Fig. 5

ACT001 suppresses the activation of STING→NF-κB pathway in microglia. (A-C) Analysis of RNA-seq of sorted single live Tmem119⁺ cells in CNS lesions from EAE-control and ACT001-treated mice at the peak of EAE (n = 3 per group). (A) Heatmap displays the expression of all differentiation (DE) genes in SCs microglia based on the RNA-seq data. (B) Volcano plot depicts DE genes in SCs microglia. Colored dots represent significant DE genes. DE genes were identified using DEseq2, applying the Wald test with Benjamini-Hochberg correction to determine the false discovery rate (FDR < 0.01). (D) Sorted single live Tmem119⁺ cells in CNS lesions were isolated and analyzed for expression of genes related to STING→NF-κB induced microglia activation (n = 6 per group). (E-F) Immunofluorescence analysis of p-STING (green) (E) and p-p65 (green) (F) levels in Iba1⁺(red) microglia within SCs tissue. DAPI (blue) was used as a nuclear marker. Scale bar represents 50 μm. (G) Phosphorylation of key proteins in STING→NF-κB pathway in the SCs tissue at Day 0, Day 10, Day 20 and Day 30 following EAE induction. (H-I) Phosphorylation of key proteins in STING→NF-κB pathway in primary microglia stimulated by LPS, 2’3’-cGAMP, and HSV-1, and the effects of ACT001. The values in the graph represent the normalized ratio of phosphorylated to total proteins. Statistics were calculated using the unpaired sample t-test (D) and one-way ANOVA with Tukey’s correction (E, F). Error bars denote the mean ± SEM, ns = not significant, *p < 0.05, **p < 0.01 and ***p < 0.001