A CytoTox 96^® Non-Radioactive Cytotoxicity Assay was obtained from Promega (Madison, Wisconsin, USA). A JC-1 MitoMP detection kit was obtained from Dojindo (Kumamoto, Kumamoto, Japan). The ER stress inhibitor 4-phenylbutyrate (4-PBA) was purchased from Sigma Chemical Co. (St. Louis, Missouri, USA).

An ER Stress Antibody Sampler Kit; anti-activating transcription factor 6 (ATF6), anti-phospho protein kinase RNA-like endoplasmic reticulum kinase (p-PERK), anti-phospho eukaryotic translation initiation factor 2α (p-eIF2α), anti-poly (ADP-ribose) polymerase (PARP), and anti-cleaved caspase-3 antibodies; and a pro-Apoptosis Bcl2 Family Member Antibody Sampler Kit and pro-Survival Bcl-2 Family Member Antibody Sampler Kit were purchased from Cell Signaling Technology Inc. (Danvers, Massachusetts, USA). Anti-CCAAT/enhancer-binding protein–homologous protein (CHOP) and anti-phospho inositol-requiring protein 1α (p-IRE1α) antibodies were obtained from Abcam (Cambridge, Massachusetts, USA). Secondary anti-rabbit-horseradish peroxidase (HRP) and anti-mouse-HRP antibodies were obtained from Jackson ImmunoResearch Laboratories (West Grove, Pennsylvania, USA).

A nontransformed human fetal small intestinal epithelial cell-line (FHs 74 Int cells) was purchased from ATCC (ATCC, Manassas, Virginia, USA) and maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS), antibiotic-antimycotic (Gibco BRL) and 30 ng/ml human epidermal growth factor (all from Gibco, Grand Island, New York, USA) at 37°C in a humidified atmosphere with 5% CO₂. The medium was changed every 2–3 days.

Tachyzoites of the T. gondii RH strain was maintained as described previously [19]. Briefly, human retinal pigment epithelial (ARPE-19) cells (ATCC) were cultured in a 1:1 (v/v) mixture of DMEM/F12 supplemented with 10% heat-inactivated FBS and an antibiotic-antimycotic solution. ARPE-19 cells were infected with T. gondii at a multiplicity of infection (MOI) of 5 for 2–3 days. After spontaneous host cell rupture, parasites and cellular debris were pelleted by centrifugation and washed in cold PBS. The final pellet was resuspended and passed through a 26-gauge needle fitted with a filter with 5.0-μm pores (Millipore, Billerica, Massachusetts, USA).

Toxoplasma gondii ESP was produced as described previously [20] with slight modifications. Briefly, freshly purified tachyzoites (1×10⁸) from the peritoneal cavity were incubated at 37°C for 3 hr under mild agitation in test tubes containing 1.0 ml Hank’s balanced salt solution (HBSS) (Gibco BRL, Rockville, Maryland, USA). After centrifugation for 5 min at 6,000 g, the supernatant, including ESP, was saved. The protein concentration was measured using a Bradford assay (Bio-Rad Laboratories, Hercules, California, USA), and samples were stored at −70°C until use.

FHs74 Int cells were seeded on 96-well plates (for LDH and MTS assays), 12-well coverslips (for immunofluorescence), and 100-mm culture dishes (for western blotting) at various densities and grown to confluence at 37°C in 5% CO₂.

FHs74 Int cells were stimulated with tachyzoites of the T. gondii RH strain at MOIs of 10 or with 1 μg/ml ESP for 0, 2, 8, and 24 hr. Then, cell cytotoxicity, apoptosis, apoptotic features, induction of ER stress and its mechanism of action, and Bcl-2 family-related protein expression were evaluated using a LDH assay, immunocytochemistry and western blotting.

To determine the role of ER stress in T. gondii- or ESP-challenged cells, FHs74 Int cells were pretreated with the ER stress inhibitor 4-PBA and then evaluated for an ER stress response, apoptotic features, MMP and Bcl-2 family protein expression. Untreated FHs74 Int cells were used as controls. Each experiment was performed at least 3 times in triplicate.

The LDH assays were performed for cytotoxicity quantification using a CytoTox 96^® Non-Radioactive Cytotoxicity Assay kit according to the manufacturer’s protocol. Briefly, 1×10⁴ FHs74 Int cells were seeded in 96-well plates and infected with T. gondii at an MOI of 10 or treated with 1 μg/ml ESP for the indicated times (0, 2, 8, and 24 hr) in an incubator (5% CO₂, 90% relative humidity, 37°C). Then, 50 μl aliquots from all infected and control wells were transferred into a new 96-well plate, and 50 μl of CytoTox 96^® reagent was added to each sample aliquot. The plate was covered with foil to protect the samples from light and incubated for 30 min at room temperature. After addition of 50 μl of stop solution to each well, the absorbance of the solution was measured immediately at 490 nm using a microplate reader (TECAN, Crailsheim, Germany). LDH release levels in the medium were quantified and compared to control values according to the kit instructions.

MMP was measured using a JC-1 MitoMP detection kit (Dojindo, Kumamoto, Japan). Briefly, FHs74 Int cells were seeded onto coverslips in 12-well plates at a density of 1×10⁴ cells/well and infected with T. gondii at an MOI of 10 or treated with 1 μg/ml ESP under various conditions with or without an ER stress inhibitor. The cells were then incubated with 4 μm JC-1 fluorescent dye at 37°C for 30 min in the dark and rinsed 3 times with HBSS. The stained cells were mounted onto microscope slides in VECTASHIELD HardSet Mounting Medium with DAPI (Vector Laboratories), and images were collected using a laser confocal microscope (Leica, TCS SP8, Wetzlar, Germany). The intensities of green (excitation/emission wavelength=485/538 nm) and red (excitation/emission wavelength=485/590 nm) fluorescence were analyzed in ≥6 microscopic fields in each sample. The intensities of red and green fluorescence were calculated using Image J software (NIH, Bethesda, Maryland, USA).

SDS-PAGE and western blotting analysis were performed to determine the expression of numerous proteins. FHs74 Int cells were cultured in 100-mm dishes and underwent serum deprivation for 4 hr to remove stimulation from serum factors. Then, the cells were stimulated with T. gondii or ESP as indicated. After washing with PBS, proteins were extracted using PRO-PREP Protein Extraction Solution (iNtRON Biotechnology, Korea). The extract was incubated with complete protease inhibitor cocktail (Roche, Basel, Switzerland) for 30 min on ice followed by boiling for 10 min and then centrifuged at 14,000 g for 15 min at 4°C. The supernatant was collected, and equal amounts of protein from each sample were separated via SDS-PAGE and transferred to a polyvinylidene difluoride (PVDF) membrane (Bio-Rad). For blocking, the membranes were immersed for 1 hr at room temperature in 5% skim milk in Tris-buffered saline (20 mM Tris, 137 mM NaCl, pH 7.6) containing 0.1% Tween-20 (TBST). After one wash in TBST, the membranes were incubated overnight at 4°C with primary antibodies diluted in TBST supplemented with 5% BSA. Following 3 consecutive washes in TBST, membranes were incubated for 90 min with HRP-conjugated anti-mouse or anti-rabbit secondary antibody diluted 1:5,000 with 5% skim milk, as described above. The membrane was soaked with Immobilon Western Chemiluminescent HRP Substrate (Jackson ImmunoResearch Laboratories), and chemiluminescence was detected using a Fusion Solo System (Vilber Lourmat, Collegien, France). These experiments were repeated at least 3 times. The band intensities were quantified using ImageJ software (NIH). The results were normalized to α-Tubulin protein levels and were expressed as fold changes over the mock-infection control group.

Mitochondrial and cytosolic fractions of FHs 74 Int cells were prepared as described previously [PMID: 32547023]. For cytosolic extracts free of nuclei and mitochondria, the cells were washed in ice-cold PBS (pH 7.2) and then in hypotonic extraction buffer (HEB; 50 mM PIPES, 50 mM KCl, 5 mM EGTA, 2 mM MgCl₂, 1 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride, pH 7.4) and harvested by centrifugation. Pellets were resuspended in HEB and lysed with a Dounce homogenizer. These cell lysates were then centrifuged at 100,000×g for 60 min at 4°C, and the supernatants were flash-frozen in cold ethanol, aliquoted, and stored at −80°C. Mitochondrial fractions were prepared by washing the cells in ice-cold PBS and then resuspending them in an isotonic homogenization buffer (10 mM Tris-HCl, pH 7.5, 250 mM sucrose, 10 mM KCl, 1 mM EDTA, 1 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride, EDTA-free complete cocktail of protease inhibitors [Roche]). After 60 strokes in a Dounce homogenizer, unbroken cells were removed by centrifugation at 1,000×g for 10 min, and the supernatants were centrifuged at 14,000×g for 20 min. The supernatants were collected as the mitochondrial fraction. The expression of cytochrome c expression was determined by western blotting. COX IV and α-tubulin were used as mitochondria and cytosol markers, respectively.

All assays were performed in triplicate, and at least 3 independent experiments were conducted per test series. The results are presented as mean±SD. Statical analysis of the data was performed using unpaired, 2-tailed Student’s t-tests. A P-value less than 0.05 indicated statistical significance.

The cytotoxicity of T. gondii RH tachyzoites to FHs 74 Int cells were 4.25±0.63%, 10.33±0.46%, 24.72±0.31%, and 44.42±1.05% at 0, 2, 8, and 24 hr postinfection, respectively. Next, we examined whether T. gondii ESP is toxic to FHs 74 Int cells. ESP (1 μg/ml) was added to the host cells for the indicated time, and the cytotoxicity was dramatically increased to 12.62±1.25%, 32.68±2.57%, and 41.98±0.94% at 2, 8, and 24 hr, respectively (Fig. 1A). To verify whether the cytotoxicity of the T. gondii RH strain or ESP to FHs 74 Int cells was associated with apoptosis, we measured the level of apoptosis indicator proteins in RH-infected or ESP-treated FHs 74 Int cells. Our results showed that the protein levels of cleaved PARP and cleaved caspase 3 were significantly elevated in response to RH and ESP, and their increase was time dependent (Fig. 1B). Similarly, T. gondii infection and ESP treatment time-dependently elevated release of cytochrome c from mitochondria into cytoplasm (Fig. 1C, D). Collectively, our data indicate that T. gondii RH strain tachyzoites and ESP induced cytotoxicity and apoptosis in FHs 74 Int cells in a time-dependent manner.

Western blot analysis data showed that the protein levels of Bax, Bak and Bim were obviously increased with T. gondii infection, whereas the protein levels of BID, Mcl-1, Bcl-2, and Bcl-xL were drastically reduced in a parasite infection time-dependent manner (Fig. 2A). JC-1 dye staining revealed that T. gondii time-dependently induced MMP depolarization (Fig. 2C).

Upon ESP treatment, the protein levels of Bax, Bak, and Bim were apparently increased and those of BID, Mcl-1, Bcl-2, and Bcl-xL were diminished in a time-dependent manner (Fig. 2B). Surprisingly, we also found that the PUMA protein level was reduced after 2 and 8 hr of T. gondii infection and then slightly increased at 24 hr (Fig. 2A), but with ESP treatment, PUMA was dramatically reduced at 2 hr and remained low until 24 hr (Fig. 2B). In addition, both T. gondii and ESP induced modest phosphorylation of Bcl-2 at 24 hr (Fig. 2A, B). ESP treatment also caused mitochondrial dysfunction in a time-dependent manner (Fig. 2D). These findings suggest that T. gondii RH strain tachyzoites derived mitochondrial dysfunction in FHs 74 Int cells in a time-dependent manner. Speculation is not Results.

The ER-resident transmembrane sensor proteins IRE1, PERK and ATF6 were activated, evidenced by the dramatic increase in phospho-IRE1α, phospho-PERK, phospho-eIF2α and cleaved ATF6 upon T. gondii infection (Fig. 3A, C). CHOP, which plays an important role in ER stress-induced apoptosis, was also elevated. However, the protein level of caspase 12, which is essential for carrying out the caspase-dependent UPR, was visibly reduced compared with that in the uninfected control group. All the above events occurred in a parasite infection time-dependent manner (Fig. 3A, C).

Similar to T. gondii infection, ESP treatment also induced elevation of p-IRE1α, p-PERK, p-eIF2α, cleaved ATF6, and CHOP and attenuation of procaspase-12 in a time-dependent manner (Fig. 3B, D). Both T. gondii infection and ESP treatment induced an obvious increase in the BIP protein level at 8 hr and maintained the high level until 24 hr. In contrast, T. gondii infection reduced the protein levels of Ero1-Lα and PDI at 2 hr, which then slightly recovered, whereas ESP treatment gradually reduced the protein levels of Ero1-Lα and PDI.

Our results showed that both T. gondii- and ESP-induced alterations in the expression of Bcl-2 family members (Fig. 4A, B) and ER stress-related proteins (Fig. 4C, D) were reversed with 4-PBA pretreatment. In addition, 4-PBA pretreatment attenuated the protein levels of cleaved PARP and cleaved caspase 3 in T. gondii-infected or ESP-treated FHs 74 Int cells (Fig. 4E, F). T. gondii infection- or ESP treatment-derived the release of cytochrome c from the mitochondria into the cytoplasm, which was also prevented by 4-PBA pretreatment (Fig. 4G and Supplementary Fig. S1A, B). Confocal microscopy imaging of JC-1 dye staining showed that T. gondii infection- or ESP treatment-induced MMP depolarization in FHs 74 Int cells was rescued by 4-PBA pretreatment (Fig. 4H, I and Supplementary Fig. S1C, D). These results demonstrate that the T. gondii RH strain and ESP regulated ER stress, which mediates host mitochondrial dysfunction and apoptosis.