Furthermore, the Mito-Tracker-labeled TCTs partially invaded the H9c2 cytosol (colocalized with Lyso-Tracker) as early as 3 h post-infection (Fig. 5B), with marked entry at 24 h (Fig.

5C). The mitochondria, which showed high activity in the amastigote stage, were analyzed and also labeled with Mito-Tracker during invasion of SVEC cells by T. theileri. TWTth1 amastigotes, identified by small Hoechst dots, were co-localized with the lysosomes in Lyso-Tracker pre-stained SVEC cells 24 h after inoculation ( Fig. 5D, arrowhead). Mito-Tracker pre-stained amastigotes were also found to be surrounded by lysosomes in Lyso-Tracker pre-stained SVEC cells at 3 h post-infection ( Fig. 5E, arrowhead). Taken together, the observations described LY2109761 order above provide evidence that the invading parasites co-localize with lysosomes, as seen in the merged image, indicating TCTs and amastigotes of T. theileri are selleck chemicals fused with lysosomes in H9c2 and SVEC cell invasion. First, endogenous autophagosomes were detected by indirect immunofluorescence using MAP1LC3 antibody in TCT-infected

H9c2 cells. The parasites were detected by labeling the nucleus and kinetoplast with DAPI (Fig. 6A, arrowhead) that were surrounded by the protein LC3 (Fig. 6B, green), indicating its association in invasion. As compared to the H9c2 control cell (Fig. 6A and B, upper right panel), the LC3 protein was not present. Second, Fig. 6C revealed that pSelect-LC3-GFP-transfected H9c2 cells expressed a stable and strong positive control image after starvation. Three hours after infection, anti-TWTth1 antibody confirmed TCTs (Fig. 6D and E, red parasite body with DAPI-labeled nucleus and kinetoplast) were colocalized with

the protein LC3 (autophagosome), indicating that the T. theileri parasitophorous vacuole (PV) is not just a consequence of protein expression ( Fig. 6D and E). These results demonstrated a more intensive interaction between autophagosomes and T. theileri at the invasion Carnitine dehydrogenase sites than was previously thought. Basal TGF-β protein expression levels are shown in the control H9c2 cells (Fig. 7A). TWTth1-infected H9c2 cells revealed significant TGF-β protein expression at the penetration sites (Fig. 7B, red) and merged with the TWTth1 body (Fig. 7B, small DAPI dots). Quantitative RT-PCR showed that T. theileri infection significantly increased TGF-β1 mRNA of infected host cells (P = 0.025) ( Fig. 7C). Calcium signaling showed onset of Ca2+ transients 3 h after T. theileri infection using Fura-2/AM staining under confocal microscopy ( Fig. 8A). TWTth1 inoculation significantly increased the cytosolic Ca2+ concentration at the attachment site ( Fig. 8A, arrowhead). Quantification of Ca2+ oscillations of Fura-2/AM-loaded H9c2 cells was immediately performed after TWTth1 inoculation from zero to 35 min. Time-lapse images of Ca2+ changes from TWTth1 infected cells and non-infected control are shown in Fig. 8B.