Fludarabine

Fluorescent dye-conjugated antibodies that are specific for CD45, CD4, CD8, and Zombie violet were obtained from Biolegend (San Diego, CA). pSTAT1 inhibitor Fludarabine [30 (link)] and pSTAT3 inhibitor Stattic [31 (link)] were obtained from Santa Cruz. The 2W1S and OVA tetramers were provided by the NIH Tetramer Core Facility (Emory University, GA). The cells were stained with 0.15 μl 2W1S and 0.25 μl OVA tetramers. All the antibodies and reagents are listed in Additional file 1 Table S1.

Human adenocarcinoma Caco-2 cells and human liver cancer HepG2 cells from ATCC were grown to confluent monolayers in Dulbecco’s modified Eagle’s medium (DMEM) (catalog number 11995-040; Thermo Fisher) with 10% serum, penicillin, and streptomycin for 3 days (modified from the procedures in references 49 (link) and 50 (link)). Cells grown in 24-well plates to 80 to 90% confluence were washed twice with DPBS, and 500 μl of DMEM medium was added per well. E. histolytica trophozoites (1 × 10⁵ per well) were treated or not with the STAT1 inhibitor fludarabine (50 μM) (catalog number Sc-204755; Santa Cruz) for 30 min prior to IFN-γ (100 ng/ml) stimulation for 20 min. For the interaction, trophozoites were added to each well for 1 h. After incubation, the cells were placed on ice, washed with DPBS, and fixed with 2.5% paraformaldehyde for 10 min. The monolayer was stained with 0.1% methylene blue in 0.1 M borate buffer (pH 8) for 10 min at room temperature. The plates were washed with 0.1 M borate buffer twice to remove excess stain. Finally, 1 ml of 1 N HCl was added to each well for 30 min at 37°C to extract the stain, and the absorbance read on a spectrometer at 655 nm (optical density at 655 nm [OD₆₅₅]). The percentage of monolayer destruction was calculated as follows: [OD₆₅₅ (control wells) − OD₆₅₅ (experimental wells)/OD₆₅₅ (control wells)] × 100.