## 2 Materials and Methods
### Reverse Transcription-Polymerase Chain Reaction (RT-PCR and qRT-PCR)  
A549 cells were seeded in a 6-well plate (\(1\times 10^{4}\)) and incubated for 24 h. The cell culture media were changed, and a new medium containing samples at various concentrations was added. After that, QIAzol lysis reagent (QIAGEN, Germantown, MD, USA) was used to extract RNA from the cells in accordance with the manufacturer's instructions. Reverse transcription was carried out using 1 ug of RNA and 20 uL of amfiRivert reverse transcription reagents (GenDepot, Barker, TX, USA). The procedure took place at the following temperatures: 25 \({}^{\circ}\)C for 5 min, 42 \({}^{\circ}\)C for 60 min, and 70 \({}^{\circ}\)C for 15 min. The RT-PCR procedure was run 35 times at 95 \({}^{\circ}\)C, 60 \({}^{\circ}\)C, and 72 \({}^{\circ}\)C for 30 s each. The 1% agarose gels with Safe Pinky DNA gel staining (GenDepot, Barker, TX, USA) were used to examine the amplified RT-PCR data and photographed under ultraviolet light. For the qRT-PCR, Enzymomics (Daejeon, Republic of Korea) SYBR TOPreal qPCR2V premix was used. In summary, the reactions were performed three times, and the ultimate 10 uL reaction volume included 1 uL of each of the forward and reverse primers, a 2\(\times\) Master Mix, and 1 uL of template cDNA. All real-time measurements were performed using a CFX Connect real-time PCR system (Bio Rad, Hercules, CA, USA). Amplification reactions were performed under the following conditions: 95 \({}^{\circ}\)C for 10 min, a total of forty cycles of 95 \({}^{\circ}\)C for 20 s, then 55 \({}^{\circ}\)C for 30 s, and 72 \({}^{\circ}\)C for 15 s. The comparative 2\({}^{-\Delta\Delta\text{Ct}}\) method was used to measure the relative amounts of mRNA, and the GAPDH gene was used to standardize the results. The lists of primer sequences (GenoTech, Daejeon, Republic of Korea) are provided in Table 1.  
\begin{table}
\begin{tabular}{l l} \hline \hline
**Gene** & **Primer Sequences (5/-3/)** \\ \hline \multirow{2}{*}{_p53_} & F: TCT TGGCCC TGT GTT ATC TCC \\  & R: CGC CCA TGC AGG AAC TGT TA \\ \hline \multirow{2}{*}{_Bcl2_} & F: GAA GGG CAG CCG TTA GGAAA \\  & R: GCG CCC AAT ACG ACC AAA TC \\ \hline \multirow{2}{*}{_BAX_} & F: GGT TGC CCT CT CTA CTTT T \\  & R: AGC CAC CCT GGT CTT G \\ \hline \multirow{2}{*}{_CASPASE 3_} & F: GAA GGA ACA CGC CAG CAA AC \\  & R: GCA AAG TGA AAT GTA GCA CCA A \\ \hline \multirow{2}{*}{_CASPASE 9_} & F: GCC CGA GTT TGA GAG GAA AA \\  & R: CAC CAG ACC AGG AC \\ \hline \multirow{2}{*}{_Cyto-c_} & F: CAGAAAGGTAAGGCC \\  & R: CGTCCAGCTGATGATG \\ \hline \multirow{2}{*}{COX-2} & F: CCT GAG CAT CTA CGG TTT GC \\  & R: ACT GCT CAT CAC CCC ATT CA \\ \hline \multirow{2}{*}{_TNF-\(\alpha\)_} & F: GCCCAAATGGCAGGCTT \\  & R: GGCCTAAGGTCCACTTTGCTG \\ \hline \multirow{2}{*}{_iNOS_} & F: CCT GAG CAT CTA CGG TTT GC \\  & R: ACT GCT CAT CAC CCC ATT CA \\ \hline \multirow{2}{*}{_IL-6_} & F: AGGGTTGCAGATGCAAATC \\  & R: AAACCAAAGGACACAGTGGAAC \\ \hline \multirow{2}{*}{_IL-8_} & F: CCCGAGAGAGAGAGACTTCACAG \\  & R: GGAAATTGGGTAGGAAGG \\ \hline \hline \end{tabular}
\end{table}
Table 1: Sequences of primers used for mRNA gene expression analysis by qRT-PCR.