Cells were fixed in 3% paraformaldehyde and permeabilized with 0.1% saponin and blocked with 3% BSA in PBS (Roth). The cells were analyzed using a Zeiss (Oberkochen, Germany) Axioplan II fluorescence microscope equipped with a cooled charge-coupled device camera (Quantix; Photometrix, Tucson, AZ). Procedures involving Toxoplasma gondii Tachyzoites from T. gondii strain ME49 were maintained by serial passage in confluent monolayers of human foreskin fibroblasts (HS27, ATCC) as described previously [11]. Mouse primary hepatocytes were either left untreated or stimulated with IFN-c (R&D Systems) at 200 U/ml for 24 h prior to infection. For immunostaining, hepatocytes were inoculated with T. gondii at a MOI of 3 for 2 h. Extracellular parasites were then removed by extensive washing with PBS and cells were processed for immunostaining as above. Vacuoles containing intracellular parasites were identified by immunostaining for the T. gondii dense granule protein GRA7 that is associated with the intravacuolar network and the PV membrane after infection (reference). Bioinformatics Public databases (NCBI (http://www.ncbi.nlm.nih.gov) and ENSEMBL (http://www.ensembl.org)) were screened for Irga6 with the Basic Local Alignment Search Tool, BLAST [33] to identify ESTs and cDNAs that could clarify Irga6 59 exon usage. The recovered sequences were aligned to distinguish ESTs based on Exon 1A and exon 1B and to identify the splice site between exon 1A or 1B and exon 2. Sequence alignments were performed either with Vector NTI (InVitrogen), or using ClustalW2 at the EBI-Sanger website (http://www.ebi.ac.uk/Tools/clustalw2/) and edited manually. Shading of alignments was conducted with GeneDoc (Version 2.6.002) (http://www.nrbsc.org) or JalView (http://www.jalview.org/). Transcriptional start points for exon 1A and exon 1B were defined informally by the following criteria and being the 59 base of the longest 59 extensions, confirmed by a second sequence and present in the genomic sequence. Since the genomic sequence standard for comparison was based on the C57BL/6 genomic sequence for chromosome 18 (contig NT_039674.7), cDNA sequences derived from C57BL/6 were chosen to define the transcriptional start although longer plausible 59 extensions were found in ESTs from other mouse strains. The regions up to 1000 bp upstream of the transcription start point were designated as the putative promoter regions for each exon 1 of Irga6 and screened for transcription factor binding sites. ISRE (IFN-stimulated response element), GAS (gamma-activated se- quence), and hepatocyte nuclear factors HNF1 and HNF3 [34] binding sites were detected with CONSITE (http://asp.ii.uib. no:8090/cgi-bin/CONSITE/consite/, [35] and were confirmed manually. HNF4 and HNF6 binding sites were identified by manual comparison to published consensus binding sequences [36–39]. Real-time PCR Real-time quantitative polymerase chain reaction (qPCR) analysis was performed in a Light Cycler I System using a LightCycler SYBR Green I PCR kit (both Roche). The 59 primers specific for Irga6 exon 1A (59-TGCTTCCTGAAGCTGAACTA- 39) and 1B exon (59-ACCGAGGGCTATTCCTCTCA-39) to- gether with a common 39 primer for the coding exon (59- CAGAGAAGGGATGATATTCAC-39) were used to detect and distinguish Irga6 transcripts containing exons 1A and 1B. Hypoxanthine-guanine phosphoribosyltransferase (HPRT) specific primers (59 primer 59-ATTAGCGATGATGAACCAGG-39, 39 Expression of Irga6 in Liver primer 59-TGGCCTATAGGCTCATAGTG-39) were used to provide an input control. cDNA synthesized from mRNA extracted from liver was used as PCR template The level of Irga6 transcript was normalized to that of HPRT in the same sample. Melting curve analysis was performed after each run. Each sample was tested in duplicate or triplicate. The quantitation of all transcripts was achieved by comparison to an external standard based on five serial tenfold dilutions of a pGEMTeasy plasmid containing a full-length construct of Irga6 carrying the 59UT exon 1A; the dilutions ranged from 106 to 102 plasmid copies per assay. Supporting Information Table S1 Promoter HNF elements Found at: doi:10.1371/journal.pone.0006787.s001 (0.03 DOC) MB Table S2 Directory of Irga gene sequence elements on chromosome 18 genomic contig, NT_039674.7 Found at: doi:10.1371/journal.pone.0006787.s002 (0.06 MB DOC) Figure S1 Mouse EST sequences corresponding to Irga6 were recovered from the public databases using Megablast. The figure shows an alignment of all ESTs with extended sequence in the 59- untranslated region. The genomic regions corresponding to exon1A and exon 1B were identified from the C57BL/6 chromosome 18 contig, NT_039674.7, and are given at top and bottom of the alignments. The splice boundary between both exon 1A and exon 1B and exon 2 is indicated by arrows above and below the alignment. The last three bases of each EST sequence in the alignment form the initial start codon of the Irga6 translated protein. We used the 59 nucleotide of the two C57BL/6-derived ESTs, AI116617 and AI662996, to define the start-point of transcription for exon 1A and exon 1B respectively. These correspond to nucleotide 57,535,681 of NT_039674.7 and for exon 1A and nucleotide 57,542342 for exon 1B (both printed in red). Non- genomic 59 sequence extensions were removed from some EST sequences before alignment. A splice variant of Irga6 was described recently in IFN-induced mouse cells which carries both exon1A and exon 1B in tandem in that order, with a short linker sequence between them (NM_001146275) [16]. This is a unique case and undoubtedly should not be considered a normal splice variant. * indicates mouse strain of origin of EST. ** indicates ESTs from libraries where the Mus domesticus strain of origin was not recorded. Found at: doi:10.1371/journal.pone.0006787.s003 (0.26 MB TIF) Figure S2 Real-time PCR to analyze Irga6 expression in RNA purified from different organs of adult C57/BL6 mice. Results are presented as means with standard deviations from 4–5 indepen- dent measurements, in each case normalised to the amount of HPRT transcript. The 1A and 1B forms of Irga6 were distinguished by 59 primers specific respectively for exon 1A and exon 1B, while a common 39 primer in exon 2 was used. Found at: doi:10.1371/journal.pone.0006787.s004 (1.24 MB TIF) Figure S3 Western blots to assay Irgm3, Irgb6 and Irgm1 in lysates of tissue culture cells and primary hepatocytes. A. From left to right: Irgm3, assayed in L929 fibroblasts and TiB75 hepatocytes after (+) and without (2) stimulation for 24 hr with 100 U/ml IFNg; whole liver, and enriched primary hepatocytes cultured for 0, 3, 6, 12 and 24 h without IFNg, and lastly for 24 h with 100 U IFNg (24+). Calnexin was assayed in each sample as a loading control. B. From left to right, Irgb6 and Irgm1 assayed in TiB hepatocytes without (2) and with (+) IFNg stimulation for 24 hr with 100 U/ml; whole liver; enriched primary hepatocytes PLoS ONE | www.plosone.org 12 August 2009 | Volume 4 | Issue 8 | e6787