Fig. 2

APOE deficiency alters neural differentiation in cerebral organoids by activating the EIF2 signaling pathway. a–h DEGs in cell clusters were identified through the scRNA-seq and subjected to the Ingenuity pathway analysis. Volcano plot and gene ontology analyses for DEGs (APOE^−/− vs. Con) in total excitatory neuron clusters (a, b), excitatory neuron cluster 3 (c, d), radial glia clusters (e, f), and astrocyte clusters (g, h) are shown. Genes significant at the P value ≤ 0.05 and fold change ≥ 1.2 are denoted in purple. Selective DEGs are labeled in the volcano plots. i Phosphorylation levels of eIF2α in the control and APOE^−/− cerebral organoids at Day 90 were quantified by Western blotting. Three cerebral organoids were pooled and analyzed as one sample (n = 3 samples/genotype). j APOE^−/− cerebral organoids at Day 60 were treated with DMSO or ISRIB (100 nM) for 30 days. Phosphorylation level of eIF2α in the cerebral organoids was quantified by Western blotting. Three cerebral organoids were pooled and analyzed as one sample (n = 6 samples/genotype). k The mRNA levels of cerebral layer markers (DCX, SLC17A7, TBR1 and CTIP2) and astrocytic markers (S100B and GFAP) were quantified by RT-qPCR. Three cerebral organoids were pooled and analyzed as one sample. All data are expressed as mean ± SEM (n = 4–5 samples/genotype). l Representative microscopy images of cerebral organoids stained with neuronal layer V marker CTIP2 and DAPI. The immunoreactivity of CTIP2 was quantified and normalized by DAPI fluorescent intensity (n = 6 organoids/genotype). Scale bar: 50 μm. Experiments were repeated in two independently differentiated batches (i–l). All data are expressed as mean ± SEM. Student’s t tests were performed to determine statistical significance. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. For raw data see Additional file 1: Figs. S7 and S8