Figure 6

Inhibition of FPRL1 or MARCO expression in astrocytes by siRNA resulted in a decrease of NM- as well as SP-induced Camp as well as IL-1β expression and ERK1/2 phosphorylation. siRNA for FPRL1 and MARCO, as well as control siRNA, was transfected in astrocytes and down-regulation of FPRL1 (A) and MARCO (B) mRNA expression was analyzed 96 h later using SYBR green real time RT-PCR and compared to the untreated sample. GAPDH was used as an internal control (housekeeping gene). Data were assessed from three independent experiments each performed in triplicate. Asterisks indicate a significant difference (*, p < 0.05) compared to control siRNA (one-way ANOVA followed by the Bonferroni test). Transfected astrocytes were incubated 96 h, then incubated with SP or NM for 24 h and Camp (C) or IL-1β (E) mRNA expression was determined using SYBR green or TaqMan real time RT-PCR and compared to the untreated sample. GAPDH or 18 s was used as an internal control (housekeeping gene). Data were assessed from three independent experiments each performed in triplicate. Asterisks indicate a significant difference (*, p < 0.05; **, p < 0.001) compared to NM or SP stimulated astrocytes transfected with control siRNA (one-way ANOVA followed by the Bonferroni test). 96 h after transfection, (D) astrocytes were treated with NM or SP for 5 min at 37°C. Levels of total ERK2 and phosphorylated ERK1/2 were determined using immunoblotting. The mean ± SEM of the three independent experiments was evaluated by densitometric quantification (F). Asterisks indicate significant difference (*p < 0.05) compared to control (one-way ANOVA followed by the Bonferroni test).