Reverse transcription from RNA to DNA was Doramapimod concentration performed with a Multiscribe Reverse Transcriptase kit from Applied Biosystem at 25°C for 10 min, at 48°C for 30 min and at 94°C for 29 sec. The PCR was performed in triplicates of each sample in a volume of 25 μL in each well containing RNA, TaqMan Universal PCR MasterMix and a primer of the target, i.e., HIF-1α (Rn00577560_m1), TGF-β (Rn00572010_m1) and VEGF-A (Rn4331348), and a primer of the housekeeping gene, 18S (4319413), all purchased from Applied Biosystems. Each RT-PCR reaction ran at 50°C for 2 min, at 95°C

for 10 min and in 40 cycles changing between 95°C for 15 sec. and 60°C for 1.30 min [27]. PCR Data analysis Data was analyzed with the ABI Prism 7000 Sequence Detector Software from Applied Biosystems. The output of amplification was measured in the TPX-0005 datasheet exponential phase of the reaction as the threshold cycle/Ct-value, which is defined as the cycle number at which amplification products are detected corresponding to the point where fluorescent intensity exceeds the background fluorescent intensity, which is 10 × the standard deviation of the baseline. The average of triplicates from each sample was used. The relative quantification of target gene was calculated using the formula: (1/2)Ct-target gene- Ct-housekeeping gene, which is described in the Users Bulletin 2,

1997 from Perkin-Elmer (Perkin-Elmer Cetus, Norwalk, CT, USA) [27]. Statistical LBH589 datasheet analysis Statistical analysis were performed by SPSS® 11.0 programs (SPSS Inc., Chicago, Illinois, USA). All data is expressed as mean ± SEM. Comparisons of data between groups were performed by non-parametric Kruskal-Wallis (ANOVA) test followed by the Mann-Whitney U-test. A p value < 0.05 was considered significant. Results Liver

parameters Blood samples showed a significant increase in ALAT in group IRI (334 ± 135 U/L), IPC (377 ± 104 U/L), IPO (1177 ± 379 U/L) and IPC+IPO (710 ± 199 U/L) compared to the control group (40 ± 2 U/L) (CG vs. IRI, IPC, IPO, and IPC+IPO, p = 0.01). No significant differences were found in ALAT between groups IRI, IPC, IPO and IPC+IPO. Alkaline phosphates and bilirubin were comparable between groups (Figure 2). Figure 2 Blood samples including ALAT (A), alkaline phosphatase (AP) (B) and bilirubin (C) levels. Samples 30 min after reperfusion in CG, Control group. IRI, 30 min of ischemia. IPC, ischemic preconditioning + 30 min of EGFR inhibitor ischemia. IPO, 30 min ischemia + ischemic postconditioning. IPC+IPO, ischemic preconditioning + 30 min of ischemia + ischemic postconditioning. * indicates p ≤ 0.01 compared to the control group. HIF-1α expression In the IRI group the expression of HIF-1α mRNA was significantly increased after 30 min of reperfusion compared to the control group (p ≤ 0.01). In the IPC group HIF-1α mRNA expression was significantly lower than the IRI group (p ≤ 0.01). In rats subjected to IPO there was a tendency towards lower HIF-1α mRNA expression compared to the IRI group (p = 0.