Figure 2B, C Kinetic change of mRNA expression of Top1, Btbd1, Tdp1, and Srsf1 in microglia following LPS/IFNγ stimulation (n = 6, technical replicates). Data information: Data are presented as the mean ± s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001; Statistical analyses were performed using two-way ANOVA with Dunnett's Multiple Comparison test for (B and C),D, E The protein expression of TOP1 was detected by Western blotting (n = 3, biological replicates). Data information: Data are presented as the mean ± s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001 Statistical analyses were performed using the student's unpaired two-tailed t-test.F-H Immunostaining of TOP1 (red) in stimulated microglia (4 h after LPS/IFNγ stimulation) or resting microglia, co-stained with Hoechst (blue) (bar = 20 μm); for the lower-left panel in (H), n = 3 technical replicates per group (2 to 5 random fields per well were analyzed); for the lower right panel, 201 cells from unstimulated microglia and 256 cells from stimulated microglia were analyzed. Data information: Data are presented as the mean ± s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001; Statistical analyses were performed using the student's unpaired two-tailed t-test.G Immunofluorescent staining of spinal cord sections from mice at EAE peak and control mice with anti-F4/80 (green), TOP1 (magenta), and nuclei with Hoechst (blue). For the left panels, bar = 400 μm; for the magnified panels, bar = 100 μm. The yellow arrowhead indicates a representative F4/80+ cell with high TOP1 expression.I A magnified image of the selected region (white box) in (G); bar = 50 F4/80+ cell with increased TOP1 expression in the nucleus in (G). J The mean fluorescent intensity of TOP1 expression in the white matter in (G) was graphed; n = 3 to 4 mice per group with 3-5 sections from each mouse. Data information: Data are presented as the mean ± s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001 Statistical analyses were performed using the student's unpaired two-tailed t-test.K TOP1 expression in the spinal cord homogenates of EAE mice dissected at disease peak (scored 3) or control mice were examined by Western blotting. L The quantification of TOP1 protein expression of Western blot bands in (K) with normalization to GAPDH and β-actin (n = 3 biological samples). Data information: Data are presented as the mean ± s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001 Statistical analyses were performed using the student's unpaired two-tailed t-test.M, Western blotting of TOP1 expression from brain homogenates of mice receiving intracisternal LPS injection or PBS after 8 h (n = 4 or 5 biological samples).TOP1 expression from brain homogenates of mice receiving intracisternal LPS injection or PBS after 8 h (n = 4 or 5 biological samples). Data information: Data are presented as the mean ± s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001 Statistical analyses were performed using the student's unpaired two-tailed t-test.O Microglia were sorted from LPS challenged mice (i.p) after 4 h or naive mice, and the mRNA expression of TOP1 was graphed (n = 5 or 6 biological samples). Data information: Data are presented as the mean ± s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001 Statistical analyses were performed using the student's unpaired two-tailed t-test.P Brain sections from healthy control and MS brain tissues, including the active MS lesion, chronic active lesion, and the normal-appearing white matter (NAWM), were stained with anti-HLA-DR, anti-TOP1, and co-stained with Hoechst (bar = 50 μm).