NECA increases leukemia inhibitory factor (LIF) expression and secretion levels in primary mouse astrocytes. (A) Primary cortical astrocytes were treated without or with NECA (1, 10 or 20 μM) for 0.5, 1, 2, 4, 8, 12 and 24 hours. Cells were then analyzed for LIF mRNA expression (relative to GAPDH) by real-time PCR. Data are normalized to the control and presented as Mean ± SEM of three independent experiments. P < 0.05. (B) shows Western blot analysis of cultured astrocytes treated without or with NECA (1 μM) for 1, 2, 4, 6, 8 and 12 hours to determine LIF protein levels. β-actin served as a loading control. (C) shows a representation of sandwich ELISA experiment performed to detect LIF content in supernatants of cultured astrocytes that were treated without or with NECA (1 μM) for 1, 2, 4, 6, 8 and 12 hours. Each bar corresponds to the mean concentration of LIF in triplicate samples; error bars indicate SEM. Observations were confirmed by repeating the experiments two additional times. P < 0.05. NECA, 5′-N-ethylcarboxamide.