Fig. 5: ADNP knockdown (KD) affects endocytic trafficking in iPSC-derived microglia (iTF-Microglia).

A Volcano plot showing the change in expression as measured by RNA-sequencing for individual genes in iTF-Microglia with ADNP KD compared to iTF-Microglia with non-targeting control (NTC) sgRNAs. RNA-sequencing data extracted from a CROP-seq dataset, see Methods for detail. Significantly differentially expressed genes (DEGs) are noted in dark gray or color-coded based on their corresponding categorization in enrichment analyses (B) and genes with no statistically significant change are noted in light gray (DEGs, Padj < 0.05, two-tailed Student’s t-test). B GO Cellular Component enrichment analyses of DEGs with increased expression in iTF-Microglia with ADNP KD. Terms representing unique gene sets and with Padj <0.05 are displayed. Terms are color-coded such that terms of the same color have many overlapping genes. C Scatterplot comparing fold changes at the transcriptomic and proteomic levels when comparing iTF-Microglia with ADNP KD to iTF-Microglia with NTC. Some significantly differentially abundant proteins are noted in black and labeled. D Venn diagrams showing the overlap in differentially expressed genes and proteins that are increased or decreased in iTF-Microglia with ADNP KD. E (Top) Schematic of the biology of dextran-Alexa488 endocytosis. (Bottom) Endocytic load within iTF-Microglia with ADNP KD or NTC and with or without endocytosis inhibitor, Dynasore, as measured by flow cytometry (N = 2–3 wells, bars represent mean +/− standard deviation, 2 way ANOVA). F (Top) Schematic of the biology of dextran-pHrodo endocytosis. (Bottom) Endocytic acidification within iTF-Microglia with ADNP KD or NTC and with or without V-ATPase inhibitor, BafilomycinA, as measured by flow cytometry (N = 3 wells, bars represent mean +/− standard deviation, 2 way ANOVA). G (Top) Schematic of the biology of Lysotracker. (Bottom) Acidic vesicles within iTF-Microglia with ADNP KD or NTC and with or without Dynasore and BafilomycinA as measured by flow cytometry (N = 3 wells, bars represent mean +/− standard deviation, 2 way ANOVA). H (Top) Schematic of the biology of lysosensor. (Bottom) Lysosomes within iTF-Microglia with ADNP KD or NTC as measured by flow cytometry (N = 3 wells, bars represent mean +/− standard deviation, two-tailed Student’s t-test). I TREM2 concentration of cell lysates or media collected from iTF-Microglia with ADNP KD or NTC measured using a Homogeneous Time Resolved Fluorescence (HTRF) assay (N = 3 wells, bars represent mean +/− standard deviation, t-test). J Relative abundances measured using the integrated intensity from a cytokine array for proteins that have a trend for differential abundance between iTF-Microglia with NTC or ADNP KD (N = 2 wells, bars represent mean +/− standard deviation).