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Figure 3: Effects of Withaferin A on LPS-induced NF-κB activity in primary spinal cord astrocytes. (a) Primary astrocytes from the spinal cord of newborn mice were transiently co-transfected with NF-κB-driven firefly and Renilla luciferase reporter vectors. Twenty-four hours after the transfection, cells were preincubated in the presence or in the absence of 0.5 μM Withaferin A (WA) for 1 hour and then treated with or without 1 μg/mL LPS for additional 6 hours ( experiments in quintuplicate). Cell lysates were assayed for luciferase enzymatic activities, and firefly luciferase levels were normalized to Renilla luciferase values. Data (mean ± s.e.m.) are expressed as percentage of luciferase enzymatic activity in control conditions ( versus control, versus LPS; one-way ANOVA followed by Bonferroni post hoc test). (b) Primary astrocytes were transiently co-transfected with the STAT3-driven firefly and Renilla luciferase reporter vectors in the presence or in the absence of a plasmid encoding the STAT3α activator protein. Cells were subsequently incubated with or without 1 μg/mL LPS for 6 hours ( experiments in triplicate). Cell lysates were assayed for luciferase enzymatic activities, as above. Data (mean ± s.e.m.) are expressed as percentage of luciferase activity in control conditions ( versus control; one-way ANOVA followed by Bonferroni post hoc test). (c) Astrocytic cultures were pre-treated in the absence or in the presence of 0.5 μM WA for 1 hour followed by incubation with 1 μg/mL LPS for 6 hours. Images are representative of cells immunolabelled for the p65 subunit of NF-κB (green) and counterstained with Hoechst 33342 (blue) to visualise the nuclei. Scale bar, 20 μm. Note that the treatment with LPS induces the nuclear translocation of p65 while this effect is strongly abrogated in the presence of Withaferin A.