Fig 3. Stable ectopic expression of miR-193b inhibits anchorage-independent growth in soft agar.

Ewing sarcoma A673 and SK-ES-1 cells were stably transduced with the pMSCV-Puro retroviral vector expressing miR-193b, or empty vector (EV) control. (A) Relative miR-193b expression levels following selection were determined using RT-qPCR, with U6 as the internal control; mean and standard error of the mean (SEM) of two independent experiments, each performed in triplicate, normalized to empty vector control. (B) For soft agar assays, cells were harvested, counted and plated at 10,000 cells per well in 6-well plates containing 0.4% agar and growth medium with 20% serum. Colonies were stained with nitroblue tetrazolium 2–3 weeks later, and quantified using Nikon digital image analysis system (NIS-Elements). Results are represented as the mean and standard error of the mean (SEM) of 3 independent experiments, each performed in triplicate (*: p<0.05, relative to empty vector control); images from one experiment are also shown.