Hsa mir 205 pre mir mirna precursor

SKBr3 were transfected with 100 nM hsa-miR-205 Pre-miR™ miRNA Precursor (Thermo Fisher, Waltham, MA, USA) or Pre-miR™ Negative Control #1 (Thermo Fisher, Waltham, MA, USA) and 50 nM siRNA HER3 or siRNA negative control. Following 24 h of transfection, cells were seeded in 96 wells coated with matrigel (Corning, New York, NY, USA) at a density of 5 × 10³ cells/well and treated or not with 1 μg and 10 μg trastuzumab for 5 days. Images of colony formation were acquired using a EVOS inverted light microscope, and quantified by a macro made with the Image-Pro Plus 7.0 software. The mean ± S.D. of three replicates is reported.

Human HER2+ BC cell lines SKBr3 and BT474 were purchased from ATCC (Rockville, MD, USA) and cultured in RPMI 1610 medium with 10 % FBS and 1 mM L-glutamine.
Cell lines were obtained between 2000 and 2010, authenticated once a year (last verification on November 2015) using the short tandem repeat profiling method in our Institute facility, and propagated in the suggested media within six months of thawing from stocks.
For transfection experiments, cells were seeded in 6-well plates at 2 × 10⁵/well and transfected with hsa-miR-205 Pre-miR™ miRNA Precursor (Thermo Fisher, Waltham, MA, USA) or Pre-miR™ Negative Control #1 (Thermo Fisher, Waltham, MA, USA). For gene knock down, specific siRNAs (Thermo Fisher, Waltham, MA, USA) were used. Cells were incubated with 100 nM miRNA precursors or 50 nM siRNAs complexed with Lipofectamine 2000 transfection reagent (Thermo Fisher, Waltham, MA, USA) according to manufacturer's instructions.

SKBr3 were transfected with 100 nM hsa-miR-205 Pre-miR™ miRNA Precursor (Thermo Fisher, Waltham, MA, USA) or Pre-miR™ Negative Control #1 (Thermo Fisher, Waltham, MA, USA) for 24 h and treated with 2 μg Trastuzumab for additional 48 h. Cells were then trypsinized, washed in PBS and fixed in 70% ethanol for 2 h at −20° C. After fixation, cells were washed in PBS and centrifuged for 5 min at 1200 rpm. Cells were resuspended in PBS containing 10 mg/ml propidium iodide (Roche, Basel, Switzerland) and incubated for 20 min at 37° C. Cells were analyzed using FACS-Calibur flow cytometer and the results were further analyzed with the ModFit software, v3.2 (Verity Software House).