Fig. 4: Dysregulated lipid metabolism supports hypermetabolism in active UC colonoids during differentiation.

A Differentially expressed lipid-related genes (FDR < 0.1) in aUC vs C epithelial colonoids differentiated for 3 days. Dots represent the Log2 fold change in aUC compared to C colonoids. n = 8 donors/group. B BODIPY⁺ neutral lipid estimation in undifferentiated (spheroids) and 2-day differentiated colonoids by flow cytometry. n = 5 donors/group C Representative confocal images of C and aUC colonoids of BODIPY⁺ neutral lipid accumulation. Scale bar, 50 µm. D LPL protein activity in C and aUC colonoids after a 2-day and 5-day differentiation. n = 5 donors/group. Boxplot represents the first, second (median), and third quartiles with whiskers representing the minimum and maximum points. E Volcano plot from lipidomics analysis of aUC vs C colonoids differentiated for 3 days showing 96 lipid metabolites differentially upregulated (red dots) in aUC vs C. n = 3 donors/group F Heatmap of the top 50 lipid classes differentially abundant in aUC vs C colonoid lipidomics analysis. n = 3 donors/group. G Schematic of Etomoxir treatment in C and aUC colonoids differentiated for 3 days. Partly Created in BioRender. Ojo, B. (2025) https://BioRender.com/nwwqemf . H Bioenergetic profile of C and aUC colonoids as in (G). n = 8 donors/group, mean ± SEM I Basal OCR, J Proton leak, and K ATP-linked OCR response of C and aUC colonoids as in G. n = 8 donors/group. L Chemokine genes in non-IBD colonoids exposed to 200 µM BSA-PA or BSA-EtOH for 3 days before further differentiation up to 5 days. n = 4 donors/group, mean ± SEM. M Schematic of colonoids from non-IBD donors exposed to BSA-EtOH control or 200 µM BSA-PA with or without etomoxir for 3 days before further differentiation up to 5 days. N Extracellular CXCL1 in the last 2 days of non-IBD colonoids treated as in M. n = 4 donors/group. Statistics: (B, D, I–K)- Two-way ANOVA with Tukey’s test in B, I–K, and Holm-Sidak test in D. E- Moderated T-test with Benjamini-Hochberg correction (L)- two-sided paired t-test. N- RM One-way ANOVA with Holm-Sidak test. P values are indicated in the figures. BSA bovine serum albumin, Eto etomoxir, OCR oxygen consumption rates, PA, Palmitic acid, Veh vehicle.