Figure 4

Disruption of AUTS2 led to the overactivation of the WNT/β-Catenin signaling. (A) The volcano plot illustrates the results of IPA, depicting the z-score of log₂ fold change in gene expression between WT and AUTS2-/- cerebral organoids against the log₁₀ p value. Pathways overactivated in WT are highlighted in red, while those overactivated in AUTS2-/- cerebral organoids are shown in blue. (B) A bar plot illustrates the outcomes of a TOP-Flash/FOP-Flash luciferase reporter assay comparing WT and AUTS2-/- HEK293T cells. Each bar represents the mean of three independent measurements, with error bars indicating the standard error. One-way ANOVA was performed using GraphPad Prism to analyze the results and statistical significance is indicated as ** (p < 0.01). (C) An immunoblotting assay showing the levels of the AUTS2 protein in both WT and AUTS2-/- HEK293T cells, with and without CHIR-99021 treatment. (D) The immunoblotting experiment reveals the protein levels of AUTS2 and MAP2 in WT, AUTS2+/-, and AUTS2-/- cerebral organoids, with and without XAV939 treatment. Beta tubulin serves as the loading control. For immunoblotting, at least three cerebral organoids for a total of three biological replicates were pooled (n = 3). (E) Heatmap of gene expression for selected ChP, neuroepithelial, and neuron markers between AUTS2-/- 75-day-old cerebral organoids with or without XAV939 treatment. Selected neuronal marker genes and ChP epithelial marker genes are annotated. (F) The RT-qPCR results indicate the rescue in the expression levels of ChP marker genes (TTR, FOLR1, and SLC4A5) and neuronal markers (FOXG1, CTIP2, and BRN2) by XAV939 in AUTS2-/- cerebral organoids. Student T-test was performed, with statistical significance indicated as * (p < 0.05) and ** (p < 0.01). For RT-qPCR and RNA-seq analyses, three biological replicates were used (n = 3), each including 3–5 organoids.