Spark 10

We used colorimetric MTT assays to study the effects of benzofuran derivatives on cell proliferation. Cells (1 × 10⁴ cells/well) were seeded onto a 96-well plate and treated with various concentrations of the compounds for 24 h. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, Amresco, OH, USA) was dissolved in PBS at 5 mg/ml then added to the well plate and incubated for 4 h at 37 °C. The supernatant was suctioned, and formazan crystals were dissolved in 100 μl DMSO. Optical density was measured using a microplate reader (SPARK10, TECAN, Switzerland) at 570 nm and cell viability was expressed as the proportion of untreated cells.

For reporter assays, cells were seeded on a 96-well plate and transfected with 50 ng firefly reporter, 5 ng renilla control, and 10 ng of the plasmid of interest in each well. Where indicated, 6 h after transfection, cells were treated with control, Wnt3a (standard dilution 1:5) or R-spondin 3 conditioned media (standard dilution 1:1000), or a combination of both (indicated as W/R). The dual luciferase assay was conducted as described previously (58 (link)) with few changes. Briefly, after overnight incubation, cells were lysed in passive lysis buffer (25 mM Tris, 2 mM DTT, 2 mM EDTA, 10% (v/v) glycerol, 1% (v/v) Triton X-100, (pH 7.8)) and agitated for 10 min. Lysates were transferred to a flat bottomed 96-well luminescence assay plate. Firefly luciferase buffer (200 μM D-luciferin in 200 mM Tris–HCl, 15 mM MgSO4, 100 μM EDTA, 1 mM ATP, 25 mM DTT, pH 8.0) was added to each well and the plate was incubated for 2 min at room temperature. Luciferase activity was measured using Spark10 (Tecan) or a SpectraMax iD3 Multi-Mode Microplate Reader (Molecular Devices). Next, Renilla luciferase buffer (4 μM coelenterazine-h in 500 mM NaCl, 500 mM Na2SO4, 10 mM NaOAc, 15 mM EDTA, 25 mM sodium pyrophosphate, 50 μM phenyl-benzothiazole, pH 5.0) was added to the plate and luminescence was measured immediately. Data were normalized to the Renilla control values, performed in triplicate.

Assessment of mitochondrial toxicity was performed using the Mitochondrial ToxGlo Assay to quantify cellular ATP levels according to the manufacturers' instructions (Promega, Germany). Briefly, for each condition a cell suspension containing 200,000 cells per ml were prepared from chopped tissue sections and supernatant. Fifty microliter cell suspension were incubated with 20 μl of cytotoxic reagent in 96-well plates for 30 min at 37°C. Subsequently, 100 μl ATPase reagent was added and plates were shaken for 1 min, before measuring the luminescence signal using a plate reader (Spark 10, Tecan).