Dual glow assay

HEK293 cells were transfected in 96-well plates with the 8xGTIIC-luciferase plasmid (firefly luciferase, # 34615, Addgene, Watertown, MA, US) and the pRL-SVl40P plasmid (Renilla luciferase, # 27163, Addgene), using 0.5 µg of each plasmid and 2 µL of the jetPRIME transfection reagent (Polyplus transfection, New York, NY, USA) in a final volume of 100 µL per well and incubated overnight at 37 °C in 5% CO₂. The next day, the spent medium was replaced with the fresh complete culture medium, and the cells were incubated for another 24 h at 37 °C in 5% CO_2. The day after, the transfected cells were challenged with S. aureus or supernatant only, as mentioned in the text. After the challenge, the cells were lysed and luminescence was quantified using the Promega dual glow assay (Promega, Madison, WI, USA) with a multimodal plate reader (TriStar, Berthold). The blank value was subtracted, and the firefly luciferase activity was divided by the Renilla luciferase activity to normalize the results according to the number of cells.

HEK293 cells were plated at 35,000 cells/well into 96 well plate. Approximately 24 h after cells were transfected using Jet prime (Polyplus transfection, New York, NY, US). Approximately 1 µg of plasmids (0.5 µg of each plasmid) with 2 µl transfection reagent were used for a 100 µl final volume 5 µl of the mixture was added to cells cultivated in 100 µl final volume in 96 well plate for 24 h. HOP flash plasmid which consist of 8× wild type TEAD binding sites with minimal promoter plus luciferase reporter gene (luciferase firefly) was transfected with pRL-SVl40P (renilla luciferase) expressing vector. TNF or IL-17 was added 24 h after transfection for 30 min, 2 h, 6 h or 24 h. After cell lysis luminescence was quantify using Promega dual glow assay (Promega, Madison, WI, US). After blank subtraction, Firefly activity was then divided by renilla activity for normalization. HOP-flash was a gift from Barry Gumbiner (Addgene plasmid # 83467, Watertown, MA, US) and pRL-SVl40P was a gift from Ron Prywes (Addgene plasmid # 27163).