Co-immunoprecipitation analysis of UT receptor and eNOS.

<p>Tissues were stimulated with either vehicle (A) or U-II (C), lysates were incubated with mouse anti-eNOS. Lanes B and D correspond to the negative control of A and C, respectively. The western blot was probed with rabbit anti-GPR14 (UT receptor). U-II but not vehicle caused the co-immunoprecipation between eNOS and UT receptor.</p

Panel A: U-II induced concentration-response curve (0.1 nM–10 µM) in HCC strips was significantly inhibited by pre-treatment with wortmannin (0.1 µM), PI3K inhibitor, or geldanamycin (1 µM) Hsp90 inhibitor (***p<0.001 and ** p<0.01 <i>versus</i> vehicle).

<p>Data were expressed as the mean ± standard error of the mean (SEM) of six different specimens. Data were analyzed by ANOVA followed by Bonferroni post test. Panel B: NOx (total nitrite) production in HCC tissue incubated with U-II (1 nM-10 µM) or vehicle for 30 min. U-II caused a significant increase in NO production compared with vehicle (*<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001). Data were expressed as mean ± standard error of the mean (SEM) from four different specimens and analyzed by ANOVA followed by Bonferroni post test.</p

Supplementary Figure 1 Legend from Synergistic Effects of Metformin Treatment in Combination with Gefitinib, a Selective EGFR Tyrosine Kinase Inhibitor, in LKB1 Wild-type NSCLC Cell Lines

Supplementary Figure 1 Legend - PDF file 52K, Effects on CALU-3, CALU-3 GEF-R, H1299, H1975 and GLC82 cell growth of the combination of metformin and gefitinib. Cell viability was determined using a 3-(4,5 methylthiazol-2-yl)-2,5- diphenyltetrazolium bromide assay. Treatment combinations and sequences are described in Materials and Methods. CI values were calculated according to the Chou and Talalay mathematical model for drug interactions using the Calcusyn software for different fractions affected (fa). CI is a quantitative measure of the degree of interaction between different drugs. If CI = 1, it denotes additivity; if CI > 1, it denotes antagonism; if 1 0.7, it denotes slight synergism; if CI = 0.7-0.3, it denotes synergism; if CI < 0.3, it denotes strong synergism. Results are the median of three independent experiments, each done in eight replicate wells for experimental point</p

Quantitative RT-PCR for U-II in HCC.

<p>U-II is expressed as mRNA in HCC samples. Human tumoural cells SW-13 were used as positive control. Data were normalized on the basis of GAPDH and expressed as mean ± standard error of the mean (SEM) of three different human specimens.</p

Western blot analysis for eNOS and p-eNOS^Ser-1177 in HCC tissue.

<p>Panel A: U-II (10 µM) caused an increase in eNOS phosphorylation expressed as p-eNOS/eNOS ratio in a time-dependent manner (*p<0.05 <i>vs</i> vehicle). Panel B: U-II-induced eNOS phosphorylation (**p<0.01 <i>vs</i> vehicle), was significantly reverted by wortmannin (0.1 µM), PI3K inhibitor, (°p<0.05 <i>vs</i> U-II) but not by geldanamicin (1 µM), Hsp90 inhibitor. Data were expressed as the mean ± standard error of the mean (SEM) of four different specimens and were analyzed by ANOVA followed by Bonferroni post test.</p

Supplementary Figure 2 Legend from Synergistic Effects of Metformin Treatment in Combination with Gefitinib, a Selective EGFR Tyrosine Kinase Inhibitor, in LKB1 Wild-type NSCLC Cell Lines

Supplementary Figure 2 Legend - PDF file 58K, Supplementary Figure 2 legend: Effects on the downstream pathways by metformin treatment in A549 and H460. Western blotting analysis of AMPK, ACC, MAPK, AKT, p70S6K, S6, 4EBP1, activation following treatment with the indicated concentration of metformin. beta-actin was included as a loading control</p

Supplementary Figure 2 from Synergistic Effects of Metformin Treatment in Combination with Gefitinib, a Selective EGFR Tyrosine Kinase Inhibitor, in LKB1 Wild-type NSCLC Cell Lines

Supplementary Figure 2 - PDF file 194K, Supplementary Figure 2. Effects on the downstream pathways by metformin treatment in A549 and H460</p

Supplementary Figure 1 from Synergistic Effects of Metformin Treatment in Combination with Gefitinib, a Selective EGFR Tyrosine Kinase Inhibitor, in LKB1 Wild-type NSCLC Cell Lines

Supplementary Figure 1 - PDF file 81K, Effects on CALU-3, CALU-3 GEF-R, H1299, H1975 and GLC82 cell growth of the combination of metformin and gefitinib</p

Supplementary Figure 1 from Increased TGF-α as a Mechanism of Acquired Resistance to the Anti-EGFR Inhibitor Cetuximab through EGFR–MET Interaction and Activation of MET Signaling in Colon Cancer Cells

PDF file 1732K, Development and characterization of cetuximab-resistant SW48 (SW48-CR) colon cancer cells</p

Supplementary Table 1 from Increased TGF-α as a Mechanism of Acquired Resistance to the Anti-EGFR Inhibitor Cetuximab through EGFR–MET Interaction and Activation of MET Signaling in Colon Cancer Cells

PDF file 184K, genes up regulated in GEO-CR versus GEO</p