Detecting AP-1 Activation in IECs by GS Isolate

To detect AP-1 activation in IECs co-incubated with the GS isolate, Caco-2 cells were transfected with the plasmid pGL4.44[luc2P/AP1 RE/Hygro] expressing the luciferase reporter gene luc2P (Photinus pyralis) under the control of a promoter that contains the transcription response element (TRE) of AP-1. Transfection of Caco-2 cells was performed as described previously (Ma'ayeh et al., 2017 (link)). Trophozoites (5 × 10⁴) were processed (as described in the host-parasite interaction experiment section) and added to transfected cells in a total volume of 75 μl DMEM per well. Trophozoites were allowed to interact with IECs for 1.5, 3, 4.5, 6, and 10 h (37°C, 10% CO₂) and cells incubated alone in DMEM served as a control. As a positive control, Phorbol 12-myristate 13-acetate (PMA) (Promega), was added to IECs at a 5 μM concentration. After interactions, luminescence reads were assayed using the Dual-Glo® Luciferase reagent following the manufacturer's instruction (Promega). In total, three biological replicates were performed separately to assess AP-1 activation.