Wound healing/scratch assay

MCF-7 cells (5 x 10⁴ cells/well) were cultured in 6 well plates with phenol red free RPMI media containing 5% charcoal stripped FBS at 37°C in CO₂ incubator. Then next day cells were transfected with 20 nM scrambled and PRL silencer select siRNAs using siPORTNeoFX reagent (Life Technologies) and grown in 1% charcoal stripped FBS till cells reach 90% confluent. Then cell monolayers were wounded by scratching with sterile 100 μL micropipette tips. Fresh medium containing 100 nM E₂ was added every 12 h. After 48 h cells were photographed under phase contrast inverted microscope and cell migration was assessed by measuring migration distance (wound gap) using ImageJ Software from National Institutes of Health. To study the effect of THZ1 inhibitor on cell migration, cells were pre-incubated with the inhibitor for 3 h prior treatment with E₂.