Fig. 2: Microglial Tbk1 deletion in vitro leads to stimulus-dependent over-activated or dampened responses.

a Setup for transcriptional analysis of neonatal mouse microglia in vitro. Microglia are isolated from P0 mouse brains with microglial specific full Tbk1 deletion (BAC-Cx3cr1-Cre^MMRRC/Tbk1^fl/fl, called Tbk1-µG-KO) and compared to microglia from littermates with only one Tbk1 copy deleted (Tbk1-µG-HET) or control littermates (Tbk1-µG-WT). Microglia are treated with pro- (LPS, poly I:C) or anti-inflammatory (IL4) stimulants, or not treated (unstimulated, Control), followed by RNAseq analysis. Note, cultures are >95% pure IBA1⁺ microglia (see Supplementary Fig. 3f, g). Created in BioRender. Lenoel, I. (2025) https://BioRender.com/ct8qnd6. b RNAseq results showing the number of genes significantly regulated (DESeq2, log₂FC ≥ 1.0, adjusted p values (FDR) < 0.05, see “Methods”) in Tbk1-µG-KO vs Tbk1-µG-WT microglial cultures after no stimulation (Control), LPS, poly I:C, or IL4 stimulation. n = 5 independent experiments. c, e Principal component analysis (PCA) of RNAseq data of Tbk1-µG-KO, Tbk1-µG-HET, and Tbk1-µG-WT microglia after LPS (c) or poly I:C stimulation (e). d, f Gene set enrichment analysis (GSEA) showing regulation of immune and other (Hallmark) pathways between Tbk1-µG-KO and Tbk1-µG-WT microglial cells, after LPS (d) or poly I:C stimulation (f) (adjusted p values < 0.05, NES normalized enrichment scores specified for each pathway and ≥ ±1.5, see “Methods”). Note, for LPS/Tbk1-µG-KO, all inflammatory pathways found were downregulated (dampened), and the 4 shown are among the top downregulated pathways. For poly I:C/Tbk1-µG-KO, all inflammatory pathways found were upregulated (overactivated), and the 4 shown are among the top upregulated pathways. Effect of each stimulus on each pathway in the control condition is represented by a red up arrow (↑) for pathways activated in Tbk1-µG-WT LPS vs unstimulated microglia (see Supplementary Fig. 3k), and a black equal sign (=) for unaltered pathways.