Supporting Information S1 - Production of α-Galactosylceramide by a Prominent Member of the Human Gut Microbiota

Contents: Section S1, materials, equipment, and general methods. Section S2, high-resolution mass spectrometry and LC-MS analysis. Section S3, in vitro titration data. Section S4, spectral data. Figure S1, B. fragilis Δ2461 is deficient in the production of sphingolipids. LC-MS trace with extracted ions shown [M-H]. See Figure 2 legend for details. Figure S2, α-GalCerBf binds CD1d in vitro. Hybridomas were stained with anti-CD3 antibodies and empty mCD1d tetramers or mCD1d tetramers loaded with α-GalCerBf or KRN7000. Flow cytometry plots representative of three independent experiments are shown. (A) Plots show forward (FSC) and side (SSC) scatter of all events. (B) Plots pre-gated as shown in (A) and further gated on DAPI-negative events show staining with tetramer and CD3 antibodies. Figure S3, KRN7000 and α-GalCerBf titration in vitro. BMDCs and NKT hybridomas were cultured at a 3∶1 hybridoma:BMDC ratio and the indicated doses of KRN7000 or α-GalCerBf in the presence of 1 µg/ml LPS. Supernatants were harvested after 24 h and IL-2 production was measured by ELISA. Table S1, Primers used in this study. (PDF)