I control

For phase-contrast microscopy, CellSens Entry (Hamburg, Germany) version 1.18 was used. BD (Franklin Lakes, NJ, USA) FACSDiva™ (6.1.3) was used to operate the BD (Franklin Lakes, NJ, USA) FACSCanto™ II system. FlowJo (Franklin Lakes, NJ, USA) version 10.7.1 was used for further analysis of flow cytometry data. The TECAN multimode-microplate reader was operated with the software i-control™ (Männedorf, Schweiz) version 3.22. Rotor-Gene Q series (Hilden, Germany) version 2.3.4) was used to operate the Rotor Gene Q PCR-cycler and to analyze the results of the RT-qPCR. Cardiomyocyte beating analysis was performed with the in-house developed software CardioVision described in Section 3.3.

The total phenolic content (TPC) of the EO sample was performed with an adapted Folin–Ciocalteu method, as previously described [40 (link),41 (link)] with minor modifications. Briefly, 15 mg of the EO sample was weighted and mixed with 1 mL methanol. The mixture was vortexed and combined in a 1:5 ratio sample with Folin–Ciocalteu reagent (diluted 1:10 in distilled water to obtain a 0.25 N concentration) and left for five minutes in the dark at room temperature; after this, an equal volume of 7.5% sodium carbonate solution with Folin–Ciocalteu reagent was added, mixed, and left for 1 h in the dark at room temperature. The samples were plated in triplicate in 96-well plates, and the absorbance was measured at 725 nm with a spectrophotometer Tecan i-control, 1.10.4.0 infinite 200Pro. TPC was expressed in gallic acid equivalents (mg GAE/g sample) calculated after a propyl gallate calibration curve with concentrations between 0.375 mg/mL to 0.732 µg/mL.

Cells were diluted and seeded into a 96-well plate, with 2000 cells per well and three replicates for each time point. Then, the CCK-8 reagent was added to each well at 24, 48, 72, and 96 h post-seeding, according to the manufacturer′s protocol. After incubation for 2 h at 37 °C, the optical densities at 450 nm and 600 nm were separately measured for individual wells using a microplate reader (Tecan i-control).
For cell cycle analysis, cells were centrifuged at 500 × g for 4 min. Then, the cell pellets were washed once using 1 × PBS and resuspended using 1 × PBS containing 0.03% Triton X-100 and 50 μg/ml propidium iodide. After incubation at room temperature for 10 min, the cell cycle assay was performed on the BD Flow Cytometer.

After selection by puromycin, the survival (transfected) cells were trypsinized and counted using hemocytometer. Then cells were diluted and seeded into a 96-well plate, with 2000 cells per well and three replicates for each time point (add CCK-8 reagent at 24 hrs, 48 hrs, 72 hrs and 96 hrs according to the manufacturer’s protocol). After each treatment, cells were incubated for two hours at 37 °C, then the optical density (OD) at 450 nm and 600 nm were measured respectively for each well by microplate reader (Tecan i-control).
For cell cycle analysis, cells were trypsinized, then centrifuged at 500g for four minutes (mins). Cell pellets were washed once with 1×PBS and resuspended with PBS containing 0.03% Triton X-100 and 50μg/ml Propidium Iodide (PI), and incubate for 10 mins. The cell cycle assay was then performed on the BD Flow Cytometer.

Cultures were lysed and homogenised by freeze-thaw with dry ice and sonication in cold buffer. COX activity was evaluated using a COX Activity Assay Kit (760151, Cayman Chemical). Samples were prepared as indicated by manufacturer’s instructions and readings were obtained with the Tecan i-control, software 1.10.4.0 (Tecan, Crailsheim, Germany).