*4.9. Gene Expression Analyses*

A549 cells were seeded in 75 cm<sup>2</sup> cell culture flasks (3 × 10<sup>6</sup> cells/flasks) and grown for 24 h. Afterward, the cells were treated with subtoxic IC20 concentrations of DOX, 20 μg/mL extracts, or combination of IC20 DOX and 20 μg/mL extracts for 24 h. The control cells were grown in the culture medium. Following incubation, cells were collected by trypsinization, washed using PBS, and the collected cell pellet was stored at −80 ◦C until further experiments.

Total RNA was extracted from cells using TRI Reagent BD kit in line with the manufacturer's recommendations. RNA bands were visualized on a UV transilluminator, and RNA concentration was determined spectrophotometrically (BioSpec Nano, Shimadzu, Kyoto, Japan). Primary cDNA was prepared with RT-PCR using random primers, and 2 μg of total RNA was used as a template for MultiScribe reverse transcriptase in a high-capacity cDNA reverse transcription kit in line with the manufacturer's instructions.
