Figure 2.

Bcl-w Is Involved in IR-Induced EMT, Migratory Potential, Angiogenesis, and the Maintenance of Stemness

H460 and MDA-MB-231 cells were transfected with sh-Bcl-w or treated with IR (5 Gy). (A) Mesenchymal marker proteins containing Vimentin, Twist, Slug, and ZEB1 were determined by western blot analysis. β-actin was used as loading control. (B) After indicated cells were grown to monolayers in 6-well plates, cells were scratched and then incubated for 18 h. The confluent cells in three fields from the scratched area (200 × 500 μm²) were stained and counted. These experiments were performed in triplicate, and the mean value is shown in the graph (scale bar, 100 μm). (C) Levels of MMP-2 and MMP-9 mRNA were verified by q-RT PCR. (D) Left, after HUVEC treatment with conditioned media (CM) of H460 and MDA-MB-231 cells (top left), which were transfected with sh-Bcl-w or exposed with IR (5 Gy), prepared cells were seeded on Matrigel-coated 96-well plates (1 × 10⁴ cells/well) and then incubated for 6 h to assess tube-formation assay (scale bar, 100 μm, bottom right). Averaged numbers of capillary tube branches in eight random fields are counted and shown as a graph. Top right, levels of Bcl-w secreted to conditioned media were measured. Ponceau S was used as loading control. Angiogenesis-related proteins Ang2 and VEGF were detected by western blot analysis. β-actin was used as loading control. (E) Indicated treated cells were seeded onto 100-mm culture dishes (1 × 10³ cells/dish) and cultured for 5∼10 days. These experiments were performed in triplicate, and the mean value was shown (scale bar, 500 μm). (F) Indicated cells were seeded on a pre-coated 24-well plate with 60 μL of Matrigel and grown for 30 days in H460 cells. Colony number was counted every 10 days. These experiments were performed in triplicate (scale bar, 500 μm). (G) Cancer stem-like cell marker proteins containing Sox2, Oct4, and Notch2 were determined in H460 and MDA-MB-231 cells by western blot analysis. β-actin was used as loading control. All data are expressed as the mean ± SD of three experiments. (*p < 0.05, **p < 0.005, ***p < 0.0005, ****p < 0.00005, Student’s t test).