Fig. 2

Brain colonization diminishes the growth and migration of metastatic lung cancer cells. A Cell growth of A549-M0 and M2 cells. Cells were counted using an automated cell counter (n = 3). Mean + SD. P, two-way ANOVA with Bonferroni post hoc tests. The adjusted P-value for the 4-day time point is < 0.0001. B Colonogenic assays of M0 and M2 cells. Colonies were stained with crystal violet at 6 days after seeding. C Soft agar colony assays of M0 and M2 cells. Colonies over 100 μm in diameter were counted at 3 weeks after seeding. Mean + SD (n = 3). P, unpaired two-tailed Student’s t-test. D Cell attachment assays of M0 and M2 cells. Cells (3 × 10⁵/well) were seeded into collagen-coated 24-well plates. Non-adherent cells were removed by washing with PBS after 30 min. Attached cells were measured by crystal violet staining. Mean ± SD (n = 3). P, two-way ANOVA with Bonferroni post hoc tests. The adjusted P-values for the 2-h and 3-h time points are 0.0038 and 0.0079, respectively. E Transwell migration assays of M0 and M2 cells. Cells (1 × 10⁵/insert) were cultured in upper wells for 24 h. Migrated cells were counted after staining with crystal violet. Mean ± SD (n = 3). P, unpaired two-tailed Student’s t-test. F Wound healing assays of M0 and M2 cells. Confluent cell cultures were subjected to scratch wounds and then incubated for 48 h in the presence of mitomycin C (1 μg/mL) to attenuate the proliferation-caused effect. Wound healing ratio (1 − [wound area ratio of 48–0 h]) was measured using ImageJ (http://imagej.nih.gov/ij). Mean ± SD (n = 12). P, unpaired two-tailed Student’s t-test. G Western blot of ZEB1, Vimentin, E-cadherin, N-cadherin, and Twist1 in M0 and M2 cells. β-actin was used as a loading control. H qRT-PCR of epithelial and mesenchymal markers in M0 and M2 cells. Expression levels were normalized to RPL32 mRNA levels. *P < 0.05, **P < 0.01, ***P < 0.001; unpaired two-tailed Student’s t-test