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. 2013 Jan 28;8(1):e55084. doi: 10.1371/journal.pone.0055084

Figure 6. IGFBP-3 inhibits NF-κB activity in an IGF-independent manner in HAECs.

Figure 6

HAEC cells were infected with Ad:EV (m.o.i.100) or Ad:IGFBP-3 (m.o.i.100) and Ad:IGFBP-3GGG (m.o.i. 100) followed by TNF-α treatment for 24 hours and were analyzed for mRNA (A) and protein expression (B) by RT-PCR and western blotting respectively. (C) A similar experiment was performed with infection with Ad:EV, Ad:IGFBP-3 or Ad:IGFBP-3GGG and TNF-α treatment and MCP-1 levels were quantified in the CM using ELISA. n = 2, in triplicates; *,#,**, p<0.001 (D) HAEC cells were infected with Ad:IGFBP-3 or Ad:IGFB3GGG and treated with 50 ng/ml of TNF-α. At the end of the incubation period, monocyte cell adhesion assay was performed. The numbers of adhered monocytes on the HAEC monolayer are depicted as a bar graph. n = 2 in triplicates; *, p = 0.05; #, **, p<0.05. (E) HAEC cells were transfected with siRNA against IGFBP-3R once or two consecutive times and cells were harvested to determine the knockdown of IGFBP-3R by western blotting. HAEC cells were transfected with either scrambled siRNA or siRNA against IGFBP-3R. (F) The cells were then infected with Ad:EV (m.o.i.100) or Ad:IGFBP-3 (m.o.i.100) followed by TNF-α treatment for 24 hours and were analyzed for mRNA and protein expression.