Figure 1.
GLI activity is high in androgen-independent cell lines.
(A) Analysis of GLI luciferase reporter activity in various androgen-independent cell lines and in comparison to the androgen-dependent LNCaP cell line. (B) Quantitative PCR analysis of GLI1 and GLI2 mRNA levels in the androgen-independent cell lines and in comparison to LNCaP cells (C) Cobblestone-like cells/colonies emerge in LNCaP cells with ectopic GLI1 or ΔNGLI2 expression (denoted by arrows). (D) qPCR analysis of PTCH1 mRNA expression in LNCaP-GLI1 and LNCaP-ΔNGLI2 cells. (E) qPCR analysis of GLI2 mRNA expression in LNCaP-GLI1 cells and GLI1 mRNA expression in LNCaP-ΔNGLI2 cells. (F) The morphology of LNCaP cells expressing EGFP does not change when co-cultured with LNCaP-GLI1 cells.
Figure 2.
GLI1 induces an androgen-independent phenotype in LNCaP cells.
(A) qPCR analysis of epithelial marker expression in LNCaP-GLI1 cells relative to LNCaP-pBP cells (n.b. the data is presented as natural logarithms so the relative induction of CD44 is almost 7000-fold). (B) Western blot analysis of AR and CD44 expression in LNCaP-pBP, LNCaP-GLI1 and DU145 cells (arrows denote CD44 isoforms common to LNCaP-GLI1 and DU145 cells). (C) FACS analysis of CD44 expression in LNCaP-pBP and LNCaP-GLI1 cells. (D) Proliferation assay to compare and to determine the effect of bicalutamide upon the proliferation rate of LNCaP-pBP and LNCaP-GLI1 cells as well as the effect of AG1478 (EGFR inhibitor) and U0126 (MEK inhibitor) upon the latter. (E) Analysis of the cell cycle by flow cytometry in LNCaP-pBP and LNCaP-GLI1 cells exposed to bicalutamide.
Figure 3.
Ectopic GLI1 induces global changes in the gene expression profile of LNCaP cells.
(A) A statistical comparison of global gene expression profiles to determine the percentage of transcripts that are expressed at significantly different levels in LNCaP-pBP, DU145 and PC-3 cells compared to LNCaP-GLI1 (Pearson correlation co-efficient ≥0.7, p<0.05) (B) Heat map denoting transcripts in LNCaP-GLI1 cells where the change in expression is both >10-fold and highly significantly different when compared to LNCaP-pBP cells (student's t-test, p<0.01): left panel lists increased genes, right panel lists decreased genes and DU145 and PC-3 cells are shown for comparison (* denotes transcript variants of the same gene). (C) Western blot analysis comparing the expression of certain signalling proteins between LNCaP-pBP and LNCaP-GLI1 cells with DU145 and PC-3 lysates included for comparison. (D) Phosphorylation of the cytoskeletal protein MLC2 is mediated by ROCK in LNCaP-GLI1 cells (n.b. the antibody for total MLC did not work in our hands).
Table 1.
Highly expressed transcripts in LNCaP-GLI1 cells.
Figure 4.
LNCaP-GLI1 cells display some stem-like characteristics.
(A) Western blot analysis comparing expression of the EMT markers E-cadherin and vimentin between LNCaP-GLI1 and LNCaP-pBP cells (n.b. the decrease of E-cadherin in LNCaP-GLI1 cells is partially reversed in the presence of the EGFR inhibitor AG1478 and to a lesser extent the MEK inhibitor U0126). (B) Transwell invasion assay comparing the invasive potential of LNCaP-pBP and LNCaP-GLI1 cells through a Matrigel substrate. (C) Clonogenicity assay assessing the colony-forming ability of LNCaP-pBP and LNCaP-GLI1 cells when seeded at low density. (D) Anchorage-independent growth is observed in LNCaP-pBP cells but not LNCaP-GLI1 cells (top panel - soft agar colony assay; bottom panel - prostasphere assay).
Figure 5.
GLI suppression does not induce a luminal-like phenotype in androgen-independent cells.
(A) The transformed morphology of LNCaP-GLI1 cells does not reverse upon transfection with GLI1 or GLI2 siRNA. (B) qPCR analysis of GLI1 and GLI2 mRNA in LNCaP-GLI1 cells transfected with GLI1 or GLI2 siRNA. (C) RT-PCR analysis of ΔNp63 and AR mRNA in LNCaP-GLI1 cells transfected with GLI1 or GLI2 siRNA. (D) qPCR analysis of GLI1 and GLI2 mRNA in DU145 and PC-3 cells transfected with GLI1 and GLI2 siRNA. (E) GLI reporter activity is suppressed in DU145 and PC-3 cells transfected with GLI1 and GLI2 siRNA (n.b. reporter activity may be influenced by GLI3 expression in PC-3 cells [17]). (F) RT-PCR analysis of ΔNp63 and AR mRNA in DU145 and PC-3 cells transfected with GLI1 and GLI2 siRNA.