Supplementary MaterialsFigure S1: Parasite-derived MTT signal and LDH levels. of cell

Supplementary MaterialsFigure S1: Parasite-derived MTT signal and LDH levels. of cell lysates added to LEW BMDMs is equivalent to the volume of parasites added at the MOI indicated in parentheses. Viability and IL-1 release were then assessed 24 h post contamination.(PDF) ppat.1003927.s001.pdf (40K) GUID:?428AA244-62D3-4D9E-8C3D-70FCF0AEFEB4 Physique S2: Activation of the NLRP3 inflammasome by nigericin in CDF and LEW rats. CDF or LEW BMDMs were pre-treated with LPS (1 g/ml, 2 h) followed by either LT (1 g/ml LF+1 g/ml PA, 90 min) or nigericin (10 M, 1 h). In a separate experiment, SD BMDMs were LPS treated (100 ng/ml, 2 h) and either infected with RH strain (MOI 0.5, 6 h or 8 h), or treated with nigericin (40 M, 4 h). Supernatants were Amicon-concentrated prior to Western blotting. The unprocessed form of IL-1 is usually 37 kD. The mature cleaved form is usually 17 kD.(PDF) ppat.1003927.s002.pdf (77K) GUID:?51D8E1DA-54C6-48BD-A5ED-48B70878552E Physique S3: Fine-mapping of the locus based on comparison of the inbred and RI rat strain SNP genotypes for the 7 rat strains BN, F344 (CDF), LEW, SHR, HXB1, HXB15 and HXB59.(PDF) ppat.1003927.s003.pdf (16K) GUID:?7EEADDBC-4E86-478E-924D-C096F430AEF1 Physique S4: Whole transcriptome analyses of LEW, SD and BN rats. Summary of genes expressed in both LPS primed and unprimed conditions are shown for which non-synonymous SNPs (NS) existed. For each SNP, comparison of to four candidates, in reddish.(PDF) ppat.1003927.s004.pdf (10K) GUID:?DEB22A67-5CA4-4478-8BB0-D49B1AE937E3 Physique S5: Parasites treated with Mycalolide B are able to secrete ROP16 and induce activation of pSTAT6. HFFs were infected with GFP-expressing type I parasites that were pretreated with 3 M Mycalolide B or vehicle control for 15 minutes. Cells were infected for Anamorelin inhibitor database four hours and then fixed with 3% formaldehyde, permeabilized with 100% ethanol and blocked. A rabbit antibody against human pSTAT6 was used as the primary antibody, followed by a goat- anti-rabbit antibody conjugated to Alexa Fluor 594. Green?=?Parasite, Blue/Pink?=?Hoechst, Red?=?p-STAT 6.(PDF) Anamorelin inhibitor database ppat.1003927.s005.pdf (310K) GUID:?98BBB5CD-6AC7-4D82-99D3-491149EF69F2 Physique S6: Parasites released from lysed macrophages can reinvade other cells. A) SD or LEW BMDMs were infected with GFP-expressing RH (2 h), washed three times with PBS and the media was replaced with fresh media made up of rabbit anti-SAG1 antibody. After 24 h cells were fixed, permeabilized and stained with Alexa Fluor 594 goat anti-rabbit antibody. Parasites are green, while SAG1 is usually reddish. The quantification of SAG1-antibody coated parasites was performed with a minimum of 50 vacuole counts per condition from 3 experiments. (B) Parasites do not shed SAG1 upon invasion of SD BMDMs. Anamorelin inhibitor database Cells were infected with GFP-expressing RH for 18 h, cells were fixed, permeabilized and stained with a rabbit anti-SAG main antibody followed by Alexa Fluor 594 goat anti-rabbit antibody. SAG1 Anamorelin inhibitor database was detected on 100% of parasites in any infected cells. Green?=?parasite, Red?=?SAG1, Blue?=?Hoechst.(PDF) ppat.1003927.s006.pdf (168K) GUID:?E1F6D057-91D2-4C93-BD51-8250B0C83E9E Physique S7: Overexpression of Nlrp1 variants confers sensitivity to infection. Infections were with Type I (RH and Type II (76K) strains (MOI 51) were performed and viability was assessed 24 h post-infection. Details on Rabbit Polyclonal to Cytochrome P450 8B1 constructions of these lines can be found in [10]. In select experiments myc-tagged caspase-1 was also transfected 24 h prior to contamination. Values graphed are mean SD, n?=?3 wells/treatment. (C) Numerous mouse macrophage cell lines and BMDMs from mouse strains were tested for susceptibility to contamination as explained above. RAW264.7 cells were not tested with the RH strain. There is absolutely no statistical difference between the combined groups or treatments in these studies.(PDF) ppat.1003927.s008.pdf (42K) GUID:?4B6F8898-7671-4D35-BDA0-4F24B83DC8C0 Dataset S1: Fresh data set from entire transcriptome analyses of LEW, SD, and BN rats. Appearance beliefs (fragments per kilobase of transcript per million mapped reads?=?FPKM) from the genes in the fine-mapped locus are shown. Genes with FPKM 2 had been considered Anamorelin inhibitor database portrayed.(XLS) ppat.1003927.s009.xls (58K) GUID:?AC3CD3F7-350C-4DBC-B86D-486834F607C6 Abstract can be an intracellular parasite that infects an array of warm-blooded types. Rats vary within their susceptibility to the parasite. The locus conferring level of resistance in rats once was mapped to an area of.