Fig 2: Functional tyrosine-mutant IDO1 inhibits multiplication of T. gondii.a Infection and followed by immunoblotting was performed with IDO1 (45 kDa), SAG1 (36 kDa) antibody in Caco2 cells. The mean ± SE of three repeats were shown by bar diagram (right panel). IFN-? was taken as an IDO1 activator. b mRNA expression of IDO1, and T. gondii was determined by qPCR after infection. In both, IFN-? was taken as IDO1 agonist. c, d Cells were over-expressed with FLAG-IDO1 plasmid (4 µg in 4 cm2 well) and immunoblot was done using FLAG (45 kDa), SAG1 (36 kDa), TDO (48 kDa), and ß-actin antibody (42 kDa). Three repeats of protein and mRNA expression was exhibited by bar diagram using ß-actin as internal control. Data represents mean ± SE of three repeats. * and # represent statistically significant (p < 0.05), comparison was made relative to 12 h infected cells. e Cells were transfected with the following plasmids (0.5 µg in 4 cm2 well): FLAG-wild-type IDO1 (Wt), tyrosine single/double mutant (FLAG-Y-115F, FLAG-Y-253-F, or FLAG-Y-115-253-F) in the presence of IFN-?, and MG132. Total cell-lysates were subjected to immunoprecipitation using FLAG antibody, followed by the immunoblot was done using ubiquitin antibody. The higher relative mobility of the bands indicates multiple tyrosine phosphorylation. Cells were transfected with the following plasmids (0.5 µg in 4 cm2 well): FLAG-tyrosine single/double mutant (FLAG-Y-115F, FLAG-Y-253-F, or FLAG-Y-115-253-F) in the presence of IFN-?. After 8 h of transfection, untransfected and transfected cells were infected with parasite. f Total cell-lysates were used for immunoblot using SAG1 antibody (36 kDa). Bar diagram represents mean ± SE of three repeats of SAG1 band. *, #, ? represent statistically significant (p < 0.05), comparison was made relative to untransfected-infected cells of each time-point. g Cells were transfected with the above-mentioned plasmids and infection was done by mCherry expressing T. gondii RH, and at the indicated times, slides were stained for nucleus (DAPI, blue) and visualized by confocal microscopy. h In similar condition, infected cells and cell supernatants were collected for measuring functional enzymatic activity of IDO1. Kynurenine production was assayed by photometric assay. Bar diagram represents mean ± SE of three separate experiment. *, and # represent statistically significant (p < 0.05), comparison was made relative to only untransfected-infected cells of each time-point

Fig 3: T. gondii infection recruits TCF to IRF3 promoter, and maintain its functional activity.a This is the hypothetical model where TCF could bind at the promoter region of IRF3 and regulates the transcription of IRF3. b IRF3 promoter sequence has two consensus sequences, CTTTGGG, and CTTTGCG where TCF prefers to bind. c There were several isomers of TCF with their binding site, present on IRF3 promoter site and the alignment score was calculated for individual isomer. d TCF4-MinP-PGL3 is (CTTTGGG/CTTTGCG) TCF binding site on IRF3 promoter with minimal promoter in pGL3 luciferase plasmid was cloned. TCF4-MinP-PGL3 was transfected with Wt-ß-catenin + TCF4 plasmids, or phopsho-mimic-ß-catenin (S552D) + TCF4 plasmids, or phopsho-mutant-ß-catenin (S552A) + TCF4 plasmids, or Wt-ß-catenin + dominant-negative (DN)-TCF4 plasmids in ß-catenin KO cells. pGL3-minimal promoter (PGL3-MinP) luciferase plasmid was taken as negative control and pCMV-Renilla-Luc plasmid was transfected in each condition. At 12 h post infection, cells were harvested for dual luciferase assay of TCF4-MinP-PGL3. The firefly luciferase units were normalized against the Renilla units and expressed in percentage. Values were averaged from four similar experiments, with error bars shown, *p < 0.001. Wild-type Caco2 cells (Wt), only ß-catenin-KO cells and ß-catenin-KO cells with FLAG-ß-catenin plasmid were infected with T. gondii and (e) phosphorylation of IRF3 and parasite growth were determined using phospho-IRF3(S396) (47 kDa), and SAG1 (36 kDa) antibody respectively and data of mean ± SE of three individual experiment was represented by bar diagram. *, #p < 0.01. f mRNA of IRF3 and T. gondii was quantified by real-time PCR. Data was represented by bar diagram after having average ± SE of three individual experiment. *, #p < 0.01. g p125-luc (IFN-ß promoter activity) in presence of IFN-ß (agonist) and IDO1 promoter activity in presence of IFN-? (agonist) was tested. In separate wells, p125-Luc or IDO1 reporter plasmid were transfected in Wt-Caco2 and ß-catenin-KO cells and parasite infection was done. IDO1 promoter activity was also checked in presence of phospho-mimic (IRF3D5), and phospho-mutant IRF3 (IRF3-S396-398A) constructs. Only cell is without plasmid, and Vector (pGL3 basic) was used as control. pCMV-Renilla Luc plasmid was transfected in each sample, and used as internal control. At indicated time post-infection, cells were harvested for dual luciferase assay. The firefly luciferase units were normalized against the Renilla units and expressed in percentage. Average values of three experiments, with error bars show mean values ± SE, *p < 0.01. h Un-transfected-uninfected cells and cells transfected with HA-STING, followed by 12 h of T. gondii infection were immunoprecipitated using HA antibody. After immunoprecipitation, all samples were separately Western blotted (WB) using phospho-STING (50 kDa), and phospho-TICAM2 antibody (24 kDa). i Caco2 cells were transfected with phospho-constitutive AKT1DD/AKT2DD or phospho-mutant AKT1AAA/AKT2AAA. After 8 h of transfection, fresh media was added. 24 h post-transfection, cell lysates were immunoblotted using P-STING(S366), and P-TICAM2 antibody. j Caco2 cells were infected with T. gondii in presence or absence of either AKT inhibitor, or TBK inhibitor. One well of Caco2 cells were only transfected with constitutive phospho-mimic AKT1 (HA-AKT1DD) plasmid without any infection or inhibitor. All samples were used for the immunoblot using phospho-STING, phospho-TICAM2, and phospho-IRF3(Serine antibody). In i, and j, we did not get dimer of STING. This blot is the average representation of three separate experiments