Fig. 6: Psen2 N141I mutation selectively elevates production of clock-controlled cytokines through the downregulation of REV-ERBα in innate immune cells.

a Analyses of REV-ERBα protein levels by western blotting in microglia (n = 3) (^##p = 0.0028) and BMDM (n = 3) (^#p = 0.042). b Primer target regions. Average fold enrichment of immunoprecipitated DNA fragments in microglia was analyzed by qRT-PCR in ChIP assay. Average fold enrichment was normalized to IgG of each gene (n = 6) (WT vs KI/+: ^#p = 0.0106 and ^#p = 0.0338; n.s., not significant; unpaired t-test). c Relative REV-ERBα expression levels in microglia transduced with lentivirus expressing shCon or shNr1d1 (n = 4) (*p = 0.0423; unpaired t-test). d, e Secretion (d; n = 3) and mRNA levels (e; n = 4) of IL-6 and TNF-α in WT microglia transduced with shNr1d1 after LPS (1 μg/mL) treatment for 12 h (untreated WT vs LPS-treated WT: ***p < 0.0001, ***p < 0.0001, ***p < 0.0001, and ***p = 0.0002; untreated KI/+ vs LPS-treated KI/+: ^†††p < 0.0001; LPS-treated WT vs LPS-treated KI/+: ^###p < 0.0001 and ^###p = 0.0001; one-way ANOVA). f Relative REV-ERBα expression levels in KI/+ microglia expressing human NR1D1 (n = 3) (^††p = 0.005; unpaired t-test). g, h Secretion (g; n = 3) and mRNA levels (h; n = 4) of IL-6 and TNF-α in NR1D1-overexpressing KI/+ microglia after LPS (1 μg/mL) treatment for 12 h (untreated WT vs LPS-treated WT: ***p < 0.0001; untreated KI/+ vs LPS-treated KI/+: ^†††p < 0.0001; LPS-treated WT vs LPS-treated KI/+: ^###p < 0.0001; one-way ANOVA). Signals on western blots were normalized to those of ACTB. mRNA levels were normalized to Actb. Data are mean ± SEM. *p < 0.05, and ***p < 0.001 vs untreated WT control. ^††p < 0.01, and ^†††p < 0.001 vs untreated KI/+ control. ^#p < 0.05, ^##p < 0.01, and ^###p < 0.001 for the indicated comparisons. Source data are provided as a Source Data file.