Results represent three independent biological replicates. h at 33C and MOI 50:1. The degree of association was determined by fluorescence microscopy. The percentage of cells with associated bacilli was plotted. Each result represents the mean SEM from three independent experiments. An ANOVA test followed by Bonferroni as a post test was performed and used for statistical analysis. ***p<0.001.(TIF) ppat.1007151.s001.tif (1.6M) GUID:?28E31CA2-9434-4FE3-90E3-72BC2348DAA4 S2 Fig: Live BCG PGL I or are more efficiently internalized by Schwann cells compared to dead bacilli. A. Internalization degree of live and dead recombinant BCG strains was determined by flow cytometry after 48 h of incubation at 33C and MOI 50:1. Bacteria were labeled with PKH67 and the degree of internalization was determined after Trypan Blue quenching. Results were represented as MFI. B and C. Internalization of Nazartinib S-enantiomer live and dead was determined by flow cytometry after 48 h of incubation at 33C and different MOIs. ST8814 SCs were either left untreated (NI) or treated with PKH67 labeled bacteria and the degree of internalization was determined after Trypan Blue quenching. A representative histogram plot of the 48 h incubation experiment is shown. Results were represented as percentage of cell population with internalized bacteria or MFI of the cell population. Each result represents the mean SEM from three independent experiments. An ANOVA test followed by Bonferroni as a post test was performed and used for statistical analysis. *p < 0.05; ***p<0.001.(TIF) ppat.1007151.s002.tif (1010K) GUID:?64FB01FE-05CA-465D-8F1B-92BD7CE70EE0 S3 Fig: The expanded phagocytic capacity induced by and BCG PGL I on SC is specific to BCG Nazartinib S-enantiomer WT and is not induced by PGL I alone. A. Flow cytometry result showing no change in the degree of internalization of PKH67 labeled BCG or latex beads when adding pure PGL I (15ng or 30ng) to the culture medium. B. Flow cytometry result showing no Mouse monoclonal to Transferrin change in the degree of internalization of green fluorescent beads after a pre-stimulus with BCG PGL I or at MOI 10:1.(TIF) ppat.1007151.s003.tif (765K) GUID:?5DA3472D-D130-44A2-8AFB-435A8A7D15B8 S4 Fig: Competition assay suggesting the mannose receptor (CD206) as a receptor candidate to mediate the internalization of BCG WT in Schwann cells. Representative images of fluorescence microscopy showing PKH 67 labeled-BCG WT association to SC after pre-infection with and in presence or absence of mannose. Cells on coverslips were fixed with paraformaldehyde and stained with DAPI (blue) for nuclear localization. The addition of mannose at 1000 g/mL to the culture medium reduced the BCG WT association rate 48 h post-infection. Results represent three independent biological replicates. Scale (white line) represents 10 m.(TIF) ppat.1007151.s004.tif (1.1M) GUID:?7328DB50-9122-4800-AA88-0163B6FD1F90 S5 Fig: Effect of GW9662 on and BCG PGL I internalization into Schwann cells. Flow cytometry results showing the degree of bacterial internalization of live PKH67 labeled bacilli after 24 h (A) and 48 h (B) of incubation with SC at 33C, MOI 50:1 in the presence or absence of GW9662 (5 M). A. A representative histogram plot of the 24 h incubation assay shows fluorescence at the FL1-A channel. The addition of GW9962 (5 M) to the culture medium had no significant effect on the internalization rate of BCG PGL I or after 24h of incubation. B. The addition of GW9962 (5 M) to the culture medium reduced BCG PGL I or internalization rate 48 h post-infection. Each bar represents the mean SEM from at least three independent experiments Nazartinib S-enantiomer in triplicate. An ANOVA test followed by Bonferroni as a post-test were performed and used for statistical analyses. *p < 0.05.(TIF) ppat.1007151.s005.tif (174K) GUID:?0115D52E-C9FD-4F94-B1B7-0B74B2E02DED S6 Fig: Effect of silencing on PGE2 production in infected and non infected Schwann cells. SCs were transfected for 24 h with control siRNA or siRNA targeting was shown to increase PGE2 production in the non infected condition.(TIF) ppat.1007151.s006.tif (226K) GUID:?7932A9D7-266D-402E-A940-E736EA1F55D8 S7 Fig: CD206 is not up-regulated nor colocalizes with Schwann cells in control nerve lesions. Serial sections of nerve biopsies from patients (n = 2) with non-leprosy peripheral neuropathies were analyzed. A. Peripheral nerve tissue was labeled with antibodies for the SC-specific marker S100 (red image), and the mannose receptor CD206 (green image) and then visualized by fluorescence microscopy. The merged images show no CD206/S100 colocalization. Nuclei were Nazartinib S-enantiomer labeled with DAPI (blue image). B. The corresponding isotype controls.