Figure 3.

Impact of SETDB1 on invasiveness and chemosensitivity. (a) Effect of SETDB1 on the invasion potential of A549 cells determined by the matrigel invasion assay. Cells were transfected with 3 μg of Flag-SETDB1 or empty vector in 60 mm dishes. After 24 h, cells were stimulated or not with phorbol myristate acetate (PMA) plus ionomycin (Io) for 30 min. Then cells were trypsinized, and 5 × 10⁴ cells were resuspended in serum-free media and added to the upper compartment of a transwell coated with 1 mg/ml Matrigel (BD Biosciences, Lexington, KY, USA). Media with 10% fetal bovine serum was added in the lower compartment and cells were incubated at 37 °C for 42 h. Invasive cells were fixed with phosphate-buffered saline 4% paraformaldehide, stained with 0.5% violet crystal and visualized and photographed under a × 10 magnification objective with a microscope. Invasive cells were counted using ImageJ 1.45s (Wayne Rasband, National Institutes of Health, Bethesda, MD, USA) and percentage of invasive cells were represented. Results are the mean of at least three experiments by duplicate and the significance was determined using analysis of variance test. *<P=0.05. (b) Cancer cells harboring the SETDB1 gene amplification are sensitive to the decrease in cell viability caused by mithramycin, a SETDB1-interfering drug. 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assays in control-scrambled shRNA DMS-273-transfected cells in comparison with three shRNA-stable downregulated SETDB1 clones (A21, A30 and A31) show enhanced inhibition of viability in cells with SETDB1 gene amplification-mediated overexpression.