RNA isolation from SK-BR-3 and MDA-MB-231 cells was performed using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.). cDNA was prepared using the ReverTra Ace RT-qPCR kit (Toyobo Life Science) with 1 µg RNA per reaction. RT was performed at 37°C for 15 min followed by 98°C for 5 min to inactivate reverse transcriptase activity. qPCR was performed with the Thunderbird SYBR qPCR mix (Toyobo Life Science) and the thermocycling conditions were as previously described (22 (link)). The primers used are listed in Table SI . Relative MMP gene expression was calculated using the 2-ΔΔCq method (23 (link)), normalized to GAPDH and compared with the MMP3 level.
For semi-qPCR, the amplification conditions were the following: Initial denaturation at 95°C for 1 min, followed by 38 cycles of 95°C for 30 sec, 58°C for 30 sec and 72°C for 1 min for MMPs, and 23 cycles under the same conditions for GAPDH. Taq polymerase (Takara Bio, Inc.) was used as the DNA polymerase. Each condition was run in triplicate with the primers listed inTable SI . Amplified products were resolved by 1% agarose gel electrophoresis and visualized by 4S green plus (Sangon Biotech Co., Ltd.), and then photographed with a Bio-Imaging system (DNR Bio Imaging Systems). The band intensity of each lane was analyzed by ImageJ software (version 1.52a; National Institutes of Health) as described previously (24 (link)).
For semi-qPCR, the amplification conditions were the following: Initial denaturation at 95°C for 1 min, followed by 38 cycles of 95°C for 30 sec, 58°C for 30 sec and 72°C for 1 min for MMPs, and 23 cycles under the same conditions for GAPDH. Taq polymerase (Takara Bio, Inc.) was used as the DNA polymerase. Each condition was run in triplicate with the primers listed in
