Fig. 2: Oncogenic truncated APC disrupts plasma membrane cholesterol homeostasis.

A Representative images of colonocytes stained with filipin III and PM stain. Scale bar: 50 µm. Quantitative analysis of cholesterol levels in B mouse-, C human CRC-cultured colonocytes, D, E their derived GPMvs and their respective representative flow cytometry images. Scale bars: 20 µm. Quantitative analysis of cholesterol levels in GPMVs. Error bars represent cells or GPMVs from n = 3 independent biological replicates (mean ± SD). Number of events analyzed using flow cytometry is shown below each bar. Statistical significance was determined by B, D two-way ANOVA or C, E one-way ANOVA and post Tukey’s multiple comparison test. RNAseq analysis from F IMCE βcat and YAMC colonocytes (n = 3 independent biological replicates per group), G mouse colonic crypts isolated from AfGC and GC mice (n = 4 mice per group, equal number of males (♂) and females (♀)), and H n = 36 human paired samples (18 normal and 18 CRC). F–H Enrichment of the cholesterol homeostasis pathway genes in AfGC compared to GC samples. I–K Volcano plot illustrating differentially expressed genes (FDR ≤ 0.05; top 15 genes are listed) (ES, enrichment score; FDR, false discovery rate; FC, fold change). L–N Gene set enrichment analysis in AfGC compared to GC samples. Gene sets were ranked by normalized enrichment score (NES). O Differentially expressed genes corresponding to Wnt/βcat signaling. For all RNA expression experiments, statistical significance was determined by EdgeR-robust in several contrasts and Benjamini-Hochberg (BH) FDR (P < 0.05). P Normalized fitted counts showing mRNA levels of differentially expressed genes associated with cholesterol efflux, uptake, and esterification assessed by RNAseq analysis. Statistical significance determined by two-way ANOVA and post Tukey’s multiple comparison test. Error bars represent n = 3 independent biological replicates (mean ± SD). Q Validation of RNAseq data via western blot normalized to β-actin. Results were used to calculate a relative ratio using YAMC (Apc +/+) as a control. In all cases, when provided, different letters indicate significant differences between WT APC (control) and treated/mutant APC (experimental) groups (P < 0.05). Source data are provided as a Source data file.