Figure 3

Citicoline increased expression of p-ERK1/2 and p-IRS-1 in citicoline-treated hCMEC/D3, and treatment with anti-sense siRNA directed against IRS-1 significantly inhibited tube-like structure formation. A and B) Representative Western blots showing the up-regulation of ERK1/2 and IRS-1 phosphorylation after 10 min stimulation of hCMEC/D3 in basal-serum-poor medium. (i) Control cells showed little p-ERK1/2/IRS-1 expression and (ii) after citicoline treatment (10 μM; 10 min) a notable increase was seen. Experiments were performed at least twice and a representative example is shown together with a bar chart showing the fold increase in expression. C) hCMEC/D3 treated with anti-sense siRNA directed against IRS-1, for 48 h prior to citicoline treatment (10 μM; 24 h), significantly reduced the ability of citicoline to induce tube-like structure formation in matrigel. Data shown are the result of a single representative experiment performed in triplicate wells. The bar graph illustrates the significant increase in the numbers of closed-loop areas in the presence of citicoline was reduced by approximately 50% in the presence of siRNA to IRS-1 (p = 0.018). Five areas from each well and 3 wells per treatment were analyzed.