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cmi12058-sup-0001-si.pdf75K

Fig. S1.B. abortus infection down-modulates the TLR agonists-induced surface expression of MHC-I. THP-1 cells were infected with B. abortus at different moi in the presence of IFN-γ (A), E. coli LPS (B), Pam3Cys (C), CpG (D) and Poly I:C (E). MHC-I expression was assessed by flow cytometry. Bars represent the arithmetic means ± SEM of three experiments. MFI, mean fluorescence intensity. Histograms correspond to one representative of three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001 versus MHC-I upregulator.

cmi12058-sup-0002-si.pdf22K

Fig. S2. MHC-I expression was decreased in cells with and without B. abortus at 48 h. THP-1 cells were infected with GFP-B. abortus at different moi in the presence of IFN-γ for 2 h, washed, and cultured in the presence of IFN-γ for 48 h. MHC-I expression was assessed by flow cytometry, using W6/32 mAb as a primary Ab and PE-labelled secondary Ab. Bars represent the arithmetic means ± SEM of three experiments. MFI, mean fluorescence intensity. **P < 0.01; ***P < 0.001 versus IFN-γ. MFI of IFN-γ: 51 ± 2.

cmi12058-sup-0003-si.pdf62K

Fig. S3. Downregulation of MHC-I surface expression is not mediated by B. abortus LPS. THP-1 cells were incubated with B. abortus LPS (BruLPS) (100 ng ml−1, 1000 ng ml−1 and 5000 ng ml−1) (A) or were infected with B. abortus or B. abortus RB51 at moi 100:1 (B), in the presence of IFN-γ for 2 h, washed, and cultured in the presence of IFN-γ for 48 h. MHC-I expression was assessed by flow cytometry. Histograms correspond to one representative of three independent experiments. Non-specific binding was determined using a control isotype Ab.

cmi12058-sup-0004-si.pdf28K

Fig. S4. Infection with a VirB mutant also inhibits MHC-I surface expression. (A) THP-1 cells were infected with B. abortus or the virB10 mutant of B. abortus at moi 100:1 in the presence of IFN-γ for 2 h, washed, and cultured in the presence of IFN-γ for 48 h. MHC-I expression was assessed by flow cytometry. Histograms correspond to one representative of three independent experiments. Bars in (B) indicate the arithmetic means ± SEM of three experiments. MFI, mean fluorescence intensity. Non-specific binding was determined using a control isotype Ab. **P < 0.01 versus IFN-γ.

cmi12058-sup-0005-si.pdf84K

Fig. S5.B. abortus infection induces retention of MHC-I molecules in Golgi apparatus. Confocal micrographs of THP-1 cells infected with B. abortus without GFP (moi 100:1) in the presence of IFN-γ. MHC-I was detected with the W6/32 mAb followed by Alexa 546-labelled secondary Ab (red). Golgi apparatus was detected using mAbs specific for GM130 followed by Alexa 488-labelled secondary Ab (green). Nucleus was detected using TOPRO-3 (blue). Results are representative of two independent experiments.

cmi12058-sup-0006-si.pdf411K

Fig. S6.B. abortus infection induces a drastic reduction of MHC-II expression.

A. THP-1 cells were infected with B. abortus at different moi in the presence of IFN-γ for 2 h, washed, and cultured in the presence of IFN-γ for 48 h. MHC-II expression was assessed by flow cytometry. Histograms correspond to one representative of three independent experiments. Non-specific binding was determined using a control isotype Ab.

B–D. Confocal micrographs of THP-1 cells treated with IFN-γ or infected with B. abortus without GFP (moi 100:1) in the presence of IFN-γ. MHC-II was detected using L243 mAb as a primary Ab and Alexa 546-labelled secondary Ab (red). Subcellular localization markers were detected using mAbs specific for EEA1 (early endosomes), LAMP-2 (late endosomes/lysosomes), GM130 (Golgi) and calnexin (ER) followed by Alexa 488-labelled secondary Ab (green) (C and D). DIC, differential interference contrast. Results are representative of three independent experiments.

E. Quantification of MHC-II expression. Data are expressed as percentage of cells with MHC-II expressed on the surface ± SEM of two independent experiments. **P < 0.01 versus IFN-γ.

cmi12058-sup-0007-si.doc31KSupporting Information

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