Fig. 1: Phosphorylation of β-catenin helps T. gondii replication. | Cell Death & Disease

Fig. 1: Phosphorylation of β-catenin helps T. gondii replication.

From: Tryptophan-kynurenine pathway attenuates β-catenin-dependent pro-parasitic role of STING-TICAM2-IRF3-IDO1 signalosome in Toxoplasma gondii infection

Fig. 1

a Human colon cell line, Caco2 was infected with T. gondii, and parasite growth at indicated time post-infection was quantified by qPCR of genomic DNA using the invariant ITS-1 primers, normalized against β-actin. Expression of β-catenin was also measured by qPCR. b Phospho-β-catenin (92 kDa) and T. gondii SAG1 (36 kDa) protein at the indicated time post-infection were analysed by immunoblotting. The intensity of each band was plotted in bar diagram after normalizing against total β-catenin (92 kDa) or β-actin (42 kDa). c Raw macrophage cell lines, mouse bone-marrow dendritic cells (BMDCs), and human colon epithelial cells (SW480) were separately infected with T. gondii (36 kDa) and phospho-β-catenin (92 kDa) was monitored by immunoblot. β-actin served as internal control. d Luciferase reporter assays of TCF/LEF was done after T. gondii infection. Caco2 cells were transfected with Vector (pGL3), or TOP-Flash, or FOP-Flash reporter plasmid with internal control pCMV-Renilla-Luc plasmid for 8 h. At 12 h post-infection, cells were harvested for dual luciferase assay. The firefly luciferase units were normalized against the Renilla units and expressed in percentage. e FLAG-TCF4 from transfected cells was immunoprecipitated with FLAG antibody after infection, and the Western blot (WB) was done with FLAG (TCF4 expression, 71 kDa), phospho-β-catenin (92 kDa), and β-actin antibody (42 kDa). In the right panel, the bar diagram represents the average expression levels of FLAG-TCF4, and phospho-β-catenin of immunoprecipitated samples of three biological repeats after normalizing with β-actin. f T. gondii growth was quantified by qPCR using the invariant ITS-1 primers in wild-type (Wt) and β-catenin knock-out (KO) cells. g Replication kinetics of T. gondii was measured in both Wt and β-catenin KO cells by immunoblot. The intensities of the SAG1 bands (36 kDa) are plotted in bar diagram (right panel). Only cells denote uninfected cells with 2 µl of vehicle (DMSO or lipofectamine), Wnt agonist = AMBMP hydrochloride (20 µM, DMSO). Mean values ± SE of three experiments are shown here. Values were averaged from three separate experiments for each diagram, with error bars shown. *p < 0.05 and #p < 0.01. h Wt and β-catenin KO cells were infected by mCherry expressing T. gondii RH at an MOI of 2, and at the indicated times (12, 18, 24 h) stained for nucleus (DAPI, blue) and visualized by confocal microscopy. Scale bar = 50 µm