Fig. 5: Lipidomic and proteomic analysis of reactive human iAstrocytes.

a, Schematic overview of experimental design. A cocktail of TNF, IL-1α and C1q was added for 24 h to make astrocytes reactive. b, The volcano plot presents the log₂ fold change of altered individual lipid species in reactive versus control iAstrocytes (Kolf2.1J set 1, ApoE3). c, Summary data of changes in all detected lipid classes in reactive versus control iAstrocytes (N = 6; three independent experiments from two ApoE3 lines). Data are shown as the median and IQR; whiskers indicate the furthest data point within 1.5× the IQR. *P < 0.05 (two-sided paired t-test or paired Mann–Whitney U-test with BH correction). d, Fold change of all phospholipid species with indicated number of double bonds (unsaturation) in reactive versus control iAstrocytes. All experiments presented in c are included here. Data are shown as the mean. e, Number of DEPs (fold change > 1.5, FDR < 0.05) in reactive versus control iAstrocytes for indicated lines. f,g, The log₂ fold changes in protein levels of reactive versus control iAstrocytes for Kolf2.1J set 1 (f) and BIONi037 (g). The top ten proteins with the highest log₂ fold change and top ten proteins with the highest P values are labeled (N = 4 wells per genotype). Statistical analysis was performed using a two-sided pairwise t-test. h,i, Venn diagram depicting the number of proteins that were significantly upregulated (h) or downregulated (i) (fold change > 1.25, log₂ fold change > 0.3) in reactive Kolf2.1J, BIONi037 and both iAstrocytes. A Reactome ORA was performed on the 275 common upregulated or 129 common downregulated proteins. No significantly enriched downregulated pathways were observed; the enrichment ratios for all significantly (FDR < 0.05) upregulated pathways are plotted in k. j, Top, heat map depicting the log₂ fold change of indicated lipid classes in ApoE4 or reactive iAstrocytes versus ApoE3 control iAstrocytes. Lipid classes that were changed in ApoE4 or reactive iAstrocytes with P < 0.05 are shown. Bottom, heat map depicting the log₂ fold change of indicated proteins from the MHC class I and immunoproteasome pathway in ApoE4 or reactive iAstrocytes versus ApoE3 control iAstrocytes (based on proteomics data). k, Relative membrane MHC I levels (stained for anti-HLA-A, anti-HLA-B and anti-HLA-C) according to flow cytometry in reactive versus control iAstrocytes (N = 9; four (Kolf2.1J) or five (Bi037) independent experiments from two isogenic sets). Data are shown as the mean. ****P < 0.0001 (two-sided one-sample t-test). l, Schematic representation of opposing lipidomic and proteomic phenotypes in ApoE4 and reactive iAstrocytes. Open circles or triangles indicate the mean per experiment, while solid dots represent all independent wells. The images in a,l were created using BioRender.com.