Fig. 5.

SPOP inhibits proliferation and migration of prostate cancer cell via CYCLIN E1. a SPOP stable knockdown cell line in DU145 was made with retroviral infection system. Cells were synchronized with double-thymidine block, and 8 h after release cells were pulse-labeled with 10 μM BrdU for 30 min and assayed with flow cytometry b Cell proliferation of SPOP knockdown cells in a, assayed with MTT. *means p-value < 0.05. c SPOP or CCNE1 was transiently knocked down with siRNAs in DU145 cells as indicated. BrdU-incorporation assay was carried out 4 h after release from synchronization. d, e SPOP and CYCLIN E1 stable expressed DU145 cells were generated with lentivirus expressing system. The real-time cell proliferation (d) and migration (e) were measured with RTCA assay according to the manufacturer’s protocol. f Plate colony formation assay of CYCLIN E1 and SPOP stable expressed DU145 cells. Colony numbers was counted and plotted as indicated. ***means p-value = 0.0005. g, h Xenograft experiments of SPOP and CYCLIN E1 stable expressed DU145 cells. In all, 5 × 10⁶ cells were injected subcutaneously into the nude mice and tumors were harvested 27 days later. Tumors were pictured (g) and tumor volumes were shown as mean ± SD, n = 9 (h). One tumor from each group was random picked and assayed with western blotting to confirm they are from original cell lines (g, right). * means p < 0.05