Changes in cytokine levels following force-expression of AIF-1. (A) TNF-α, IL1β, IL1RA, IL6, IL12, IL15 and IL18 mRNA levels assayed by RT-qPCR at 0, 24, 48, 72 and 96 h after transfection with AIF-1-expression vector. All the cytokines increased significantly (P < 0.05 or P < 0.01), except for IL6. (B) Levels of TNF-α, IL1β, IL1RA, IL12, IL15 and IL18 mRNA after 72 h of transfection with AIF-1-expression vector (0.5 or 1 μg). TNF-α, IL1β and IL1RA mRNA levels were significantly higher than the control (P < 0.01). (C) Changes in TNF-α, IL1β and IL1RA protein levels analyzed by Western blotting at 96 h after transfection with the AIF-1-expression plasmid in Ishikawa cells. Only TNF-α was significantly increased (P < 0.05). (D) Changes of TNF-α protein level analyzed by Western blotting at 96 h after transfection with the AIF-1-expression plasmid in primary endothelial and stromal cells. TNF-α was significantly increased after AIF-1 plasmid transfection (P < 0.05). All data are presented as the mean ± SEM. The differences between groups of all data were analyzed by ANOVA, followed by multiple comparisons using Bonferroni test (except AIF-1 expression in Figure 4B, which was analyzed by Kruskal–Wallis test, followed by multiple comparisons using Mann–Whitney U-test). Data were considered statistically significant if P < 0.05.