Fig. 6
GKT137831 improves neuronal survivability in co-culture with activated microglia. a Primary neurons were co-cultured with microglia exposed to FeSO4, DMSO, LPS, GKT, or in combination. Immunostaining of neurons after 24 h co-culture showed a reduction in the expression of NeuN in groups co-cultured with microglia treated with FeSO4 & LPS & DMSO. b Improved survivability among all cell types present in the neuronal culture were observed by the increased cell counts in groups treated with GKT137831. c Quantitative analysis of NeuN counts revealed a significant reduction in the presence of neurons after co-culture with microglia treated with FeSO4 & LPS & DMSO. GKT137831 reduced neuronal toxicity and prevented neuronal loss after co-culture. d PC12 cells were differentiated to a neuronal like phenotype for one week prior to microglia co-culture. Immunocytochemistry revealed increased staining among PC12 co-cultured with microglia treated with LPS, or in combination with FeSO4. The addition of GKT137831 to the microglia treatment groups reduced the presence of cells positive for cleaved caspase-3. e Quantitative analysis of PC12 cells positive with cleaved caspase-3 were significantly increased when co-cultured with microglia treated with FeSO4 & LPS & DMSO. However, this increased apoptosis among PC12 cells was reversed by the addition of GKT137831. All immunostaining images are representative of one trial. All groups were compared using one-way ANOVA with Tukey post-test. In all graphs, symbols representing significance were assigned according to comparisons: DMSO group (*); FeSO4 & DMSO (#); LPS & DMSO (!); and FeSO4 & LPS & DMSO ($). **p < 0.01, ****p < 0.0001, ####p < 0.0001,!!p < 0.01, and $$$$p < 0.0001. All graphs represent n = 3. Bars represent mean ± SEM. Size bars represent 50 μm