FIGURE 4. Cav1.2 knock-down prevents astrocyte activation by LPS.

After 3DIV, astrocytes were transfected with siRNA duplexes specific for Cav1.2 and Cav1.3 (siCav1.2/1.3). (A) Cells were treated with fluorescein-labeled dsRNA oligomers to determine siRNA transfection efficiency. Scale bar = 60μm. (B) Three days after transfection, semi-quantitative RT-PCR and western blot analysis of Cav1.2 and Cav1.3 expression in astrocytes was performed using β-actin and GAPDH as internal standards. Data from three independent experiments are summarized based on the relative spot intensities and plotted as percent of controls. (C) At the same time, VOCC activity was examined in cultured astrocytes using fura-2 as intracellular Ca⁺⁺ indicator. Note that each trace corresponds to a single cell and the horizontal bars indicate the time of addition of external solution containing high K⁺. (D) The bar graph shows the average amplitude of the Ca⁺⁺ response, calculated from the responding cells expressed as a percentage of change of the emission intensities. (E–F) Three days after siRNA transfection, astrocytes were treated with LPS (1μg/ml) for 3 consecutive days and were stained with antibodies against GFAP, s100β and Ki67. The percentage of positive cells in each experimental condition was examined by confocal microscopy. Scale bar = 160μm. Values are expressed as mean ± SEM of at least six independent experiments. ***p<0.001 vs. corresponding controls.