Fig. 3: INPP5D inhibition induces changes in pathways associated with innate immunity. | Nature Communications

Fig. 3: INPP5D inhibition induces changes in pathways associated with innate immunity.

From: INPP5D regulates inflammasome activation in human microglia

Fig. 3

a, b iMGs were treated with vehicle (ethanol) or 3AC (1.25 μM) for 6 h. a Cells were then lysed, RNA purified, and RNAseq performed, n = 6 per condition. Volcano plot of DEGs comparing vehicle and 3AC treatment conditions. b Cells were lysed in urea and TMT-MS performed. n = 4 per condition. Volcano plot of DEPs; For a, b differential expression was calculated using a linear modeling with empirical Bayesian statistical analysis using the limma package in R. All p-values are adjusted using the Benjamini Hochberg (BH) procedure. c Heatmap of expression of DEGs that showed concordant differential expression at the RNA and protein level. d Heatmap of relative expression of DEPs encoded by LOAD GWAS candidate genes19 between vehicle and 3AC-treated iMGs. e. Relative RNA and protein levels of CD33 and PTK2B between vehicle and 3AC-treated microglia, as quantified by RNAseq and TMT-MS. Mean ± SEM. Two-sided Welch’s t-test. f, g. A variety of microglial subtypes previously have been defined through single-cell sequencing, with specific subtypes implicated to be altered in AD brain and model systems49,50,51,52,53. Volcano plots comparing 3AC vs vehicle treatment for genes defining these subtypes within the transcriptomic and proteomic datasets are shown; significance determined by BH FDR < 0.05. h Heatmap of relative protein-levels of scavenger receptors in vehicle and 3AC-treated iMGs. i, j. Enriched GO terms of biological processes for the differentially expressed genes that are elevated with 3AC treatment (geneontology.com54); Fisher’s exact test, Bonferroni multiple comparison’s test, adj p-value as shown by size of circles. j Heatmap of relative expression between vehicle and 3AC treatment of DEGs associated with NFκB signaling and JAK-STAT signaling. k Relative abundance measures of RNA or protein levels of inflammasome related components in vehicle and 3AC-treated iMGs. Mean ± SEM. Two-sided Welch’s t-test. l Representative western blot of iMGs treated with either vehicle (ethanol) or 3AC (1.25 μM) for 6 h showing protein expression of INPP5D and inflammasome related proteins: CASP1, PYCARD/ASC, GSDMD and NLRP3, as well as GAPDH. Similar WB results obtain in 3 separate differentiations. Cell lines used for experiments and full datasets for can be found in Supplementary Data 4 and 5, Supplementary Data 14. For all graphs, the number of biological replicates is represented by dots. For all panels: **p < 0.01, ***p < 0.001, ****p < 0.0001; ns = p < 0.05. See also Supplementary Figs. 5 and 6.

Back to article page