*2.2. Cell Maintenance*

A-549 cells were grown in DMEM high-glucose medium (Gibco, Oslo, Norway) with 10% FBS (Gibco, Oslo, Norway) supplementation, penicillin–streptomycin 2% (Gibco, Oslo, Norway), and amphotericin B 0.5% (Gibco, Oslo, Norway) in T25/T75 flasks (Greiner, Frickenhausen, Germany) and then stored in an incubator (Sanyo, Tokyo, Japan) at 37 ◦C, with 5% CO2. The medium was changed every three days and subcultured when in confluent conditions. Cells were harvested by accutase cell detachment (0.5 mM EDTA.4Na) (Gibco, Oslo, Norway) and grown in new flasks. Confluent cells that were not used for the experiment were stored frozen with a composition of 10% DMSO (Santa Cruz Biotechnology, Dallas, TX, USA) and 90% medium in a 1 mL cryo-vial, and then stored in a −80 ◦C freezer or cryotank.

#### *2.3. Adhesion Assay Using Cell Counting Kit-8 Assay*

The CCK test was carried out according to the manual of the CCK-8 Kit (Abbkine, Hubei, China). A quantity of 1.5 × 104 A-549 cells/100 mL was grown on a culture test 96-well plate (Greiner, Frickenhausen, Germany). Cells were incubated for 24 h, then divided into five groups, including the non-treated group (NT); AP3 80 μg/mL; ethanol extract of *Ocimum sanctum* Linn. (EEOS) at 50, 70, 100, or 200 g/mL; and cisplatin 9 g/mL. Each treatment was replicated three times. The treatments were incubated for 24 h, then 100 mL of water-soluble tetrazolium (WST-8) reagent was added to each sample and incubated for 4 h (in the dark). Then, the reaction was stopped by adding 100 mL/well of DMSO (Santa Cruz Biotechnology, Dallas, TX, USA). The results were read using an ELISA Reader (BioRad, Hercules, CA, USA) at a wavelength (λ) of 460 nm. The absorbance results obtained were then calculated using the following formula to obtain the percentage of viability.

$$\text{cell viability} (\%) = \frac{\text{treatment absorption} - \text{media absorption}}{\text{absorption cell control} - \text{media absorption}} \times 100\%$$

The final data were analyzed via one-way ANOVA using GraphPad Prism 7 software (La Jolla, CA, USA).

#### *2.4. A549 Cell Lysate Preparation*

A-549 cell lysate preparation was carried out according to the kit manual (Biomol, Hamburg, Germany). A total of 5 × <sup>10</sup><sup>5</sup> A-549 cells/mL were grown in each well on a tissue culture test 6-well plate and then incubated for 1 hour. The treatments in each well consisted of non-treatment (NT); AP3 80 μg/mL; ethanol extract of *Ocimum sanctum* Linn. (EEOS) at 50, 70, 100, or 200 g/mL; or cisplatin 9 g/mL, all followed by incubation for

24 h. The medium was aspirated and the plate was washed using Dulbecco's PBS (Gibco, Oslo, Norway), then 900 L of RIPA lysis buffer was added (Santa Cruz Biotechnology, Dallas, TX, USA), and the plate was shaken for 15 min. A cell scraper was used to remove the cells from the bottom of the plate. The lysate was transferred to 1.5 mL microtubes (Eppendorf, Hamburg, Germany). The lysate was centrifuged at 10,000× *g* for 10 min at 4 ◦C. The supernatant was transferred to 1.5 mL microtubes.
