Figure 5

GSK3 promotes IL-6 production. A, Quantitation of the effects of lithium treatment on inflammatory molecules produced by primary enriched astrocytes stimulated with LPS (100 ng/mL) and IFNγ (1 ng/mL) for 6 h and analyzed by cytokine array, as described in Figure 3C. B, Time-dependent production of IL-6 by primary enriched astrocytes treated with vehicle (0), LPS (100 ng/mL), IFNγ (1 ng/mL), or both, in the absence and presence of 20 mM lithium. White bars indicate that no GSK3 inhibitor was added. *p < 0.05 compared to LPS plus IFNγ. C, IL-6 production by primary enriched astrocytes (n = 7) treated with 1 ng/mL IFNγ, 100 ng/mL LPS, or both, without or with 20 mM lithium or 10 μM other GSK3 inhibitors, for 6 h. White bars indicate DMSO vehicle with no GSK3 inhibitor. *p < 0.05 compared to LPS plus IFNγ without any GSK3 inhibitor. D, Cell viability: Primary enriched astrocytes were treated with 100 ng/mL LPS, 1 ng/mL IFNγ, or both, and with 20 mM lithium, 10 μM 6-bromoindirubin-3'-oxime (BIO), 10 μM kenpaullone, 10 μM GSK3 inhibitor II, or 10 μM SB216763 for 6 h and viability was assessed by a MTT assay. White bars indicate DMSO vehicle with no GSK3 inhibitor. Means ± SEM; n = 3. E, IL-6 production by primary microglia (n = 4) treated with 1 ng/mL IFNγ, 100 ng/mL LPS, or both, without or with 20 mM lithium or 10 μM SB216763, for 6 h. White bars indicate that no GSK3 inhibitor was added. *p < 0.05 compared to LPS plus IFNγ without any GSK3 inhibitor. F, IL-6 production by primary enriched astrocytes after 48 h siRNA-mediated knockdown of GSK3α, GSK3β, or both, and stimulation with LPS (100 ng/mL), IFNγ (1 ng/mL), or both, for 6 h (n = 5, *p < 0.05 compared to corresponding control siRNA SNC values). G, Cooperation between GSK3 and STAT3 to induce IL-6 production by primary enriched astrocytes, after 36 h expression of control GFP, S21A-GSK3α, or S9A-GSK3β, with or without expression of STAT3C (n = 3;*p < 0.05). Means ± SEM.