09 M Tris borate, 2 mM EDTA, pH 7.8) at 90 mV for 60 min. cDNA was prepared from 2 μg of total RNA using the Superscript™ First-Strand Synthesis System (Invitrogen, Paisley, UK) with random hexamer primers according to the manufacturer’s protocol. Samples were incubated at 65 °C for 5 min then held

on ice for 1 min before the addition of Superscript III reverse transcriptase. Samples were then incubated at 25 °C for 10 min followed by reverse transcription at 50 °C for 50 min. The reaction was terminated by heating to 85 °C for 5 min to inactivate the enzyme. Quantitative PCR was carried out on a 7500 Real Time PCR Sequence Detection System (Applied Biosystems, Foster City, CA). TaqMan analysis was performed in a 25 μl reaction mixture containing 30 ng CB-839 datasheet cDNA, TaqMan Universal PCR Master Mix (comprising AmpliTaq Gold DNA polymerase, dNTPs with dUTP, passive reference and optimised selleck products buffer) and Assay-on-demand™ gene expression assay mixes containing specific primers and probes (all from Applied Biosystems). The PCR conditions comprised a 2 min incubation at 50 °C followed by a 10 min polymerase activation at 95 °C. This was followed by 40 cycles alternating between 95 °C for 15 sections and 60 °C for 1 min each.

Amplification curves were analysed using the SDS version 3.2 software (Applied Biosystems, Foster City, CA). The baseline and threshold values were set and the Ct values extracted for each gene of interest. Relative quantification was calculated using the geometric mean of two selected house-keeping genes, gapdh and mvp. Relative gene expression

levels were calculated using the equation 2−ΔCt. An arbitrary classification system was applied to the data quantifying relative expression levels secondly as ‘high’ >0.5, ‘moderate’ between 0.02 and 0.5, ‘low’ between 0.001–0.02 and ‘negligible’ <0.001. All transport experiments were conducted in standard buffer solution (SBS) comprising Hank’s Balanced Salt Solution (HBSS) supplemented with 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES). Cell layers were allowed to equilibrate in SBS for 60 min at 37 °C before TEER measurements were taken. Each condition was carried out in quadruplicate and only layers with a resistance >250 Ω cm2 were accepted for experimentation. For transport studies with radiolabelled markers, donor compartments were filled with 0.51 ml (apical to basolateral (AB) transport) or 1.51 ml (basolateral to apical (BA) transport) of SBS containing 25 nM 3H-digoxin and/or 6.55 μM 14C-mannitol. Receiver compartments were filled with 1.5 ml (AB transport) or 0.5 ml (BA transport) of SBS. At the start and end of the experiment, 10 μl samples were taken from the donor compartments for determination of the initial and final concentration. Every 30 min over a 2 h period, 300 μl samples (AB transport) and 100 μl samples (BA transport) were taken from the respective receiver chambers and replaced with the same volume of SBS.