ETBF, through BFT, increases glucosylceramide levels in mice and in colonoids. Colonization with C57BL/6J mice for 1 week with ETBF increases glucosylceramide levels in the distal colon (A). Treatment of colonoids with concentrated bacterial culture supernatant from ETBF (BFT) significantly increases glucosylceramide levels at 24 h when compared to concentrated bacterial supernatant from ETBFΔbft (Control) (B). Addition of purified Bacteroides fragilis toxin (Purified BFT2, isolated from ETBF strain 86-5443-2-2, see Materials and Methods for details) to colonoids increases glucosylceramide production (C). Treatment of colonoids with BFT increases mRNA expression of glucosylceramide synthase (GCS) at 3 and 6 h, as measured by qPCR (D). Group comparisons were performed using a one-way ANOVA and Tukey’s multiple comparisons test, while single comparisons were made using an unpaired t-test. Statistical significance is indicated by asterisks: *P < .05, **P < .01, or ***P < .001. Error bars represent the standard deviation of the mean. Sham represents PBS control. NT indicates no treatment was added. Control represents concentrated bacterial culture supernatant from ETBFΔbft, BFT represents concentrated bacterial culture supernatant from ETBF, and Purified BFT2 represents purified BFT from ETBF strain 86-5443-2-2 (see Materials and Methods)