Figure 6. Cloning efficiency of i-SOX2-T3M4 cells is reduced by treatment with trametinib or MK-2206 and is partially reversed by overexpression of SOX2.

A. Clonal growth was determined 8 days after plating i-SOX2-T3M4 cells at 80 cells per cm². The cells were treated for 8 days with trametinib (40 nM) or MK-2206 (2 μM) in the presence or absence of Dox (300 ng/ml) where indicated. Colony number was determined by an observer unaware of sample designation in 15 random fields. These studies were repeated and similar results were obtained. B. i-SOX2-T3M4 cells seeded at 1.2×10⁴/cm² were treated for 6 days with trametinib (40 nM) in the presence or absence of Dox (300 ng/ml) and representative photomicrographs were taken at the same magnification. C. After 6 days, the pre-treated cells were subcultured and plated in monolayer culture at 400 cells per cm² without trametinib or Dox. After 7 days the number of colonies formed was determined as described in the Materials and Methods and representative photomicrographs were taken at the same magnification. D. After 6 days, the pre-treated cells were subcultured at 800 cells per cm² and grown in soft agar containing serum-free, stem cell medium without trametinib and Dox. After 10 days colony numbers were determined as described in the Materials and Methods. Statistical significance was determined by student's t-test (*p<0.05 and ***p<0.005). The studies shown in A were repeated multiple times and similar results were obtained.