A) Pim-2 inhibits cell proliferation. HCT116 cells were transfected with Pim-2 (WT or KD) only or co-transfected with wild-type p21 and Pim-2 (WT or KD). Forty hours post transfection, cells were trypsinized and used for the proliferation assay as described in the Materials and Methods. Relative proliferation rates were calculated as percentage of the sample with the highest luminescence reading. B) Pim-2 inhibits cell proliferation via phosphorylation of p21 on Thr145. HCT116 cells were co-transfected with Pim-2 and p21 (either T/D or T/A). Forty hours post transfection, the cell proliferation rate was determined as above. C) Phosphorylation of p21 on Thr145 is required for the inhibition of cell proliferation. HCT116 cells were transfected with different forms of p21 (WT, T/D, T/A, DD, or AA). Forty hours post transfection, cell proliferation rate were determined as above. Data represent means ± SD for four independent experiments, *=P< 0.01 as determined by student’s t-test.