Fig. 7

AhR-SYK signaling mediates phagocytic uptake of microglia. A ChIP-qPCR analysis was performed in WT microglia to detect the binding of promoter sequences for AhR to SYK (n = 3 biological replicates). The precipitated chromosome segment was PCR-amplified with the use of 5 pairs of specific primers in the SYK promoter. The rlgG is normal rabbit IgG, the homotype control antibody of AhR antibody. Data are shown as mean ± SEM and analyzed by unpaired two-tailed t-test. B mRNA expression of Syk in primary microglia cultured in vitro and treated with DMSO or AhR agonist I3S (n = 4 biological replicates). Data are shown as mean ± SEM and analyzed by unpaired two-tailed t-test. C Western blot analysis of SYK protein level in primary microglia treated with DMSO or AhR agonist I3S. 30 μg protein was loaded per well. D The quantification of SYK protein expression in C (n = 4 biological replicates). Data are shown as mean ± SEM and analyzed by unpaired two-tailed t-test. E Representative confocal immunofluorescent images displaying SYK expression in the corpus callosum of control mice and cuprizone-treated mice (after a 5-week cuprizone administration). Scale bar: 40 μm. F Representative western blot analysis of SYK protein level in the corpus callosum of control mice and cuprizone-treated mice (in the context of the end of week 5 in the cuprizone model). 30 μg protein was loaded per well. G The quantification of SYK protein expression in F (n = 4 mice per group). Data are shown as mean ± SEM and analyzed by unpaired two-tailed t-test. H Representative confocal immunofluorescent images displaying co-staining of SYK (green) and Iba1 (red) in the corpus callosum of mice after 5 weeks of cuprizone treatment. Scale bar: 40 μm. I Flow cytometry analysis of the phagocytosed pHrodo green bioparticles by HMC3 cells treated as indicated (n = 3 biological replicates). J The quantification of positive cells engulfing pHrodo green bioparticles and FITC fluorescent intensity in each group (n = 3 biological replicates). Data are shown as mean ± SEM and analyzed by one-way ANOVA with Tukey's multiple comparisons test. ***p < 0.001