Fig. 5: Oncogenic truncated APC modifies the organization of Wnt signaling plasma membrane domains.
A Di-4-stained crypts from AfGC mice. B Quantitative analysis of di-4 from crypts and C their derived GPMVs (blebbing, left white arrow; GPMVs, right white arrow; whole cells green arrow), D CSCs, E human CRC cells and F their derived GPMVs, and G PDOs. For all d-i4 experiments, error bars represent B n = 3–6 crypts (16♂,15♀), C n = 3–15 GPMVs (15♂,14♀), D n = 5–13 CSCs (15♂,14♀) from mice or E cells and F their derived GPMVs from n = 3 independent biological replicates or G n = 17–31 FOV (2–3 PDOs per FOV) (mean ± SD, n value shown in each graph). Number of crypts, CSCs, cultured colonocytes, and their GPMVs, and FOVs analyzed are provided below each bar. Statistical significance was determined by one-way ANOVA and post hoc Tukey’s test. Different letters indicate significant differences between WT and mutant APC groups (P < 0.05). H Effects of oncogenic APC on plasma membrane cholesterol organization. Cells co-expressing fluorescently-labeled I D4H and tH or J Lypd6 were used to for FLIM-FRET analyses. K Quantitative analysis of IL cholesterol. D4H-EGFP plasma membrane fluorescence intensity was normalized to total D4H-EGFP fluorescence intensity. Cells were pre-treated with mevastatin (5 µM, 24 h), MβCD (10 mM, 30 min), or cholesterol (2 mM, 30 min) and subsequently incubated with control or Wnt3a-conditioned media (30 min). FRET efficiency was calculated from FLIM data averaged per FOV (mean ± SD, n = # FOVs provided below each bar, FOV containing 2–6 cells). For flow cytometry IL cholesterol experiments, error bars represent n = 3 independent biological replicates (mean ± SEM, cell number provided below each bar). Statistical significance determined by H–K two-way ANOVA and post hoc Tukey’s. Different letters indicate significant differences between control and experimental groups (P < 0.05). L Quantitative analysis of Ld and Lo ___domain stability. The percentage of phase separated GPMVs was determined by GPMVseparated/GPMVtotal ratio (mean ± SEM, n = 12 FOVs, analyzed GPMV number shown in graph). Representative images and scale bars are provided for microscopy data. A sigmoidal four parameter logistic regression model was used to determine Tmisc. Source data file provided.
