*2.7. Quantitative Real Time PCR (qPCR)*

The total RNA was extracted using Trizol reagent (Omgea, Norcross, GA, USA) according to the manufacturer's instructions. Next, the samples of RNA were reverse transcribed to generate cDNA using the Reverse Transcription Kit (TRAN, Beijing, China). Each 25 μL application contained 4 μL diluted cDNA, 10 μL qPCR PowerUp SYBR Green Master Mix (TRAN, Beijing, China), 1 μL forward and reverse primers (10 μM), and 4 μL RNA-free water. Two tests were performed for each gene in each biological replicate. The qPCR condition was as follows: 94 ◦C for 30 s (1 cycle), 94 ◦C for 5 s, 60 ◦C for 15 s, and 72 ◦C for 10 s (45 cycles). The results were subjected to relative quantitative analysis using *β-actin* as an endogenous control gene [26]. The primers used for qPCR are listed in the Table S1. The expression of relative genes was calculated by the 2–ΔΔCT method [18].

#### *2.8. Statistical Analysis*

SPSS version 20.0 (SPSS Inc., Chicago, IL, USA) was used for statistical analysis. LC50 values were determined using GraphPad Prism software version 9.0 (GraphPad Software, San Diego, CA, USA). The data were expressed as mean ± standard error of means (SEM). The results were demonstrated through three independent experiments. One-way analysis of variance (ANOVA) with Tukey's multiple range test was used to analyze cell activity differences. Differences among two groups were analyzed by unpaired *t*-test with Welch's correction. \* *p* < 0.05 was considered significant, and \*\* *p* < 0.01 was considered highly significant.
