Figure 6.

KLF5 is also crucial for androgen/AR signaling to promote cell proliferation and tumor growth in PCa cells. (a–d) Knockdown of KLF5 by siRNA in LNCaP cells or by shRNA in C4-2B cells reduced colony forming efficiency in 2-D culture (a, b) and sphere formation in Matrigel (c, d). Cells with KLF5 knockdown were seeded onto 6-well plates at 2000 cells/well for LNCaP and 1000 cells/well for C4-2B in regular medium for colony formation assay, and at 4000 cells/well for LNCaP and 2000 cells/well for C4-2B cells for sphere formation assay. Regular media were used, and enzalutamide (10 µM) treatment was applied. The culture time was 2 weeks for C4-2B and 3 weeks for LNCaP in both assays. Images of colonies or spheres were taken (left), and their numbers were counted (right). Only spheres with a diameter greater than 80 µm were counted. Scale bars, 200 μm. (e,f) Knockdown of KLF5 attenuated tumor growth of C4-2B cells in nude mice, as indicated by tumor images (e) and tumor weights at excision (f). (g,h) Knockdown of KLF5 reduced AR expression in xenograft tumors of C4-2B cells, as detected by immunohistochemical (IHC) staining with anti-KLF5 (g) and anti-AR (h) antibodies. (i–j) Knockdown of KLF5 reduced cell proliferation, as indicated by the Ki67 index, and the expression of cyclin D1 and MYC in tumor xenografts of C4-2B cells, as detected by IHC staining (i) and quantitation of positive cells (j). Scale bars, 100 μm. ns, not significant; *, p < 0.05; **, p < 0.01; ***p < 0.001.