The Caco-2 and HT-29 cell lines were gained from Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and maintained in a humidified atmosphere at 37°C with 5% CO₂ in this study. Caco-2 cells were grown in DMEM (Hyclone, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS; Cellmax, Beijing, China), 1% nonessential amino acids (Alphabio, Tianjin, China), 1% glutamine (Alphabio, Tianjin, China), and 1% penicillin/streptomycin. HT-29 cells were cultured in DMEM/F12 (Hyclone, Logan, UT, USA) supplemented with 10% FBS and 1% penicillin/streptomycin.

The Giardia duodenalis WB isolate typed as assemblage A was used in this study (ATCC30957, Manassas, VA, USA). Giardia trophozoites were grown in sterilized modified TYI-S-33 medium with 10% FBS, 0.1% bovine bile, 0.1% gentamycin and 1% penicillin/streptomycin at 37°C under microaerophilic conditions. Giardia was collected by replacing the culturing medium with fresh medium to eliminate dead parasites. The tubes were placed on ice for 10 to 20 minutes, centrifuged (2000 × g for 8 min at 4°C) for parasite collection. Trophozoites were then counted and diluted.

CCK-8 (K1018; ApexBio, Houston, TX, USA) assay was performed according to the manufacturer’s instructions to assess cell viability. Cells were seeded in 96-well plates with 1 × 10⁴ cells per well. The cells cultured under identical growth conditions were exposed with Giardia at different ratios (3, 5 and 10 trophozoites/cell) for different time durations (0, 3, 6, 12 and 24 h). Following adding 10 μl of the CCK-8 solution to each well of the plate, the optical density was measured at 450 nm.

Caco-2 and HT-29 cells were exposed with Giardia trophozoites at a ratio of 1:10 for different time durations (0, 3, 6 or 12 h). IRE1 inhibitor MKC3946 (10 nM, MCE, Beijing, China), PERK inhibitor GSK2606414 (5 μM, Abmole, Shanghai, China), ATF6 inhibitor AEBSF (200 μM, Abmole, Shanghai, China), ROS inhibitor NAC (1 mM, ApexBio, Houston, TX, USA), AKT inhibitor MK2206 2HCl (50 μM, Abmole, Shanghai, China), and JNK inhibitor SP600125 (50 μM, Abcom, Atlanta, GA, USA) were used in this study. Caco-2 and HT-29 cells were pre-treated with inhibitors for 2 h, followed by exposure to Giardia for 6 h.

Cell cycle was analyzed with propidium iodide (PI) staining by flow cytometry (34). Briefly, Caco-2 and HT-29 cells were exposed to Giardia for the indicated time periods. After incubation, media was removed and cells were washed three times with cold PBS to remove Giardia trophozoites. After this treatment, the cultures were observed by microscopy for the presence of the trophozoites. Cells were fixed with cold 70% ethanol overnight at 4°C. Cells were then washed three times with PBS and incubated with PI and RNase for 30 minutes at 37°C. Cell cycle distributions were assayed by flow cytometry on a BD FACS Canto II. The data were analyzed with Modfit LT software version 3.1 (Verity Software House, Topsham, ME, USA).

Total RNA was extracted using the Trizol reagent (Invitrogen, Carlsbad, CA, USA). The quality of RNA was determined by the ratios 260 nm/280 nm. Then cDNA was synthesized from 1 μg of total RNA using a HiScript II 1st Strand cDNA Synthesis Kit (Vazyme, Nanjing, China) as instructions. qPCR was performed using SYBR Green qPCR Master Mix (Bimake, Houston, TX, USA). Primer sets ( Supplementary Table 1 ) used for qPCR analysis were designed and synthesized by Sangon Biotech (Shanghai, China). GAPDH was an internal control and chosen for normalizing, and the 2^−ΔΔCt method was used to calculate the relative expression of genes.

Western blotting was performed as previously described (36). Equal amounts of protein per sample were separated by 12% SDS-PAGE and transferred to the PVDF membranes (Thermo Fisher Scientific, Waltham, MA, USA). Protein bands were visualized with an enhanced chemiluminescence assay kit (Syngene, Cambridge, UK), and signal intensity of protein bands was analyzed by the use of Image J software. The immunoblots were incubated with primary antibodies against cyclin A (1:1000 dilution in PBST), cyclin B (1:1000), cyclin D (1:500), cyclin E (1:750), CDK1 (1:500), CDK2 (1:1500), CDK4 (1:500), CDK6 (1:500), E2F1 (1:500), E2F2 (1:1000), E2F3 (1:500), E2F4 (1:1000), RB (1:500), p21 (1:1000), p27 (1:1000), p53 (1:500), GRP78 (1:2000), IRE1 (1:500), p-IRE1 (1:500), XBP1s (1:1000), PERK (1:1000), p-PERK (1:500), ATF4 (1:1000), CHOP (1:500), ATF6 (1:500), Skp2 (1:500), pro-CASP3/cl-CASP3 (1:500), pro-PARP/cl-PARP (1:500), AKT (1:500), p-AKT (1:500), JNK (1:500), p-JNK (1:500) and β-actin (1:8000) probed with the secondary antibody HRP conjugated goat anti-rabbit IgG (1:5000; Bioss, Beijing, China), and visualized by diaminobenzidine (DAB) (ZSGB-BIO, Beijing, China). Primary antibodies were obtained from two commercial sources (ABclonal, Wuhan, China; Bioss, Beijing, China).

Caco-2 and HT-29 cells were seeded at a density of 1 × 10⁵ cells/well in 24-well plates. Cells were exposed to Giardia for the indicated time periods. After incubation, media was removed and cells were washed three times with cold PBS. After confirmation of parasite removal by microscopy, the apoptosis of cells induced by Giardia was evaluated by dual staining with fluorescent dyes acridine orange (AO) and ethidium bromide (EB) (BestBio, Shanghai, China). Cells were observed under a Lionheart FX Automated Microscope (BioTek, Winooski, VT, USA) and photographed at 4× magnification. Dead or late apoptotic cells (with compromised membranes) stain orange-red, while normal cells stain green. Image J software (National Institutes of Health, Bethesda, MD, USA) was used to quantify fluorescence intensities.

Protein-protein interactions were analyzed by co-immunoprecipitation (co-IP) as previously described (37). In brief, anti-E2F1 antibody and protein A/G magnetic beads (Bimake, Houston, TX, USA) were used to immunoprecipitate RB in cell lysates overnight at 4°C. Then the immunoprecipitated beats were collected and washed three times with lysis buffer. The proteins were separated by SDS-PAGE and analyzed by western blotting using anti-RB antibody.

Caco-2 and HT-29 cells were seeded at a density of 2 × 10⁵ cells/well in 12-well plates for Skp2 knockdown, cells were transfected with scramble control siRNA (si-NC) or siRNA targeting Skp2 (50 nM, Comate, Changchun, China) using lipo3000 (Invitrogen, Carlsbad, CA, USA) according to the instructions. Total RNA and protein were extracted after 48 h of transfection.

Intracellular ROS was measured using the ROS assay kit (Beyotime, Shanghai, China). In brief, cells were stained with the 2’,7’-dichlorofluorescin diacetate (DCFH-DA) at 37°C for 30 min, washed with PBS for three times. The fluorescence intensity of DCF was detected by the Lionheart FX Automated Microscope mentioned earlier and photographed at 20× magnification, and also measured with a FLUOstar Omega reader (BMG Labtech GmbH, Ortenberg, Germany).

Data distribution was assumed to be normal. Statistical analysis was completed using GraphPad Prism software v8.0 (GraphPad Software; La Jolla, CA, USA). Data were expressed as mean ± SD. The statistical significance of the differences between two groups was measured by an unpaired Student’s t test, and among three or more groups by one-way ANOVA. A value of p < 0.05 was considered statistically significant (*p < 0.05, **p < 0.01).

The effect of Giardia on the cell viability of Caco-2 and HT-29 cells was evaluated by CCK-8 analysis. The results indicated that the cell viability of both of the Caco-2 and HT-29 cell lines was decreased in a dose-dependent manner following Giardia exposure and bottomed out at a ratio of 10 parasites/cell (thus was applied hereinafter) ( Figure 1A ). The cell cycle distribution after Giardia exposure for 0, 3, 6 and 12 h was detected using PI staining and flow cytometry. As shown in Figure 1B , the percentage of cells in the G0/G1 phase was increased, and the percentage of cells in the S phase was decreased following Giardia exposure. In order to determine the molecular mechanism of Giardia-induced cell cycle arrest, levels of cyclins and CDK proteins reported to play important roles in cell cycle progression were determined by qPCR and western blot analysis. The mRNA and protein expression levels of cyclins (cyclin A, cyclin B, cyclin D, and cyclin E) and CDK proteins (CDK1, CDK2, CDK4 and CDK6) showed a decreased trend at 6 h with Giardia exposure in both Caco-2 and HT-29 cells ( Figures 1C, D ). The effect of Giardia on the cell apoptosis was determined by detecting apoptosis markers (cleaved CASP3 and cleaved PARP). As shown in Figure 1E , CASP3 was cleaved, and CASP3-mediated PARP was also cleaved subsequently after Giardia exposure. Furthermore, AO/EB double staining test was used to investigate the effect of Giardia on Caco-2 and HT-29 cells. Giardia -treated groups displayed more apoptotic cells compared to the controls ( Figure 1F ).

To further investigate the mechanism of Giardia on the cell cycle arrest, the expressions of E2Fs, p21 and p27 were examined in Caco-2 and HT-29 cells exposed to Giardia. For the mRNA level, E2F1, E2F2 and E2F3 was decreased at both of the 6 h and 12 h post-exposure, E2F4-E2F8 of which was increased at 6 h post-exposure ( Figure 2A ). For the protein expression level, E2F1 and E2F3 was decreased after Giardia exposure. The expression of E2F2 was increased at 3 h and then decreased at 6 h and 12 h under Giardia exposure in Caco-2 cells, but the expression of E2F2 was decreased in HT-29 cells. The expression of E2F4 was increased after Giardia exposure ( Figure 2B ). Although the mRNA and protein expression levels of E2Fs varied at different time points between Caco-2 and HT-29 cells, the expressions of E2Fs were indeed changed due to the exposure with Giardia. To assess whether Giardia exposure could increase the E2F1-RB interaction resulting low levels of E2F1, immunoprecipitation of cell extracts was conducted with E2F1 and RB polyclonal antibodies. The expression of RB in E2F1 immunoprecipitates was increased after Giardia exposure ( Figure 2C ). To determine if Giardia-induced G0/G1 cell cycle arrest was regulated by p21 and p27 expressions in Caco-2 and HT-29 cells, mRNA and protein levels of p21 and p27 were tested. The results showed that mRNA and protein levels of p21 and p27 were both increased with Giardia exposure ( Figures 2D, E ). The mRNA and protein levels of p53 were decreased with Giardia exposure ( Figures 2D, E ), suggesting that the p21 or p27 induction is probably p53-independent.

The main proteins related to ER stress were assessed in order to determine whether ER stress could be induced and UPR and ERAD pathways were activated by Giardia exposure. The results showed that the mRNA levels of ER chaperone proteins and ERAD-related genes were increased with Giardia exposure ( Figure 3A ). As shown in Figure 3B , ER stress signaling pathways were significantly activated by Giardia exposure, as demonstrated by the increased expression levels of GRP78 and CHOP. Three ER stress signaling pathways IRE1-XBP1s, PERK-ATF4 and ATF6 were activated at 6 h post-exposure. It was demonstrated by higher-level expressions of XBP1s, ATF4 and ATF6, and activation of IRE1 and PERK phosphorylation following Giardia exposure ( Figure 3B ). IRE1-XBP1s is active in the adaptive phase and attenuated in the apoptotic phase (10). This might be the reason for the decrease in p-IRE1 expression in HT-29 cells at 12 h after exposure. In all, these findings suggest that Giardia exposure activated three UPR pathways (IRE1-XBP1s, PERK-ATF4 and ATF6) and ERAD pathway.

To further investigate the role of UPR signaling pathways in cell cycle progression, the effects of cell cycle and the expression levels of G0/G1 phase-associated cyclins and CDKs were determined after treatment by combination of inhibitors (IRE1 inhibitor MKC3946, PERK inhibitor GSK2606414, ATF6 inhibitor AEBSF) and Giardia. As compared with cells treated by Giardia alone, the combination of inhibitors with Giardia significantly decreased the percentage of G0/G1 phase ( Figure 4A ), whereas increased the expression of cyclins, CDKs and E2F1-E2F3, and downregulated the expression of E2F4 ( Figures 4B–D ), but inhibitors alone had no effects on the cell cycle. Three inhibitors of UPR pathways inhibited the formation of E2F1-RB complex after Giardia exposure in Caco-2 cells ( Figure 4E ).

In order to determine the relationship between UPR signaling pathways and p21 and p27 induction, MKC3946 (inhibitor of IRE1), GSK2606414 (inhibitor of PERK) and AEBSF (inhibitor of ATF6) was used to block the Giardia-activated UPR signaling pathways. The results indicated that the expressions of p21 and p27 were decreased when Giardia-induced upregulation of XBP1s, PERK and ATF6 was inhibited by MKC3946, GSK2606414 and AEBSF ( Figure 5A ), respectively. Meanwhile, the inhibitors alone had no effects on the expressions of p21 and p27. In all, p21 and p27 might be tightly linked to the activation of UPR pathways. The mRNA levels of Ufd1 and Skp2 were both decreased and the protein expression level of Skp2 was decreased under Giardia exposure ( Figure 5B ). To further investigate the role of Skp2 in regulating p21 and p27, siRNA was used to knockdown Skp2 and the protein expression levels of p21 and p27 were measured. Loss of Skp2 did not change mRNA levels of p21 and p27, but upregulated protein expressions of p21 and p27 ( Figure 5C ).

As a significant regulator of several physiological and pathophysiological processes, ROS is essential for cell proliferation, survival and apoptosis. The study indicated that ROS level was increased during Giardia exposure ( Figure 6A ). Furthermore, ROS was examined for its role in Giardia-induced ER stress and cell cycle arrest. The percentage of G0/G1 phase was significantly decreased by the combination of NAC (ROS inhibitor) with Giardia ( Figure 6B ), but inhibitor alone had no effects on cell cycle. As shown in Figure 6C , the expressions of UPR-related proteins, p21, p27 and E2F4, were decreased by the combination of NAC with Giardia. The expressions of cyclins, CDKs and E2F1-E2F3 were increased and E2F1-RB complex formation was reduced by the combination of NAC with Giardia ( Figure 6C ).

To further clarify the role of UPR pathways in apoptosis caused by Giardia exposure, MKC3946 (inhibitor of IRE1), GSK2606414 (inhibitor of PERK) and AEBSF (inhibitor of ATF6) were used to block the Giardia-activated UPR signaling pathways. The results showed that the expression levels of cleaved CASP3 and cleaved PARP were decreased in cells pre-treated with GSK2606414 and AEBSF, but were increased pre-treated with MKC3946 under Giardia exposure ( Figure 7A ). AO/EB double staining test was used to investigate the effect of UPR signaling on apoptosis. The result obtained from AO/EB double staining was showed in Figure 7B . The number of apoptotic cells was decreased by the use of GSK2606414 and AEBSF, and increased by the use of MKC3946 under Giardia exposure. To further investigate the mechanism of IRE1 signaling pathway regulating apoptosis, the phosphorylation levels of AKT and JNK were measured. AKT phosphorylation could be detected at a significantly low level in Caco-2 cells when MKC3946 was used. On the other hand, JNK phosphorylation level was increased in Caco-2 cells when MKC3946 was used ( Figure 7C ). As shown in Figure 7D , the phosphorylation levels of AKT and JNK were upregulated by Giardia exposure. Considering the role of the phosphorylation of AKT and JNK in regulating apoptosis, the AKT inhibitor MK2206 2HCl and JNK inhibitor SP600125 were applied. The AKT phosphorylation suppression by MK2206 2HCl upregulated the expression levels of cleaved CASP3 and cleaved PARP at least partially, and JNK phosphorylation suppression by SP600125 downregulated expression levels of cleaved CASP3 and cleaved PARP at least partially ( Figure 7E ). AO/EB double staining was used to investigate the role of AKT and JNK pathways in regulating apoptosis. AKT phosphorylation suppressed apoptosis and JNK phosphorylation promoted apoptosis induced by Giardia ( Figure 7F ). It is possible that the hypophosphorylation of AKT and hyperphosphorylation of JNK could be contributors to the increased Giardia-induced apoptosis observed in cells pre-treated with MKC3946.