Fig. 2. SL production by BTWT regulates SL metabolism genes.

a Incorporation of palmitic acid alkyne (PAA) into complex SLs allows fluorescent detection of bacterially-derived lipids using an azide (N₃) conjugated fluorophore (Alexa Fluor 647). b Metabolism of PAA in BTWT vs. SLMUT. SPT = serine palmitoyltransferase. c Illustration of the transwell coculture system, showing transfer of PAA derivatives from bacterial cells in upper well to Caco-2 cells in lower well. d Confocal image showing alkyne-tagged lipids detected in BTWT cells grown with PAA (red - Alexa Fluor 647; detection by click chemistry). Scale bar is 20 μm. Confocal microscopy image of Caco-2 cells in the lower transwell after 6-h exposure to (e) cell culture media (f) BTWT-PAA or (G) SLMUT-PAA. e–g Alkyne-tagged lipids were detected by Alexa Fluor 647 (red) and DNA was stained using DAPI (blue). Images are representative of three independent experiments. Scale bar is 20 μm. f Heatmap showing the average time-zero normalized log₂ change in gene expression between BTWT and SLMUT in transwell with Caco-2 cells for two biological replicate 8-h time course experiments (yellow = higher, blue = lower for log₂ BTWT/SLMUT relative expression). Source data are provided as a source data file.