Human breast cancer cell lines MDA-MB-231 (derived from pleural effusion), MDA-MB-468 (derived from pleural effusion), MDA-MB-453 (derived from pericardial effusion), and T47D (derived from pleural effusion) were obtained from American Type Culture Collection (ATCC), and cultured in either α-MEM or RPMI 1640 supplemented with 10% fetal bovine serum (FBS). Human normal mammary gland epithelial cells (MCF-10A) were obtained from Dr. Linda Penn (Princess Margaret Cancer Centre Research Institute) and cultured in DMEM/HAM F12 supplemented with 5% horse serum, insulin, hEGF, hydrocortisone (Clonetics), and cholera toxin (Sigma-Aldrich). All cell lines were maintained at 37°C and 5% CO2, authenticated using Short Tandem Repeat analyses, and tested to be free from mycoplasma contamination.
Anti-miR-449a or pre-miR-449a mimic (Ambion) were reverse transfected into cells using Lipofectamine 2000 (Invitrogen) at a final concentration of 40 nM (unless otherwise indicated), according to the manufacturer's instructions.
CRIP2-expressing vectors were purchased (Applied Biological Materials) and transfected into MDA-MB-231 cells using Lipofectamine 2000. Independent single clones were obtained after 2 weeks of drug selection. qRT-PCR and Western blot were used to screen for positive clones.
Anti-miR-449a or pre-miR-449a mimic (Ambion) were reverse transfected into cells using Lipofectamine 2000 (Invitrogen) at a final concentration of 40 nM (unless otherwise indicated), according to the manufacturer's instructions.
CRIP2-expressing vectors were purchased (Applied Biological Materials) and transfected into MDA-MB-231 cells using Lipofectamine 2000. Independent single clones were obtained after 2 weeks of drug selection. qRT-PCR and Western blot were used to screen for positive clones.