Figure 2.

Testing the effect of SU086 on prostate cancer cell growth, migration, and invasion in vitro

(A) Proliferation of C4-2, DU145, and 22RV1 cells over 6 days in the presence of 1 μM SU086 or vehicle control measured by viable cell number via trypan blue exclusion assay.

(B) Colony-formation assay of C4-2, DU145, or 22RV1 cells grown for 9 days in the presence of 1 μM SU086 or vehicle control, graphed as colony-formation rate (colony number/500 cells plated ×100). Scale bar, 100 mm.

(C) LuCaP 136 and LuCaP 147 cells grown as tumoroids for 15 days in presence of 1 μM SU086 or vehicle control, with medium and compounds changed every other day. Graph represents area of well covered by tumoroids as an average of three wells ±SD. Scale bar (top), 300 μm; enlarged, 50 μm.

(D) Migration assay of C4-2, DU145, and 22RV1 cells following 72 h pretreatment with 1 μM SU086 or DMSO control. After pretreatment, viable cells were counted and the same number of cells were plated and cultured in transwell chambers for 20 h, with continued treatment of SU086 or DMSO control in serum-free (top of transwell), or serum-supplemented medium (bottom of transwell). Cells per field are graphed. Scale bar, 250 μm.

(E) Invasion assay of C4-2, DU145, and 22RV1 cells following 72 h pretreatment with 1 μM SU086 or DMSO (vehicle). Post-pretreatment, live cells were counted, and the same number of cells were plated and cultured in Matrigel-coated invasion transwell chambers for 20 h, with continued treatment of SU086 or vehicle. Scale bar, 100 μm.

(F) 3D Matrigel drop invasion assay of C4-2 and DU145 cells in presence of 1 μM SU086 or vehicle control measuring distance migrated after escape from the Matrigel dot, graphed as distance (mm). Scale bar, 250 μm. All experiments were performed in triplicate with triplicate wells. Representative experiments are shown.

For all, error bars represent standard deviation. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.005, ∗∗∗∗p < 0.001 determined by Student’s t test.