Pierce coomassie plus assay

AhR reporter cells were stimulated as depicted in figure legends. For competition assays, AhR reporter cells were pre-incubated with TB drugs for 1 h prior to stimulation with 50 mM Pht. After stimulation, cells were washed using sterile Dulbecco's phosphate-buffered saline (DPBS, GIBCO) and subsequently lysed using 1x concentrated Reporter Lysis Buffer (Promega). Cell lysates were used to determine luciferase activity by Luciferase Assay System (Promega) according to the manufacturer's instructions and luminescence was measured with an Infinite M200 pro reader platform (TECAN). Luciferase activity was normalized to the protein concentration measured by Bradford reaction (Pierce TM Coomassie Plus Assay, Thermo Fisher Scientific). Results are shown as log2 fold induction normalized to the solvent control of the respective time point.
siRNA Knockdown of AhR THP-1 AhR reporter cells were treated with ON-TARGET plus siRNA AHR or ON-TARGETplus Non-targeting Pool (Dharmacon) for 24 h prior to stimulation with RFB, according to manufacturer's instructions.

CYP1A1 is under transcriptional control of AhR (Nebert, Goujon and Gielen, 1972; Poland, Glover and Kende, 1976; Poland and Knutson, 1982) . The EROD assay measures the conversion of non-fluorescent ethoxyresorufin by CYP1A1 to the fluorescent product resorufin, where the amount of resorufin-fluorescence is proportional to the enzymatic activity of CYP1A1 (Mohammadi-Bardbori and Mohammadi-Bardbori, 2014). Cells were stimulated as depicted in the figures. After stimulation, cells were washed once using sterile DPBS (GIBCO) and 5 mM ethoxyresorufin (EROD, Sigma-Aldrich) and 10 mM dicoumarol (Sigma-Aldrich) were added to the cells for 1 h. Subsequently, relative fluorescence of resorufin (excitation 535nm/emission 590nm) was measured either in form of an endpoint assay or as kinetic (kinetic reads every 30 min at 37 C, 5% CO 2 ) using an Infinite M200 pro reader platform (TECAN). EROD activity was corrected to the protein concentration measured by Bradford reaction (Pierce TM Coomassie Plus Assay, Thermo Fisher Scientific) and normalized to the solvent control of the respective time point for end point assay. Endpoint assays are shown as Log2 activity fold induction ans kinetic measurements are shown as total well fluorescence over time.