Dual glo luciferase reagent

Reporter gene activity assays were performed essentially as described previously64 (link). A miR-451a reporter construct was created by inserting a sequence complementary to hsa-miR-451 into the Xho I and Not I sites of psiCHECK-2 vector (Promega, Madison, WI), downstream of the Renilla luciferase (Rluc) reporter gene. Firefly luciferase (Fluc) was used as a normalization control. Constructs were transfected into HEK293 cells 24 h before incubation with EVs for up to 48 h. Rluc and Fluc activities were measured with Dual Glo-luciferase reagents (Promega) using a luminometer (Dynex Technologies, Chantilly, VA).

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.

The luciferase-based replication assays of the C33a cells were essentially conducted as previously described [33 (link)], with a slight modification. Briefly, 80 ng each of the HPV-31 E1, E2, and pFLORI31 constructs was co-transfected along with Renilla luciferase at 16 ng per well on a 12-well plate. Twenty-four h post-transfection, the cells were exposed to 2 mM caffeine (dissolved in PBS; Sigma-Aldrich, St. Louis, USA) for the indicated times. The cells were lysed in Promega Dual-Glo luciferase reagent, and both the firefly and Renilla luciferase activity were determined using PHERAStar FS (BMG Labtech, Ortenberg, Germany). The firefly luciferase activity was normalized to the Renilla luciferase activity, and the value for the control was set as 100%.

iPSC-derived neurons (2 × 10⁴ cells) were transfected with 10 μg of 8xGTIIC-luciferase reporter (34615, Addgene, Watertown, MA, USA) and 10 μg of pGL4.74[hRluc/TK] Vector (E6921, Promega, Madison, WI, USA) using Lipofectamine LTX with Plus Reagent (14338100, Thermo Fisher Scientific, Waltham, MA, USA). The 8xGTIIC-luciferase reporter plasmid possesses eight synthetic TEAD-binding sites upstream of the luciferase gene, making it YAP/TAZ-responsive; this construct was generated by adding four more TEAD-binding sites to 4XGTIIC-Lux, originally created by Ian Farrance^{58 (link)} (https://www.addgene.org/34615/). pGL4.74[hRluc/TK] encodes the luciferase reporter gene hRluc (Renilla reniformis). After 48 h transfection, an equal volume of Dual-Glo Luciferase Reagent (E2920, Promega, Madison, WI, USA) was added to each well. Firefly luminescence was measured on a Spark 10 M multimode microplate reader (TECAN, Männedorf, Switzerland) after incubation for 20 min. For Renilla luminescence, an equal volume of Dual-Glo Stop & Glo Reagent (E2920, Promega, Madison, WI, USA) was added to each well before Dual-Glo Luciferase Reagent, mixed, and measured on a Spark 10 M. For the recovery experiments, AAV-CMV-YAPdeltaC-ins61 or AAV-CMV-NINS (MOI: 5000) or 20 nM S1P (S9666, Sigma-Aldrich, St. Louis, MO, USA) was added to the culture medium 4 days before plasmid transfection.

Cells were transiently transfected, using calcium-phosphate transfection as described above, with firefly luciferase promoter vectors (1 μg) and an SV40-promoter driven renilla luciferase vector (0.5 μg). Cells were harvested and snap frozen 24 h post transfection. Pellets were lysed in Dual-Glo Luciferase Reagent (Promega, E2920) and firefly luciferase activity was analyzed by luminometer Microbeta II (PerkinElmer, Waltham, MA, United States). Renilla luciferase activity was recorded by the instrument after subsequent addition of 1:1 volume Dual-Glo Stop & Glo (Promega, E2920). To correct for transfection efficiency, firefly luciferase signals were normalized to SV40 renilla luciferase signals of corresponding sample.