Ldh cytotoxicity assay

We assessed the plasma membrane integrity of HeLa cells by estimating the amount of lactate dehydrogenase (LDH) present in the culture medium. Briefly, we cultured HeLa cells in 96-well plates at a density of 1 × 10⁴ cells/well and then exposed them to six different concentrations of compound 1 (15, 10, 5, 1, 0.75, and 0.5 µg/mL) for 24 h. After treatment, we performed LDH cytotoxicity assay according to the manufacturer’s instructions (Abcam). Briefly, the culture plates were centrifuged at 600 × g for 10 min to precipitate the cells, 50 μL of clear cell culture supernatant was transferred from each well to a 96-well plate, and 100 μL of freshly prepared LDH reaction mixture was added to each well. After 30 min incubation at room temperature in the dark, absorbance was measured at a wavelength of 450 nm using a Synergy microplate reader (BioTek, Winooski, VA, USA). The LDH content was expressed as a percentage compared to control cells, which was considered 100%.

We performed the fluorometric LDH-cytotoxicity assay (Abcam, Boston, MA, USA ab197004) to assess the degree of cell death on spheroids in response to the exposure to our treatments. We collected medium samples (5 µL) from wells containing spheroids corresponding to the treatments. According to the manufacturer’s instructions, we assayed our samples plus negative and positive controls to determine the cytotoxicity percent. We used a Synergy HT microplate reader (Agilent, Santa Clara, CA, USA) to obtain the 535 and 587 nm fluorescence readings corresponding to the wavelengths for excitation and emission, respectively.

All cell lines were plated into a 96-well plate with growth media at 1 × 10⁴ cells/well and allowed to grow for 24 h, the cells were treated with nodakenin for 24 h. An LDH cytotoxicity assay (Abcam, Milpitas, CA, USA) was performed and analyzed according to the manufacturer’s protocol.