Fig. 4: Proteomic and transcriptomic analysis of human isogenic ApoE3 and ApoE4 iAstrocytes.

a,b, Volcano plots present log₂ fold changes in protein levels in ApoE4 versus ApoE3 iAstrocytes from Kolf2.1J set 1 (a) and BIONi037 (b). The top ten proteins with the highest log₂ fold changes and top ten proteins with the most significant P values are labeled (N = 4 wells per genotype). Statistical analysis was performed using a two-sided pairwise t-test. c, Number of DEPs (fold change > 1.5, FDR < 0.05) detected in ApoE4 versus ApoE3 iAstrocytes of Kolf2.1J (set 1) and BIONi037 isogenic sets. d, Relative ApoE protein levels in ApoE3 and ApoE4 iAstrocytes (from proteomic analysis) from BIONi037 and Kolf2.1J background. Data are shown as the mean and s.d. *P < 0.05 (two-sided Mann–Whitney U-test). e,f, Venn diagrams depicting the number of DEPs significantly upregulated (e) or downregulated (f) (fold change > 1.25, log₂ fold change > 0.3) in Kolf2.1J, BIONi037 or both ApoE4 iAstrocytes. A Reactome ORA was performed on the 105 common upregulated (e) or 109 common downregulated (f) proteins and the enrichment ratio was plotted for all significant pathways (FDR < 0.05). g, Schematic overview of interferon-dependent regulation of MHC class I antigen presentation (in blue) and immunoproteasome (in green) pathways. The heat map indicates the log₂ fold change of indicated proteins in ApoE4 versus ApoE3 iAstrocytes (proteomics). PM, plasma membrane. This image was created using BioRender.com. h,i, Representative western blot (h) and quantification (i) of MHC I levels (stained for HLA class I heavy chain) in ApoE4 versus ApoE3 iAstrocytes (N = 10; five independent experiments from two isogenic sets). Data are shown as the mean. *P < 0.05 (one-sample t-test). j,k, Representative histogram (BIONi037) (j) and quantification (k) of plasma membrane MHC I levels (stained for HLA-A, HLA-B and HLA-C) by flow cytometry (N = 9; five (Kolf2.1J) or four (Bi037) independent experiments from two isogenic sets). Data are shown as the mean. Unst, unstained control. l,m, Example images (l) and quantification (m) of MHC I levels as measured by immunofluorescence microscopy (stained for HLA class I heavy chain) (N = 14; seven independent experiments from two isogenic sets). Data are shown as the mean. *P < 0.05 (two-sided one-sample t-test). Scale bar, 50 μm. n, Comparison of significant Reactome pathways (by gene set enrichment analysis) from our transcriptomic analysis of ApoE4 versus ApoE3 astrocytes (BIONi037) with previously published datasets. Shown is the average log₂ fold change of all measured genes in the indicated pathway in each isogenic set or case–control set. Tcw et al. ind1–ind4 (four different isogenic sets) and population (control versus ApoE4) represent iAstrocytes from a previous study¹⁰. Lin et al. represents one isogenic set of ApoE4 versus ApoE3 iAstrocytes from a previous study⁸. F, female; M, male. o, Heat map shows the log₂ fold change in individual genes in the MHC I and immunoproteasome pathway across indicated studies, including our data here. Open triangles indicate the mean per experiment, while solid dots represent all independent wells.

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