Renilla luciferase

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.

The Wnt-induced and basal luciferase activity was analyzed as described (8 (link), 20 (link)). Briefly, 50 μl of indicated cell lines at 60,000/ml for HCT116, U87, U118, U251, or 120,000/ml for KURAMOCHI, OVSAHO, or 84,000/ml for Huh7, or 96,000/ml for DLD1, SNU398, LS174T, or 144,000/ml for HepG2, Hep3B, SW620, OVCAR3, SW48, PEO1, BT20 were distributed in a white opaque 384-well plate. The cells were maintained in their respective culture mediums and incubated at 37°C, 5% CO2 overnight for attachment. Afterwards, the cells were transfected by the 1:1 mixture of the plasmid constitutively (under the CMV promoter) expressing Renilla luciferase (Addgene, Cambridge, MA, USA), and a reporter plasmid (Syngene). Transfection was carried out as described in the manufacturer’s protocol using 12 μg/ml of DNA and 40 μl/ml XtremeGENE 9 reagent (Roche). The next day, the medium of each well was replaced 30 μl fresh medium containing Wnt3a (500 ng/ml) (purified as described by (21 (link)) or GSK3β inhibitor (CHIR99021, Sigma-Aldrich) and clofazimine at 5, 10, and 15 µM concentrations (following 1h of pre-incubation with the compound), or MSAB at the indicated concentration. After overnight incubation, the supernatant in each well was removed and the luciferase activity was measured as described (21 (link)).