p19^Arf inhibits tumor cell invasion. (A) Immunoblot analysis of BL185 HCC cells infected with retroviral vectors targeting luciferase or p19^Arf. β-actin serves as a control. (B) Immunoblot detection of p19^Arf in HCC cell lines infected with either pBabe puro retrovirus (PB) or pBabe puro retrovirus encoding p19^Arf (WT). β-actin serves as a control. (C) Migration (clear bars) and invasion (grey bars) activity of BL185-MLP-Luci and BL185-MLP-p19 cells. Data are from a representative experiment performed in triplicate. Error bars represent standard error of the mean (S.E.M.). The cell number is the number of migrated or invaded cells in five 50X microscopic fields per insert. The invasion index was calculated as described in the methods. (D) Migration (clear bars) and invasion (grey bars) activity of MM189-PB and MM189-WT cells. Data are from a representative experiment performed in duplicate. Error bars represent S.E.M. (E) Cell proliferation assay for MM189-PB and MM189-WT cells. (F) Anchorage-independent growth assay for MM189-PB and MM189-WT cells. The colony number is the total number for twenty 100 X microscopic fields in every plate. Data are from a representative experiment performed in duplicate. Error bars are S.E.M. (G) Migration and invasion assay for BL322-PB and BL322-WT cells. Error bars are S.E.M.

p19^Arf inhibition of cell invasion is dependent on the CtBP binding domain. (A) Schematic illustration of the p19^Arf mutants used in this study. (B) Immunoblot detection of expression of wild-type or mutant p19^Arf proteins in MM189 HCC cells. (C) Migration (clear bars) and invasion (grey bars) activity of MM189 cells infected with retroviruses encoding the indicated p19^Arf mutant proteins.

p19^Arf does not influence cell morphology or EMT. (A) Immunoblot analysis of epithelial and mesenchymal proteins in MM189 cells expressing either wild-type p19^Arf or the indicated p19^Arf mutant proteins. 3T3L1 serves as a positive control for expression of the mesenchymal marker vimentin. β-actin serves as a loading control. (B) Phase contrast image of MM189-PB and MM189-WT cells grown for 3 days on a collagen matrix. (C) Immunofluorescent images of MM189 cells expressing p19^Arf and vector controls. Left panel: p19^Arf positive cells (arrows) are labeled with FITC-conjugated antibodies, while p19^Arf negative cells (arrowheads) are not. Right panel: Rhodamine-conjugated phalloidin labels the actin cytoskeleton.

CtBPs regulate tumor cell invasion. (A) Detection of CtBP1 and CtBP2 by immunoblot after introduction of lentiviral vectors encoding shRNAs specific for either family member. Empty pLKO vector was introduced as a control. Protein bands corresponding to CtBP1 or CtBP2 are indicated by arrowheads. β-actin serves a a loading control (indicated by arrows). (B) Detection of ectopic V5 epitope-tagged CtBP2 by immunoblot with anti-V5 antibodies in MM189 cells transfected with pcDNA3-CtBP2 (PC/Ct) or empty vector control (PC). β-actin serves as a loading control. The band shown indicated by the arrow is non-specific. (C) Invasion activity, represented by invasion index (see methods) for MM189 cells infected with either control lentivirus or lentiviruses encoding shRNAs targeting either CtBP1 or CtBP2. (D) Invasion activity of MM189 cells expressing the p19^Arf L46D mutant and infected with either control lentivirus or lentiviruses encoding shRNAs specific for either CtBP1 or CtBP2. (E) Invasion activity of MM189 cells transfected with pcDNA3-CtBP2 or empty vector.