Pcmv6 ac gfp vector backbone

MCF7 and MCF7-TAMR cells were cultured in Dulbecco's modified Eagle's medium (WelGENE Inc., Daegu, Korea). The medium was supplemented with heat-inactivated 10% FBS (WelGENE Inc.) and 1% penicillin-streptomycin (WelGENE Inc.). In case of the MCF7-TAMR cells, the medium additionally contained 1 μM 4-hydroxytamoxifen (Sigma, MO, USA). All cells were grown at 37 °C in a 5% CO₂ incubator. TAMR cells were generated as in vitro models of acquired tamoxifen resistance by exposing the parent cell lines MCF7 for a long-term with 1 μM 4-hydroxytamoxifen (Sigma) treatment. For overexpression studies, MCF7 cells were transfected with the HOXA5 plasmid (pCMV6-AC-GFP vector backbone; OriGene Technologies, Inc., Rockville, MD, USA) for 24 h using the Attractene transfection reagent (Qiagen, Hilden, Germany) according to the manufacturer's protocol. Knockdown studies were performed by transfecting TAMR cells for 24-48 h with 40 nM siHOXA5 using G-fectin (Genolution, Seoul, Korea) following the manufacturer's protocol. A pool of 5 individual siRNAs targeting exons 1 and 2 of HOXA5 were used for the knockdown.

Parental BT474, MCF7, T47D, MDA-MB-453, and MDA-MB-231 cell lines were provided by Drs. Yong Nyun Kim and Kyung Tae Kim (National Cancer Center, Korea). MDA-MB-157 and MDA-MB-468 cell lines were provided by Prof. Jong Hoon Park (Sookmyung women's University, Korea). These breast cancer cell lines were cultured as previously described26 (link), and periodically tested for mycoplasma contamination. For overexpression studies, cells were transfected with the GFP-HOXB2 plasmid (pCMV6-AC-GFP vector backbone; Origene, Rockville, MD, USA) for 24 h using the Attractene Transfection Reagent (Qiagen, Hilden, Germany) according to the manufacturer's protocol. GFP-HOXB2 plasmid-transfected cells were treated with neomycin (0.5 mg/ml; Thermo Fisher Scientific, Waltham, MA, USA) for 2 weeks to generate HOXB2-overexpressing stable cell lines. For transient knockdown studies, HOXB2-siRNA (Genolution, Seoul, Korea), HOXB-AS1-siRNA (Genolution), MATN3-siRNA (Genolution), SMOC2-siRNA (Genolution), ECM2-siRNA (Genolution), COL8A2-siRNA (Genolution), and negative control siRNA were transfected at a final concentration of 40 nM for 48 h. Sequences of siRNAs are available in Table S1.