Extended Data Fig. 4: Profiling of A. muciniphila supernatant fractions that induced glucagon-like peptide-1 (GLP-1) secretion.
a, Schematic workflow used to identify GLP-1-inducing fractions derived from A. muciniphila. b, The cell-free supernatant (CFS) of A. muciniphila was fractionated using molecular size cut-off filters, as indicated. A volume of each fraction containing 5 mg of protein was used to treat NCI-H716 cells. c, 100–300 kDa filtrate and the 30–100 kDa filtrate of A. muciniphila were treated with proteinase K (100 µg ml−1) as indicated (+PK). A control sample was used that comprised BHI broth containing 5% FBS and was treated with proteinase K. The effects of each on GLP-1 expression was then analyzed. d, 100 K filtrates of control media and A. muciniphila were separated by ion exchange fast protein liquid chromatography (FPLC) and 26 fractions (m1–m26) were obtained. Each fraction was used to treat NCI-H716 cells and GLP-1 secretion was analyzed by ELISA. e, Concentrates of the m2–m4 fractions of Control and SNUG-61027 were separated by size columns (to generate fractions G1–G34) using the FPLC system, then the procedures listed above were followed. f, Venn diagram of the identified proteins. Sample 1, Sample 2, and Sample 3 are the 100 K concentrates obtained from size cut-off filtration, concentrates from the ion exchange chromatography, and concentrates from the size-exclusion chromatography, respectively. g, SDS-PAGE gel of the expressed proteins were processed in parallel in the same gel. h, Nine proteins identified by LC-MS/MS from Sample 3. Nine candidate proteins were cloned using an E.coli expression system according to the protein sequences for the SNUG-61027 strain. The sequence similarity for each protein between SNUG-61027 and ATCC-BAA-835 was analyzed. Data are presented as the means ± SEMs. The data in b–e represent the results of triplicate experiments and were analyzed using the Kruskal-Wallis test, followed by Dunn’s post-hoc test.
