Figure 5

Caspase processing of p27 abolishes RhoA binding and inhibition, and p27 dependent induction of cell migration and cell invasion. (a) Flag-tagged p27 proteins (wt or 1–176) were co-expressed with HA-tagged RhoA in HeLa cells. The immunoprecipitation of Flag-tagged proteins was subjected to western blot analysis for Flag- and potentially co-immunoprecipitated HA-tagged proteins (FLAG-IP, upper panels). The abundance of expressed proteins in total extracts is shown in western blot analysis (Input, lower panels). A representative experiment of three independent biological experiments is shown. (b) Pull-down assay for active GTP-bound RhoA from HeLa T-REx cells stably expressing p27 wild-type or a mutant p27 truncated at residue 176. The input and the pull-down fractions were blotted for p27, RhoA and β-tubulin. A representative blot of three independent biological experiments is shown. (c) Migration analysis of lentivirally transfected MEFs (GFP, p27 wt, p27-1-176) by time-lapse video microscopy. Pictures taken at 0 and 24 h are shown. Cells were serum starved for 36 h (0.1% FCS). At 0 h, cells were stimulated with 10% FCS and scratches performed as described in the methods. The border of adherent cells is marked by the dotted line. After 24 h, migration into the gap was determined and the novel border was indicated with a second dashed line (lower panels, right lines). The distance of cell migration by adherent cells was determined and compared to a GFP-transfected control. One out of three independent biological experiments summarized in (d, Exeriment #2) is shown. (d) Comparison of cell migration of p27 wt and p27-1–176 in comparison to GFP-transfected control cells. The relative increase or decrease compared to GFP-transfected cells is shown in percent. Three independent experiments (Ex.#1, Ex#2, Ex#3) as in c are shown. (e) MDA-MB-231 breast cancer cells were lentivirally transduced with a control plasmid (C), p27CK⁻ or p27CK⁻-1-176. Extracts were blotted for p27 and β-tubulin. ‘C’ indicates MDA-MB-231 cells transduced with a p27 construct in which codon 2 was mutated to a stop codon, which results in no exogenous expression of p27. (f) Transduced MDA-MB-231 cells were analyzed in transwell cell invasion assays and analyzed for cell invasion after 20 h. The changes of invasion in percent compared to cells transduced with a control plasmid are shown.