Mtt dye

In total, 10,000 cells/well were seeded in 96 well-plates 24 h prior to UVC. Initial dosis-finding with increasing UVC irradiation was performed with A375 wildtype cells in between 0 and 500 J/m². Non-UVC-treated cells and wells containing only DMEM served as control and blank, respectively. After 48 h incubation in 100 µL DMEM, 15 µL of MTT dye solution per well were added. After 4 h incubation 100 µL stop solution (MTT dye and stop solution from Promega, Madison, WI, USA) were added, and plates were incubated overnight. Absorptions were captured with Tecan Sunrise (Tecan Trading AG, Männedorf, Switzerland) and the differences between the readings of two wavelengths (550 nm and 650 nm) were indicative of measured cellular metabolic activity. All results were set in relation to the respective non-irradiated cells (set as value 1).

RAW264.7 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% fetal bovine serum at 37 °C humidified air and 5% CO₂. Cells were seeded (2 × 10⁴/well) in 96-well plates and allowed to attach for 24 h before being treated with the compounds (1 and 2) or the solvent control (0.5% DMSO) for 1 h, followed by the addition of LPS at 1 μg/mL. Non-stimulated cells (no LPS) were tested simultaneously. The production of NO in the cell supernatant was measured after 24 h by measuring the nitrite concentration, which is an oxidative product of NO. A total of 50 μL of the supernatant was mixed with the Griess reagent using the manufacturer’s protocol (Promega) and the absorbance was measured at 540 nm. The nitrite concentration was derived from a calibration curve generated from a fresh nitrite standard solution. Cell viability was measured under the same seeding conditions and time points, using MTT dye following the manufacturer’s protocol (Promega).

Human colon cancer cells HCT116 were used as a representative model system to probe for antiproliferative activity. HCT116 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% fetal bovine serum at 37 °C humidified air and 5% CO₂. Cells were seeded (8000 cells/well) in 96-well plates, allowed to attach overnight and treated with (4E)-R-dysidazirine carboxylic acid (1), methyl ester (2), and the solvent control (0.5% DMSO). Cell viability was measured after 48 h, following treatment with MTT dye using the manufacturer’s protocol (Promega, Madison, WI, USA). IC₅₀ values were calculated from variable slope fitting for a dose response curve using GraphPad Prism software. Gatorbulin-1 was tested at the same time (IC₅₀ 0.80 μM) [11 (link)] and served as a positive control.