Extended Data Fig. 7: Microglial Hex secretion and uptake by other cell types.
From: Microglia–neuron crosstalk through Hex–GM2–MGL2 maintains brain homeostasis
A) Experimental scheme of microglia culture and Hex activity measurement in cell culture supernatants. B) Immunoblot of culture supernatant and exosome isolates for TSG101 (exosomal marker). C) Diagram of major protein secretion pathways and their molecular inhibitors. ER: endoplasmic reticulum, EGTA: ethylene glycol-bis(β-aminoethyl ether)-N,N,N’,N’-tetraacetic acid, MVB: multivesicular body. D) D)-F) Hex activity in microglial supernatants treated with indicated concentrations of Brefeldin A (D), Vacuolin-1 and/or Ionomycin (E), or EGTA, Thapsigargin, and BAPTA-AM (F). G) Enzyme activity in conditioned media (CM), heat-inactivated CM (hiCM), and unconditioned media (non-CM). H) Hex activity in lysates of Hexb+/− NPCs treated with CM, hiCM, non-CM, or CM added to wells without cells (“no cells”) for 24 h. I) Top: Experimental setup using transwell inserts. Bottom: Ηεξ activity in lysates of Hexb−/− and Hexb+/− NPCs after co-culture with Hexb+/+ microglia ± Brefeldin A (BFA) J) Immunoblots of NPC lysates after 6 h treatment with recombinant Hex-His. K) GM2 levels in NPC lysates after CM treatment. L) Experimental scheme for primary Hexb−/− fibroblast culture. M) Immunoblots of fibroblast lysates after 6 h treatment with recombinant Hex-His. N-O) Enzyme activity in fibroblast lysates ± recombinant enzyme and following pretreatment with mannose-6-phosphate (M6P), M6P receptor (M6PR) antibody, wortmannin, or EIPA. P) Immunocytochemistry if His-tagged enzyme in Hexb−/− fibroblasts (vimentin+, His+). Triangles point to intracellular His+ inclusions. Q) Schematic of chimeric organotypic hippocampal slice cultures (OHSCs). R) Hex activity in OHSC supernatants, exosomes, and exosome-depleted fractions from Hexb+/− and Hexb−/− slices ± clodronate treatment and microglia transplantation. S) Correlation of enzyme activity with microglia density. (Spearman r, p values from two-tailed t-test). T) Immunohistochemistry for NeuN (red) and HEXB (brown) counterstained with hematoxylin (Htx) in microglia-transplanted Hexb−/− slices at d17. Graphs display greyscale intensity profiles. U) Immunohistochemistry and quantification of IBA1+ microglia in OHSCs at d17. Data shown as mean ± s.e.m. Statistical analyses: one-way ANOVA with Tukey’s post hoc test (D-I,K,O); two-way ANOVA with Sidak’s test (O,R). Illustrations in a, c, i, l and q were created using BioRender (https://biorender.com).
