Figure 3

Overexpression of miR-9 enhances invasion of malignant mast cells and has no effect on cell proliferation or apoptosis. (A) Mouse P815 and C57 mast cells transduced with pre-miR-9-3 lentivirus or empty vector control were sorted to greater than 95% purity based on GFP expression. MiR-9 levels were assessed by real-time PCR in wild-type, empty vector, and miR-9 expressing cells (*p < 0.05). Three independent experiments were performed and all reactions were performed in triplicate. (B) Mouse P185 and C57 mast cells transduced with either empty vector or pre-miR-9-3 lentivirus were transferred onto cell culture inserts coated with Matrigel® for 24 hrs. After incubation, membranes were stained and cells that had invaded the membrane were counted in ten independent 20x hpf for each sample. Three independent experiments were performed and all assays were performed in triplicate wells (*p < 0.05). (C) Mouse P185 and C57 mast cells were transduced with either empty vector or pre-miR-9-3 lentivirus vector and cell proliferation was analyzed at 24, 48, and 72 hours using the CyQUANT method. Nontransduced P815 and C57 cells served as non-treated controls. Three independent experiments were performed and all samples were seeded in triplicate wells. Values are reported as percentage of untransduced control cells. (D) Mouse P185 and C57 mast cells transduced with either empty vector or pre-miR-9-3 lentivirus were assessed for apoptosis at 24 and 48 hours by measuring active caspase-3/7 using the SensoLyte® Homogeneous AMC Caspase-3/7 Assay kit. Relative fluorescence units are reported after subtraction of fluorescence levels of wells with medium only.