Spectramax id3 reader

A FAD assay was performed using a FAD assay kit (Abcam) according to the manufacturer's instructions. Briefly, 1× 10⁶ cells were lysed with the assay buffer and deproteinated with 50% trichloroacetic acid. FAD in lysate functions as a cofactor in an enzymatic reaction and produces fluorescence with OxiRed probe. Fluorescence (Ex/Em = 535/587 nm) was measured by SpectraMax iD3 Reader (Molecular Devices).

Hep3B and HepG2 cells were seeded in a 96-pillar tray at a density of 4000 cells per pillar in triplicate for each treatment. One day after seeding, cells were treated with drugs in a two-fold and seven dose point serial dilution from 100 µM to 3.12 µM. After six days of incubation at 37 °C in a 5%-CO₂ humidified incubator, cell viability was determined using an adenosine triphosphate (ATP) monitoring system based on firefly luciferase (CellTiter-Glo^® 3D Cell Viability Assay, Promega, Madison, WI, USA) and 3D live-cell staining (Calcein AM, Invitrogen, Carlsbad, CA, USA), according to manufacturer’s protocol. Briefly, the assay mixture was prepared in an ATP monitoring system by adding 20 µL of CellTiter-Glo^® 3D reagent into 60 µL of medium per well. Cells were lysed with an assay mixture by shaking for 5 min, followed by incubation for 25 min at room temperature. Viable cells were estimated using the SpectraMax iD3 Reader (Molecular Devices LLC, San Jose, CA, USA). For 3D live-cell staining, the staining solution was prepared by adding 1 µL of Calcein AM into 7 mL of DMEM medium. Cells were incubated with staining solution for 1 h at 37 °C in a 5% CO₂-humidified atmosphere. Live-cell images were acquired using an automatic fluorescence microscope scanner (ASTA Scanner™, Medical & Bio Decision, Suwon, South Korea)

For 2D-HTS analysis, HCC cell lines were mixed with fresh cell culture medium containing no ECM, and then samples prepared for 2D cell culture at a concentration of 2000 cells per 40uL volume were manually dispensed using a pipette. One day after seeding, using an ASFA Spotter DZ, drugs were dispensed in the same drug layout and concentration gradient as the 3D-HTS experimental conditions on a 384 well plate for 2D-HTS. After six days of incubation at 37 °C in a 5% CO₂ humidified incubator, cell viability was determined using the adenosine triphosphate (ATP) monitoring system based on firefly luciferase (CellTiter-Glo® Cell Viability Assay, Promega, Madison, WI), according to manufacturer’s protocol. The ATP assay mixture was prepared by adding 10 µL of CellTiter-Glo reagent to 40 µL of the medium per well. Cells were lysed with an assay mixture by shaking for 2 min, and luminescence was recorded 10 min after reagent addition using the SpectraMax iD3 Reader (Molecular Devices LLC, San Jose, CA).

The cardiolipin assay kit (ab241036, Abcam) was used to quantify the cardiolipin according to the manufacturer's instructions. Briefly, for cultured HEK293 cells, cells were trypsin digested and washed with PBS. Cells were then suspended in CL assay buffer and lysed by using sonicator. For mice heart issue, samples were homogenized in CL assay buffer and further lysed by using sonicator. After Centrifuge at 10000 g for 10 min at 4°C, the supernatant was transferred to a fresh tube and the protein concentration of that was quantified. Each sample containing 20 μg protein were mixed with 50 μL probe then incubated at room temperature for 10 min. Finally, fluorescence can be recorded at Ex/Em = 340/480 nm using a SpectraMax iD3 Reader (Molecular Devices).

LDH activity was measured using CyQuant LDH Cytotoxicity Assay kit (Invitrogen AB, Sweden) according to the manufacturer’s instructions.
CellTiter-Fluor™ Cell Viability Assay kit (Promega Corporation, Madison, WI, USA) was used to determine the relative cell numbers from each cell treatment. The companys’ instructions were used to determine the relative cell number by measuring the fluorescence, excitation 390 nm, and emission 505 nm with a SpectraMax^® iD3 reader (Molecular Devices, San Jose, CA, USA).
Lactate determinations were performed according to the manufacturer’s instructions using Lactate- Glo™ Assay kit (Promega Corporation, Madison, WI, USA). The lactate concentrations were measured by recording the luminescence using a SpectraMax^® iD3 reader with an open wavelength setting and 1000 ms integration time. To obtain an optimal readout, white opaque optiPlate-96-well plates (PerkinElmer, Waltham, MA, USA) were used.
The lactate concentrations from the different treated cells were calculated using a lactate standard from 0–100 µM, and all samples were diluted to fit the standard.