Fig. 2

Bin1 KD in primary microglia dysregulates proinflammatory and PU.1-dependent genes. A Bin1 siRNA transfection resulted in > 80% reduction in Bin1 transcripts, as confirmed by qRT-PCR. B PCA identified two PCs, which accounted for 71% of the variance in the dataset. PC1 captured the effect of Bin1 loss (42%), while PC3 captured the LPS effect (29%). The LPS effect shown by PC2 was blunted in the absence of Bin1. C Both PCs were increased by LPS stimulation. Bin1 KD caused a significant increase in PC1 in resting and LPS-stimulated microglia; Bin1 KD only decreased PC2 during LPS stimulation (* p < 0.05, **p < 0.01, ***p < 0.001, Dunn’s). D K-means clustering identified six gene clusters, of which five showed distinct patterns of expression based on in vitro manipulations. Cluster 1 was positively regulated by BIN1 in homeostasis, and LPS-stimulated up-regulation was BIN1-dependent. Cluster 2 was positively regulated by BIN1 during LPS stimulation, but its homeostatic regulation was not affected by BIN1. Cluster 3 was positively regulated by BIN1 (during homeostasis and LPS stimulation) but downregulated during LPS stimulation. Cluster 5 was negatively regulated by BIN1 and unaffected by LPS stimulation. Cluster 4 was not regulated by BIN1 but was upregulated during LPS stimulation (not shown in the figure). E Gene ontology enrichment analyses (GO, KEGG, Wikipathways included) identified key inflammatory and immune (clusters 1 & 2), homeostatic microglial (cluster 3), and non-microglial-specific (cluster 5) pathways affected by in vitro manipulation of primary microglial cultures. Predicted upstream transcriptional regulators for each cluster are shown, among which Sfpi1 (PU.1) was shared across clusters 1, 2, and 3