Total RNA was isolated from MDA-MB-231 cells using RNeasy Plus Mini Kit (QIAGEN, Mississauga ON). Total RNA (1.0 μg) was used to synthesize complementary DNA (cDNA) using Bio-Rad Iscript cDNA Synthesis Kit (Bio-Rad, USA). TaqMan probes Hs00176619_m1, Hs00950344-m1, Hs 00195591_m1, and Hs02758991-g1 were used to quantify the expression of FRK, SLUG, SNAIL, and glyceraldehyde-3-phosphate dehydrogenase, as recommended by the manufacturer (Life Technologies, Burlington, ON, Canada). Briefly, 0.6 μl of cDNA, 0.5 μl of probes for each target and housekeeping genes, and 5 μl of TaqMan(R) Master Mix were added in each well. Distilled H2O was added in each well to make the volume of 10 μl. Probes for target genes and housekeeping genes were labeled with FAM and VIC dyes, respectively. The expression of both genes was measured within the same well using an Applied Biosystems, Step One Plus qRT-PCR machine (Life Technologies, Burlington, ON, Canada). In addition, total RNA was also prepared from the whole cell extract using Direct-zolTM RNA Miniprep plus kits (Zymo Research, Irvine, CA) according to the manufacturer’s instructions. Residual DNA was removed using deoxyribonuclease I. cDNA was synthesized using iScript Reverse Transcription Supermix (Bio-Rad), and the resulting cDNA was analyzed by qPCR using a MyiQ real-time detection system (Bio-Rad). The primers were used for PCR are listed in table S3.

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