GSK-3β inhibitors inhibit LPS-induced TNF-α production in microglia. (A) Cells were preincubated with vehicle or GSK-3β inhibitors 10 μM TDZD-8 (TDZD), 10 μM AR-A014418 (AR), 10 μM L803-mts (L803), or 5 μM TWS119 (TWS) for 30 min, and subsequently treated with 100 ng/ml LPS for 6 h. Released TNF-α was measured by ELISA. Data are presented as mean ± SEM for three independent experiments. The TNF-α content in untreated cultures or cultures treated with inhibitor alone was not detectable. The levels of TNF-α in LPS-treated alone BV-2 cells and primary microglia were 3.78 ± 0.27 and 3.49 ± 0.22 ng/ml, respectively. (B) TWS119 inhibits activation of GSK-3β. BV-2 cells were exposed to DMSO or TWS119 (2.5 μM) for 60 min and analyzed by western blot, using the indicated specific antibodies. (C and D) Cells were pretreated with vehicle or various concentrations of TWS119 for 30 min followed by treatment with 100 ng/ml LPS for 6 h (C) or 2 h (D). Released TNF-α (C) is expressed as mean ± SEM for three independent experiments. The levels of TNF-α in LPS-treated alone BV-2 cells and primary microglia were 3.95 ± 0.32 and 3.62 ± 0.3 ng/ml, respectively. Expression of TNF-α mRNA (D) was quantified by real-time RT-PCR as described in Methods. The relative differences in expression between groups were analyzed on the basis of cycle time values normalized with β-actin. The relative differences between control and TWS119 pretreated groups were calculated and expressed as % of control. Data are presented as mean ± SEM for three independent experiments. *p < 0.05; **p < 0.01 compared with control.