Fig 1: (A) MCM Tat caused a significant increase in MCP-1/CCL2 levels compared to MCM Control treatment while incubation with PF3845 blocked this increase and further decreased MCP-1/CCL2 levels compared to control. Antagonizing GPR18 with CID blocked these PF3845-mediated decreases in MCP-1/CCL2 as seen in the MCM CID + PF3845 + Tat condition. (B) MCM Tat caused a significant increase in proMMP-9 levels compared to control and incubating with PF3845 blocked this effect. Antagonizing GPR18 with CID blocked this PF3845-mediated decrease in proMMP-9 as seen in the MCM CID + PF3845 + Tat condition. (C) MCM Tat caused a significant increase in MMP-9 levels compared to control and incubation with PF3845 blocked this effect. Antagonizing GPR18 with CID blocked the PF3845-mediated decrease in MMP-9 as seen in the MCM CID + PF3845 + Tat condition. Collectively, these data suggest that Tat enhanced the excretion of neurotoxins, while PF3845 blunted this effect at least partially through GPR18-related activity. Statistical significance was assessed by ANOVAs followed by Bonferroni’s post hoc tests; *p < 0.05 vs. MCM Control, #p < 0.05 vs. MCM Tat, $p < 0.05 vs. MCM CID + PF3845 + Tat. MCM, microglial conditioned media; CID, CID-85469571.
Fig 2: (A) Shows the effects of treatment conditions on microglia morphology. Tat does not show any effects on fractal dimention (DF) and lacunarity (?) whereas PF3845 via GPR18 increases DF and decreases ?, which is associated with a less activated microglia state and more overall morphological homogeneity, respectively. (B) Shows the soluble factors released from treated microglia into the microglial conditioned media (MCM). MCM conditions derived from Tat treated microglia (MCM Tat) show upregulated proinflammatory responses with increased levels in MCP-1/CCL2, MMP-9, and proMMP-9. In contrast, MCMs derived from microglia pretreated with PF3845 (MCM PF3845) show increased AEA levels by inhibiting the catabolic FAAH enzyme via GPR18-related mechanisms, which results in anti-inflammatory responses by decreasing MCP-1/CCL2, MMP-9, and proMMP-9 levels. (C) Shows neuronal changes that occur when frontal cortex neurons are treated with MCM conditions derived from treated microglia. MCM Tat increases neuronal Ca2+ dysregulation and synaptodendritic damage whereas MCMs derived from microglia pretreated with PF3845 alongside Tat (MCM PF3845 + Tat) results in reduction of Ca2+ dysregulation and synaptodendritic damage via GPR18-related mechanisms. ?, lacunarity; Ca2+, calcium; CID-85469571, GPR18 antagonist; DF, fractal dimension.
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