Figure 9.

Role of Rho/ROCK pathway in the LCN2-induced morphological changes of astrocytes. Primary astrocytes were pretreated with Y27632 (ROCK inhibitor; 100 μm) in the absence or presence of recombinant LCN2 protein (10 μg/ml) for 24 h. Cells were double-stained for GFAP (green) and Hoechst 33342 (cell nuclei; blue) (magnification, ×400) (A). Measurement of the length of the longest astrocyte processes for each treatment was done (B). Error bars indicate SD. *p < 0.05 compared with LCN2 treatment alone. GFAP mRNA (C) or protein levels (D) were also evaluated by RT-PCR or Western blot analysis, respectively, after stimulation with LCN2 in the absence or presence of Rho kinase inhibitor Y27632 (100 μm) for 24 h. β-Actin or α-tubulin was detected to confirm the equal loading of the samples. Results of densitometric analysis are shown below (C, D). After primary astrocytes were treated with LCN2 protein (100 ng/ml or 10 μg/ml) for the indicated time periods or 24 h, the activation state of Rho was assessed by a pulldown assay (E). The results are representative of three or more independent experiments.