4.4.9. Quantitative Real-Time RT-PCR

Cells were grown with or without different doses of genistein and solvent control for 5 h. Total RNA was isolated using the RNeasy Plus Mini Kit. The cDNA was synthesized using a reverse transcription reagent kit (Selleck, Shanghai, China). Target genes were amplified with the following specific primers in the Light Cycler® 96 Real-Time PCR System (Roche, Indianapolis, IN, USA):

GAPDH: 5- -GGAGCGAGATCCCTCCAAAAT-3- (forward) and 5- -GGCTGTTGTCATACTTCTCATGG-3- (reverse); FAK: 5- -TGGTGCAATGGAGCGAGTATT-3- (forward) and 5- -CAGTGAACCTCCTCTGACCG-3- (reverse); Paxillin: 5- -CTGCTGGAACTGAACGCTGTA-3- (forward) and 5- -GGGGCTGTTAGTCTCTGGGA-3- (reverse); Snail: 5- -TCGGAAGCCTAACTACAGCGA-3- (forward) and 5- -AGATGAGCATTGGCAGCGAG-3- (reverse); Twist: 5- -GTCCGCAGTCTTACGAGGAG-3- (forward) and 5- -GCTTGAGGGTCTGAATCTTGCT-3-(reverse).

GAPDH was used as a normalization control. Each treatment was tested in triplicate. The relative expression levels of genes were normalized using the 2−ΔΔCt method.

#### *4.5. Statistical Analyses*

Data are reported as the mean ± S.D. from at least three independent experiments. A *p* < 0.05 was used to measure statistical significance. Student's *t*-tests were used to compare statistical significance between the treated groups.

**Author Contributions:** T.C.: Data curation; Writing—Original draft preparation. J.W.: Data curation; Writing—Original draft preparation. M.L.: Data curation. Q.W.: Data curation; Reviewing and Editing. S.C.: Conceptualization; Supervision; Writing—Original draft preparation; Reviewing and Editing. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the National Natural Science Foundation of China (NSFC) (81703969), Scientific Research Foundation of Jiangsu Province, China (BK20211327), The Natural Science Foundation of Jiangsu Higher Education Institutions of China (21KJA360004) Jiangsu Province "Qinglan" project (2022), and Yangzhou University "Qinglan" project (2018).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author.

**Acknowledgments:** We acknowledge the National Natural Science Foundation of China, Scientific Research Foundation of Jiangsu Province, The Natural Science Foundation of Jiangsu Higher Education Institutions of China, Yangzhou University "Qinglan" project, and Jiangsu Province "Qinglan" project for the financial support.

**Conflicts of Interest:** The authors declare no conflict of interest.

**Sample Availability:** Samples of the compounds are not available from the authors.

#### **References**


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