Constitutive activation of Notch1 pathway enhances melanoma growth in vitro. (A) Cells were transfected with either N^IC-GFP or GFP. Equal numbers of stable transfectants were plated and photographed after 4 hours under fluorescence microscopy. Scale bar: 20 μm. More than 95% of green cells were observed in all other melanoma cell lines. (B) Upper panel shows COS7 cells transfected with N^IC-GFP or GFP lentiviruses were analyzed by Northern (left) and Western (right) blot. N^IC gene and protein expression are shown. 28S rRNA and β-actin were used as controls for Northern and Western blots, respectively. Lower panel shows expression of activated Notch1 in melanoma transfectants as detected by Western blot. (C) Cell growth was measured by ³[H]-thymidine incorporation assays. Ectopic expression of N^IC accelerated primary melanoma but did not affect metastatic melanoma cell growth. Data were normalized by setting the activities of GFP-transfected control cells at 100. Results are mean ± SD from triplicates in 3 independent experiments. *P < 0.005, Student’s t test. (D) Percentage of colonies formed in soft agar. N^IC enhances colony formation of primary but not metastatic melanoma cells. **Note that colonies formed by WM3248-N^IC-GFP cells are larger than those of control, although the numbers are not significantly increased. Data were normalized by setting the activities of GFP-transfected control cells as 100. Results are mean ± SD from independent experiments. ^#P < 0.001, Student’s t test. (E) Representative fields in soft agar plates. Scale bar: 500 μm.