Figure 6.
Microglial activation by extracellular HMGB1. A, Primary microglial cultures (1 × 104 cells) were incubated with media collected from NMDA- or staurosporine (stauros)-treated primary cortical cultures for 24 h in the presence or absence of anti-HMGB1 antibody, and NO production was determined using the Griess method. shHMGB1-pU6 or MshHMGB1-pU6 was transiently transfected into primary cortical cells 24 h before NMDA treatment. Con, Control. B, The amounts of HMGB1 in NMDA-treated cells and in medium bathing were determined by RT-PCR after 24 h of NMDA treatment. C, Primary microglia cultures were treated with media collected from NMDA-treated primary cortical cultures, transfected or not transfected with shRNA-expressing plasmid or mutant shRNA-expressing plasmid, and NO production was determined using the Griess method. D, Changes in the RNA levels of the proinflammatory markers TNF-α, COX-2, and IL-1β in the presence of HMGB1 shRNA-expressing plasmid or of mutant shRNA-expressing plasmid were followed by RT-PCR. LPS (100 ng/ml) treatment was used as a positive control. GAPDH, Glyceraldehyde-3-phosphate dehydrogenase. The results of four independent experiments are presented as means ± SEM.