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tra12159-sup-0001-FigureS1.tifTIFF image527KFigure S1. E. coli, which must be translationally active, rapidly inhibits RBL-2H3 mast cell degranulation. A) RBLs were plated in 96-well plates 24 h prior to being cultured with increasing MOIs of E. coli for 2 h. Following sensitization with anti-DNP IgE for 1 h and stimulation with DNP-BSA for the indicated times, β-hexosaminidase release was quantified. Total intracellular β-hexosaminidase content was determined after E. coli co-culture for each MOI (inset). B) RBLs were cultured with or without E. coli as described in panel A, but stimulated with 1 µm PMA/1 µm ionomycin and β-hexosaminidase release was quantified. Graphs depict the mean ± standard deviation of five independent experiments conducted in duplicate. The amount of β-hexosaminidase released at 60 min in the control population was considered 100%. C) RBLs were plated in 96-well plates 24 h prior to being cultured with or without (control) heat-killed E. coli (left), chloramphenicol-treated E. coli (center) or E. coli-conditioned medium (right) for 2 h. The cells were then sensitized with anti-DNP IgE for 1 h, stimulated with DNP-BSA for 1 h and β-hexosaminidase release was quantified. The amount of β-hexosaminidase released at 60 min in the control population was considered 100%. Graphs depict the mean ± standard deviation of three independent experiments conducted in triplicate. In all graphs, asterisks denote statistically significant p values, where * ≤ 0.05 and ** ≤ 0.01.
tra12159-sup-0002-FigureS2.tifTIFF image551KFigure S2. FcεRI surface expression is not affected by E. coli exposure. A) RBLs were seeded on coverslips 24 hr prior to being incubated with (right panel) or without (left panel, control) E. coli at an MOI of 10 000 for 2 h. The cells were then washed, fixed and stained with anti-FcεRI and anti-mouse IgG1 AlexaFluor488-conjugated antibodies. Nuclei were stained with Hoechst. Surface levels of FcεRI were determined using immunofluorescence microscopy. Scale bars = 20 µm, representative of three independent experiments. B) RBLs were cultured with or without E. coli at an MOI of 10 000 either once (left panel) or thrice (right panel), labeled as described in panel A and surface levels were quantified using flow cytometry. Graphs represent the mean fluorescence intensity of FcεRI staining from three independent experiments ± standard deviation.

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