Fig. 6

CD146 expression is up-regulated by stimulation with TNFα and IL-1β through NFκB activation. A, B CD146⁺ pericytes and BECs were left untreated or stimulated with TNFα, IL-1β, and IL-6 for 12 h, 24 h, and 48 h. Western blot analysis shows up-regulation of CD146 expression in response to TNFα and IL-1β treatment compared to unstimulated cells, while no changes in the protein levels of CD146 are observed after IL-6 stimulation. Parallel evaluation of p65 phosphorylation shows that only TNFα and IL-1β treatment induce p65 phosphorylation/activation in both pericytes and BECs. The equal loading was assessed using anti-GAPDH antibody for CD146 and anti-p65 for phospho-p65. For the densitometric analysis, the values from three independent experiments were normalized and expressed as fold increases and are reported as mean values ± standard deviations (SD). Unpaired Student’s t-test was performed, and significance levels are defined as * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. NS, not significant. C, D CD146.⁺ pericytes and BECs were left untreated or stimulated with TNFα or IL-1β in the presence or absence of the NFκB inhibitor, BAY 11–7082 (Bay) for 24 h. Western blot analysis shows that the increase in the levels of CD146 upon TNFα or IL-1β stimulation is abolished by treatment with NFκB inhibitor. The equal loading was assessed using anti-GAPDH antibody for CD146 and anti-p65 for phospho-p65. The densitometric analysis and unpaired Student’s t-test were performed as reported above. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001