Additional file 1: of MFG-E8 mediates arterial aging by promoting the proinflammatory phenotype of vascular smooth muscle cells

Figure S1. MFG-E8 abundantly expresses in aged human aortas and augments transactivation of NF-κB p65 in human VSMCs. (a–j) Representative IHC images display increased coexpression of MFG-E8 and phosphorylated NF-κB p65 (Ser536) as well as elevated ICAM-1 intensity in aged aortas. Arrows indicate the autofluorescence of the elastic laminae in the vessels; bar, 100 μm. (k–l) After treatment with rMFG-E8 (250 ng/mL) for 24 h, hAoSMCs were stimulated with Ang II (1 μM) for 24 h to mimic aging. (k) Cell lysates were analyzed through immunoblotting with antibodies specific for NF-κB p65 phosphorylated at Ser536 and NF-κB p65. Levels of phosphorylated p65, normalized to that of total p65 (n = 3), were analyzed. (l) The protein expression of VCAM-1 in hAoSMCs was evaluated through immunoblotting; the quantitative analysis results for VCAM-1, normalized to that of Gapdh, are displayed (n = 3). Data are presented as mean ± standard deviation. *P < 0.05 and **P < 0.01, one-way analysis of variance followed by Tukey’s multiple comparison test. (PDF 474 kb)