Fig. 2. The metastasis-suppressive effect of the PRAK inhibitor is dose- and time-dependent.

a Mice received an intravenous injection of B16 cells. Different doses of GLPG0259 were administrated daily from day 0 to 15 with vehicle control. Tumor development in the lung was examined at day 16. Representative imaging (left) and quantification of tumor colonies (right) are shown. b Mice received subcutaneous inoculation of B16 cells. GLPG0259 was given on day 10 at a daily dose of 2 mg/kg. The tumor volume was recorded at different time points. Data are presented as mean ±  s.d. with five mice for each group (left). The tumor weight was measured on day 16 (right). ns, not significant. c After intravenous injection of B16 cells, mice were treated with GLPG0259 between day 0–4 or day 5–15 with a daily dose of 2 mg/kg. The number of tumor colonies in the lung are shown with nine mice for each group. d, e B16 cells were transfected with a lentiviral vector expressing shPRAK under the control of a Tet-On promoter. Inducible knockdown of PRAK following doxycycline treatment was confirmed by Immunoblotting (d). The formation of lung metastases was examined at day 15 after intravenous injection of B16 cells bearing inducible shPRAK and shSCR. PRAK knockdown was induced by supplying 2 mg/ml doxycycline in drinking water 1 day before or 4 days after tumor cell inoculation. Data are present as mean ± s.d. with five mice for each group (e). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.001, ns no significant. p-value was determined by a two-tailed, unpaired t-test (a–c, e).