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. 2020 Jan 20;19(4):479–491. doi: 10.1080/15384101.2020.1717025

Figure 4.

Figure 4.

The p27Kip1 S140A overexpressing cells have a growth advantage over p27Kip1 WT overexpressing cells. (a) To determine the effects of p27 S140 phosphorylation on cell growth, HEK293 cells were plated in 6 cm dishes and transfected with p27-WT and p27-WT plus WIP1-WT and on the next day non-transfected cells were irradiated with 5 Gy IR. Each cell was trypsinized and plated into 96 well cell culture. Growth of cells was monitored up to 4 days by cell proliferation assay using the CCK-8 kit. Left panel, Each time point was analyzed at N = 6.*** p < 0.001. Central panel, Bar graph of day 4 from left panel. Right panel, Expression of p27Kip1 and WIP1 protein in cells. Western blot analysis of cells from left panel were tested for p27Kip1 and WIP1 protein expression. After 24 h of transfection, cells in each condition were lysed with SDS-PAGE treatment buffer and subjected to immunoblot analysis with the indicated antibodies. GAPDH served as a loading control. (b) p27Kip1 S140 status affects cell growth. To determine the effect of phosphorylation of p27Kip1 S140 on cell growth, HEK293 cells containing stable inducible p27Kip1 WT, p27Kip1 S140D and p27Kip1 S140A expression constructs were plated 96 well plate and incubated with treatment of doxycycline (0.1 ug/ml) for 4 days. Relative cell numbers were determined by the CCK-8 kit to compare differences of cell growth in each cell line (upper panel). Expression of p27Kip1 in cells is shown in the lower panel. (c) Colony formation is reduced for cells transfected with WT p27Kip1 and p27Kip1 S140 vectors, but not p27Kip1 S140A vectors. In a clonogenic assay 1000 stable HEK293 cells containing inducible p27Kip1 WT, p27Kip1 S140D and p27Kip1 S140A expression system were plated in 6 well plates and grown for 2 weeks in the presence or absence of doxycycline. Colonies were stained with 0.5% crystal violet. (d) Bar graph quantitating the relative intensity of colonies formed in the clonogenic assay two weeks after plating. *, p < 0.05 and **, p < 0.01 (n = 3). P values were determined by t test.