Fig. 3

SP140 knockdown reduces the activity of the inflammatory macrophages. a Scheme of polarization protocol of human CD14⁺ monocytes to “M0,” “M1,” and “M2” macrophage phenotypes, with indicated cytokines as described in the “Methods” section. b Relative gene expression of SP140 in “M0,” “M1,” and “M2” macrophages, n=6. c Immunofluorescence staining of SP140 speckles in “M1” and “M2” macrophages imaged by microscopy (left) and quantified per nuclei as nuclear bodies count (right). Images were counted in 150 cells selected randomly from 3 different staining per condition (Total number of SP140 speckled in 150 cells/150 = average per cell), scale bar: 30 μm. d Scheme of SP140 silencing protocol as described in “Methods” section. e The efficiency of SP140 silencing was assessed by measuring relative gene expression (qPCR) of SP140 (n=6) and f immunofluorescence staining of SP140 speckles nuclear bodies (left) and SP140 speckled nuclear bodies count (counted as described above) (right), scale bar: 3 μm. g Relative gene expression after LPS stimulation: (top, n=6), and LPS-induced protein levels (bottom, n=3) n=3, of TNF, IL-6, and IL-8. h PCA of gene expression dataset of LPS induced genes in scrambled- or SP140 siRNA-treated “M1” macrophages (unstimulated or stimulated with 4 h 100 ng/mL LPS) assessed through microarray; PC1 represents most variance associated with the data (LPS stimulation) and PC2 represents second most variance (siRNA), n=3. i Heatmap of top 50 DEGs from microarray gene expression dataset of LPS induced genes in scrambled- or SP140 siRNA-treated “M1” macrophages (unstimulated or stimulated with 4 h 100 ng/mL LPS) (non-annotated genes were not included). j Hallmark pathways analysis and k Reactome pathway analysis were carried out using ShinyGO v0.60 illustrating the most impacted pathways by SP140 siRNA in unstimulated “M1” macrophages (bottom) or after 4 h of 100 ng/mL LPS stimulation (bottom). In all assays, statistical significance is indicated as follows: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001