FIGURE 2.

A selective inhibitor of glucosylceramide synthase (GCS) decreases glucosylceramide lipid levels. The treatment regimen for the administration of 5 μmol/L ibiglustat to colonoids is depicted (A). Addition of 5 μmol/L ibiglustat effectively reduced total levels of glucosylceramide (B). Colonoids were pre-treated with 5 μmol/L ibiglustat for 24 h and then treated with C6-ceramide nanoliposomes (CNL) for 1 hour to determine the effectiveness of ibiglustat in blocking GCS activity. Colonoids were collected and lipids were extracted. Presence of ibiglustat completely blocks the ability for colonoids to process C6-ceramide into C6-glucosylceramide, demonstrating the efficacy of ibiglustat at inhibiting GCS activity (C). Pre-treatment of colonoids with 5 μmol/L ibiglustat followed by the addition of concentrated bacterial supernatant from ETBFΔbft does not alter colonoid morphology, visualized by confocal microscopy (D). The treatment regimen for the administration of 5 μmol/L ibiglustat and concentrated bacterial culture supernatant is depicted (E). Single comparisons were made using an unpaired t-test. Statistical significance is indicated by asterisks: ***P < .001. Error bars represent the standard deviation of the mean. Vehicle represents vehicle control (see Materials and Methods). Confocal images were captured using 10x magnification. Scale bar indicates a distance of 100 μm