Slug sirna

Cells were transfected separately with 300 pmol of E-cadherin shRNA (Addgene, Cambridge, MA, USA) or Slug siRNA (Santa Cruz Biotechnology, Inc.) by using Lipofectamine 2000 (Invitrogen). The levels of respective proteins were estimated by Western blotting. The Slug cDNA (Addgene) plasmid was used for overexpression studies. The Slug cDNA clone was introduced in cells by using Lipofectamine 2000. Stably expressing clones were isolated by limiting dilution and selection with G418 sulphate (Cellgro, a brand of Mediatech, Inc., Manassas, VA, USA) at a concentration of 400 μg/mL, and cells surviving this treatment were cloned and screened by Western blot analysis with specific antibodies.

Human ER+ breast cancer cell line MCF-7 was purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA). The adriamycin (ADR)-resistant MCF-7/ADR cells were generated by a conventional stepwise method that gradually increases the concentration of ADR over 8 months. The cancer cells were maintained in RPMI 1640 medium supplemented with 10% FBS, 2mM glutamine, 100 units of penicillin/ml and 100 μg of streptomycin/ml at 37°C and 5% CO₂.
Human Slug (Snai2/Slug, NM_003068) cDNA ORF clone (cat. RG202363), Human MMP1 (NM_002421) cDNA ORF Clone (cat. RG202460) and the empty control pCMV6-AC-GFP vector (cat. PS100010) were purchased from OriGene (Rockville, MD, USA). MCF-7 cells were transiently transfected with either the Slug cDNA ORF clone, MMP1 cDNA ORF clone or pCMV6-AC-GFP vector using Lipofectamine 2000 (Invitrogen).
The ready-to-use MMP1 siRNA was purchased from Santa Cruz (sc-41552), while Slug siRNA (5’-GCUACCCAAUGGCCUCUCU-3’) [17 (link)] were chemically synthesized by Ribobio (Guangzhou, China). MCF-7/ADR cells were transfected with 40 nM MMP1 siRNA or Slug siRNA using Lipofectamine 2000 (Invitrogen).