Infinite m2000 protm

NCM460 cells were transfected with signal AP-1 reporter kit (Qiagen) according to manufacturer’s instructions. In brief, cells were suspended and added to a mixture of reporter and transfection reagent. After 24 h of transfection, medium was changed to assay medium (Opti-MEM + 0.5% FBS + 0.1mM NEAA + 1 mM Sodium pyruvate + 100 U/ml penicillin + 100 μg/ml streptomycin) and cells were treated with 10 ng/ml of PMA or CM in mentioned concentrations. PMA is a known inducer of AP-1 activity and was used as a positive control. The AP-1 reporter contains a mixture of inducible AP-1-responsive firefly luciferase construct and constitutively expressing renilla luciferase construct (40:1). The assay was developed by using Dual-Luciferase Reporter Assay System from Promega. Cells were lysed within the wells and luciferase assay reagent was added. Luminescence was measured on an Infinite M2000 Pro^TM plate reader (Tecan). After measuring firefly luciferase activity, the stop solution was added and the measurement was repeated to measure renilla luciferase activity. Relative Luciferase activity was determined as luminescence signal of firefly luciferase activity normalized to the signal of renilla luciferase activity.

resazurin reduction assay was conducted to test the cytotoxicity of the compounds. This assay is based on the reduction of resazurin to resorufin by viable cells [64 (link)]. Non-viable cells do not show a blue staining because they lost their metabolic capacity which causing reduction of resazurin. Briefly, aliquots of 0.5×10⁴ adherent cells which were allowed to attach overnight and 1×10⁴ suspension cells per well were seeded in 96-well-plates with or without the addition of varying concentrations of the test substance to get a total volume of 200 μL/well. After 72 h incubation and addition of resazurin (Sigma-Aldrich) for 4 h, staining was measured by an Infinite M2000 ProTM plate reader (Tecan, Germany) using an excitation wavelength of 544 nm and an emission wavelength of 590 nm. Each assay was independently performed for at least three times, with six parallel replicates each. The protocol has been recently reported [65 (link)]. Fifty percent inhibition concentrations (IC₅₀) represent the drug concentrations required to inhibit 50% of cell proliferation, which were fitted with nonlinear regression using GraphPad^® Prism7.

Different concentrations of HRB and compound 1 were used to treat CCRF-CEM cells for 6 h. The activities of caspases were determined using Caspase-Glo 3/7, Caspase-Glo 8, and Caspase-Glo 9 Assay kits (Promega, Mannheim, Germany) by measuring the luminescence using an Infinite M2000 ProTM plate reader (Tecan), as reported previously [13 (link)].