Fig. 1

RIZ2 overexpression in DLD1 cell line. A Immunofluorescence analysis of GFP in DLD1-pEGFP and DLD1-pEGFP_hRIZ2. Nuclei are stained in blue. Images were captured using a DC480 camera (Leica) and acquired by Application Suite (Leica) software and are representative of three independent experiments. B Western blot analysis with an antibody that recognizes both RIZ2 (185 kDa) and RIZ1 proteins. C Effects of RIZ2 overexpression on DLD1 cell viability. Cell number was obtained by microscopy count or extrapolated by WST-1 assay. DLD1 cells stable transfected with pEGFP_hRIZ2 or with pEGFP alone, as control, were cultured for 36- and 72 h and counted in the Bürker chamber. Fold increase vs the control cells is also illustrated. DLD1 cells stable transfected with pEGFP_hRIZ2 or with pEGFP alone, as control, were cultured for 24-, 48-, and 72 h and assayed with WST-1. Graphs represent the extrapolated cell number at the different time points. Three independent experiments were performed. *p < 0.05 and **p < 0.0001 for the indicated experimental points vs. the corresponding untreated control. D Representative images of clonogenic assay. Ten days after seeding, clones were counted, and their cellularity was evaluated by phase contrast microscopy. The histogram represents the average number of colonies of at least three independent experiments, each performed in triplicate (**p < 0.0001 vs. empty vector); on the right the fold change in colony number formation between pEGFP_hRIZ2 cells and pEGFP control cells (**p < 0.0001 vs. empty vector)