Figure 5

SOD2 is not involved in cytokine-mediated protection of astrocytes. (A) The expression of SOD2 was analyzed in resting (C) and cytokine-activated (CK) astrocytes by RT-qPCR. Statistical analysis was calculated by using paired two-tailed Student’s t-test. (*p < 0.05). (B) Resting astrocytes were incubated with lipofectamine alone (controls, for this and the following experiments), or transfected for 90 min with either a SOD2-specific siRNA or a control siRNA (against the Dmt1 transcript). After at least 6 h from transfection, astrocytes were treated with cytokines to induce activation. Efficiency of silencing was assessed by Western blot analysis. (C) Acute iron overload was performed on fura-2 loaded monolayers of either resting (C) or activated (CK) astrocytes with or without SOD2 silencing. Oxidative stress (recovery of fura-2 signal) and cell death (dye loss) were monitored by a fluorescence plate reader. (D) Ninety minutes after iron overload, cell viability (MTT assay) was measured on resting astrocytes (C) or activated (CK) astrocytes, with or without SOD2 silencing. Statistical analysis was performed by using one-way ANOVA followed by Bonferroni post hoc test. (**p < 0.01; ***p < 0.001).