Withaferin A Inhibits Nuclear Factor-κB-Dependent Pro-Inflammatory and Stress Response Pathways in the Astrocytes

Figure 2

Impact of Withaferin A on LPS-induced NF-κB activity in immortalized P0-17D cortical astrocytes. (a) P0-17D astrocytic cells were transiently co-transfected with the NF-κB-RE-Luc transgene and the Renilla luciferase encoding plasmid, used as transfection control. Twenty-four hours after the transfection, cells were pre-treated for 1 hour with increasing concentrations of Withaferin A (WA) and then incubated in the presence or in the absence of 1 μg/mL LPS for additional 6 hours ( experiments in triplicate). 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 activity in control conditions, that is, the corresponding culture type challenged with saline ( versus control, versus LPS; one-way ANOVA followed by Bonferroni post hoc test). (b) P0-17D astrocytes were transiently co-transfected with STAT3-driven firefly luciferase and Renilla luciferase reporter vectors in the absence or in the presence of a plasmid encoding the constitutively active STAT3α protein. Cells were subsequently incubated with or without 1 μg/mL LPS for 6 hours ( experiments in triplicate) and 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, that is, the corresponding culture type challenged with saline ( versus control; one-way ANOVA followed by Bonferroni post hoc test).

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