Xtt kit

Cells were seeded in 96-well culture plates at a concentration of 5×10³ cells/well. Cell viability was assessed for Day 2, 3 and 4 using the XTT kit (Biotium) according to manufacturer’s protocol. Absorbance was measured by ELISA plate reader (Perkin Elmer Envision, Waltham, MA, United States) at 500 nm with a reference wavelength at 650 nm.

For cytotoxicity assay, HepG2 cells were seeded in 96-well plates at a final density of 5 × 10⁴ per well and incubated at 37 °C for 24 h before drug treatment. The cells were treated with CDD-1733 or CDD-1819 or CDD-1845 at the final concentrations of 0, 50, 75, or 100 μM (3 replicates per concentration level per drug). After 24 h incubation at 37 °C, the medium in the plate was decanted. The cell viability was measured with an XTT kit (Biotium) by mixing 100 μL of DMEM (without phenol red) with 25 μL of XTT reagent and adding it into each well. The absorbance at 475 nm was read with a Tecan M1000 pro plate reader, with a reference wavelength of 660 nm. The reading was normalized by vehicle with the final 0.5% dimethylsulfoxide (DMSO) for each drug.

Cell proliferation was determined using XTT kit (Biotium) and cell viability kit (Enzo biosciences). Cultured EC were trypsinized, counted, and plated in a 96-well plate (1000–2000 cells/well). 24–48 h post plating, media was removed and cells were treated with GSK1016790A (GSK) (10–500 nM) in serum free media for overnight. Cells were washed once with PBS; and XTT or Calcein dye was added to the cells and incubated for 30 min-24 h. Wells containing no cells with the dye served as controls to determine the level of the background signal. Absorbance was read at 485–535 nm or 450–500 nm, respectively for Calcein and XTT.