b was about 20 million IU/m² dependent on the clinically observed side effects. Only days of IFNα treatment are shown. (B) mRNA expression of TAP1 in PBMCs of patients treated with adjuvant high-dose IFNα-2b. Statistical analysis of TAP expression was performed using the <i>Statistical Analysis System</i> of the SAS Institute Inc. (Cary, NC, USA).</p

Stimulatory effects of IFNα on TAP expression, peptide binding and transport.

<p>THP1 cells and A375 cells were treated with the indicated concentrations of IFNα for 3 hours (A, B). After stimulation, TAP1 mRNA and 18SrRNA mRNA (as internal reference) were measured by qRT-PCR analysis. Depicted are -fold changes relative to untreated cells = standard error of mean (SEM) of three independent experiments. (C) TAP1 protein expression is induced by IFNα in THP1 cells measured by western blot analysis. (D) TAP dependent peptide-binding sites are increased significantly by IFNα in THP1 cells (p = 0.0072). (E) ATP-dependent peptide transport is stimulated significantly by IFNα in THP1 cells (p = 0.0006). Statistical analysis was performed using unpaired Student’s t-test (** p<0.01, *** p<0.001).</p