Fig. 1

6-MP attenuates TNF-α production in microglia. a Cells were pretreated (P) with vehicle or 50 μM 6-MP for the indicated times, cells were then stimulated with 100 ng/ml LPS for 6 h. TNF-α secretion into the culture medium was analyzed by ELISA. The relative differences between control and 6-MP pretreated groups were calculated and expressed as percent (%) of control. Data are presented as mean ± SEM for three independent experiments. *p < 0.05; **p < 0.01 compared with control. b, c Cells were pretreated with vehicle or various concentrations of 6-MP for 16 h followed by treatment with 100 ng/ml LPS for 6 h (b) or 2 h (c). Released TNF-α (b) was measured by ELISA. Expression of TNF-α mRNA (c) was quantified by real-time RT-PCR as described in the “Methods” section. Data are presented as mean ± SEM for three independent experiments. *p < 0.05; **p < 0.01 compared with control. The TNF-α content in untreated cultures was not detectable. The levels of TNF-α in LPS-treated alone BV-2 cells and primary microglia were 16.0 ± 0.4 and 2.8 ± 0.3 ng/ml, respectively. d The suppression of TNF-α mRNA by 6-MP requires de novo protein synthesis. BV-2 cells were pretreated with cyclohexamide (CHX, 0.5 μg/ml) or its vehicle 1 h before 16-h incubation without or with 6-MP (50 μM) and subsequent LPS stimulation (100 ng/ml) for 2 h. TNF-α mRNA expression was analyzed by real-time RT-PCR. Data are presented as mean ± SEM for three independent experiments. **p < 0.01 compared with control. e BV-2 cells were pretreated (P) with various concentrations of 6-MP for 4 h or 16 h followed by exposure to 100 ng/ml LPS for 2 h. Total RNA was then extracted for real-time RT-PCR analysis. Data are presented as mean ± SEM for three independent experiments. **p < 0.01 compared with respective control