Mitopotential assay

For the various flow cytometric analyses, 1 × 10⁵ cells per well were seeded in triplicate in 6-well cell culture plates and treated with 5 μM of 4,5-diCQA for 72 hours. Cells were then harvested and stained as per the MUSE cell kit protocols. The various flow cytometric analyses performed are MUSE Annexin V & Dead cell assay (MCH100105), Mitopotential assay (MCH100110), Bcl2 Activation Dual detection assay (MCH200105), PI3K-MAPK Dual Activation detection assay (MCH200108), and Cell Cycle analysis (MCH100106) (EMD Millipore). The cells were analyzed as per the manufacturer's instructions provided in the kit (assay kit reference numbers provided in parenthesis above) using a MUSE cell analyzer. Each experiment was done at least 3 times independently.

The mitochondrial membrane potential changes were measured with the Muse^® Cell Analyzer (Merck Millipore, Burlington, MA, USA) using MitoPotential Assay (Merck Millipore, Burlington, MA, USA) according to the manufacturer’s protocol. Briefly, U-118 MG cells were treated with increasing concentrations of lycopene, [6]-gingerol, silymarin and DMSO for 24 and 48 h. Adherent cells were collected, washed and incubated with the Muse^TM Mitopotential Dye, a cationic, lipophilic dye to detect changes in the mitochondrial membrane potential for 20 min at 37 °C in the thermoblock (Eppendorf, Hamburg, Germany). Next, cells were incubated with 7-AAD, an indicator of cell death for 5 min at room temperature.