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

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

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