The β-actin gene was utilized as an internal control and was chosen as a reference gene because it is a housekeeping gene. Real-time PCRs were performed in 25 μl of final volume containing

2 μl of cDNA, master mix with SYBR Green (iQ SYBR Green Supermix Bio-Rad, Milan, Italy) and sense and antisense primers for the ZO-1, Claudin-1, Occludin and the β-actin gene (Table 1). Table 1 Sequences of amplification primers Gene   Primer ZO-1 Sense 5′- ATCCCTCAAGGAGCCATTC-3′ Antisense 5′- CACTTGTTTTGCCAGGTTTTA-3′ Claudin-1 Sense 5′- AAGTGCTTGGAAGACGATGA-3′ Antisense 5′- CTTGGTGTTGGGTAAGAGGTT-3′ Occludin Sense 5′-CCAATGTCGAGGAGTGGG-3′ Antisense 5′-CGCTGCTGTAACGAGGCT-3′ β-actin Sense 5′-AAAGACCTGTACGCCAACACAGTGCTGTCTGG-3′   Antisense 5′-CGTCATACTCCTGCTTGCT

GATCCACATCTGC-3 Real-time PCRs were carried out in a CFX96 Real-Time PCR Detection System (Bio-Rad Laboratories, Batimastat cost Inc.) using the following protocol: EPZ015666 clinical trial 45 cycles at 95°C for 3 min, 95°C for 10 s, 55°C for 30 s followed by a melting curve step at 65 – 95°C with a heating rate of 0.5°C per cycle for 80 cycles. The PCR products were quantified by external calibration curves, one for each tested gene, obtained with serial dilutions of known copy number of molecules (102-107 molecules). All expression data were normalized by dividing the target amount by the amount of β-actin used as internal control for each sample. The specificity of the PCR product was confirmed by gel electrophoresis. As Western Blot concerns, Caco-2 cells were collected and lysed on ice Carnitine palmitoyltransferase II in RIPA buffer (Pierce Ripa buffer, Thermo Scientific, Rockford, IL, USA). After homogenization and centrifugation at 14000 rpm for 15 min at 4°C, protein concentration was measured by a standard Bradford assay (Bio-Rad Laboratories, Milan, Italy). Aliquots of 50 μg of total proteins were separated in 4-12% pre-cast polyacrylamide gels (Invitrogen, Life Technologies, OR, USA) and transferred onto a PVDF membrane (Bio-Rad Laboratories, Milan, Italy) with Transblot Turbo (Bio-Rad Laboratories). ZO-1, Claudin-1, Occludin

and β-actin protein expressions were evaluated by 1:500 diluted ZO-1 (H-300), Claudin-1 (D-4), Occludin (N-19) and β-actin antibody, respectively (Santa Cruz Biotechnology, Santa Cruz, CA, USA). After overnight incubation, the membranes were further incubated with a horseradish peroxidase-conjugated goat secondary antibody (Bio-Rad Laboratories). The proteins were detected by chemiluminescence (ECL, Thermo Scientific, Rockford, IL, USA) and the densitometric Ferrostatin-1 chemical structure analysis of each protein-related signal was obtained using the Molecular Imager Chemidoc™ (Bio-Rad Laboratories) and normalized against β-actin expression. Statistical analysis Due to the non-normal distribution of the data, non-parametric tests were performed.