Each cDNA sample was run in technical triplicate using gene-specific primers. Therefore each gene set included 36 target and 36 reference cDNA samples. Each gene

set also contained a 5-point standard curve for the reference and target genes, a no-template control, an extraction negative find more and a reverse transcriptase (RT) negative as controls. mRNA expression was analysed independently by one-way analysis of variance (ANOVA) using Satistica 6 software (StatSoft Inc., USA). Data analysis was carried out using Sequence Detection Systems software (Applied Biosystems). For quantification of gene expression changes, the ‘relative standard curve method’ was used to calculate relative fold changes normalised against the GAPDH gene (endogenous Cilengitide chemical structure control) using Eqs. (5) and (6). No significant differences were observed between vials within a batch (for all genes) and the vials behaved consistently across the two batches tested (batch 1 and 2). Therefore, data from all vials were pooled to increase the level of replication for each condition. Fold differences were calculated using Eq. (7) to compare batch-to-batch differences and primary vs. P.1 gene expression levels. A 2-fold difference is considered significant. equation(5) Normalisedtarget(test),NTT=Target/endogenouscontrol equation(6) Normalisedtarget(calibrator),NTC=Target/endogenouscontrol equation(7)

Folddifferenceintarget=NTT/NTC Permeability assays (apical to basal direction) were performed on 10 radio-labelled compounds covering passive permeation ([3H]diazepam, [3H]naloxone, [3H]propranolol, [14C]sucrose), uptake ([14C]caffeine, [3H]L-glutamic acid (as Na glutamate in saline), [3H]L-leucine) and efflux ([3H]colchicine, [3H]digoxin, [3H]vinblastine) transporters, as described for [14C]sucrose in Section 4.8. The apparent

permeability Papp was calculated according to Eq. (2) and plotted against Amrubicin the calculated Log Poctanol as a measure of lipophilicity of the compound. Log Poctanol estimation was obtained from http://www.syrres.com/eSc/est_kowdemo.htm. Data were expressed as mean±standard error of the mean (SEM) and analysed and presented using Microsoft Excel or GraphPad Prism (version 4.0). Groups of two were analysed using Student’s t-test, groups of three or more were analysed using one-way analysis of variance (ANOVA) with a Dunnett’s post-hoc test. Values were considered to be significantly different when the probability that differences were not due to chance alone was less than 5% (p <0.05). The authors thank Dr. Gavin Nixon from LGC Ltd., Teddington, UK for assisting with TaqMan real-time RT-PCR assays, Dr. Diana Dolman for the advice on functional assays, Dr. Siti Yusof for technical help with permeability assays and Professor Nancy Rothwell for support.