Fig. 5: PPM1D activity impairs OIS and promotes cell transformation.

A BJ-hTert-HRASV12^ER-TAM, BJ-hTert-HRASV12^ER-TAM-PPM1D-T1 and BJ-hTert-HRASV12^ER-TAM-PPM1D-T2 cells were induced or not with 4OHT for 20 d and stained for β-galactosidase activity. Plotted is the fraction of β-gal positive cells, bars indicate SD, n = 3. Statistical significance was calculated by Student’s t test (***p ≤ 0.001, ****p ≤ 0.0001). B Cells grown as in A were fixed and stained for p16. Plotted is the mean nuclear p16 intensity, bars indicate SD. More than 300 cells of each condition were quantified per experiment, n = 3. Statistical significance was calculated by Student’s t test (**p ≤ 0.01, ***p ≤ 0.001). C Cells as in A were induced with 4OHT for 5 to 20 days were incubated with EdU 24 h prior fixation followed by CLICK-IT reaction. Fraction of EdU positive cells was analysed using FACS. Bars indicate SD, n = 3. Statistical significance was calculated by Student’s t test (*p ≤ 0.05, **p ≤ 0.01, ****p ≤ 0.0001). D Whole cell lysates from cells from (C) were analyzed by immunoblotting using indicated antibodies. E BJ-hTert-HRASV12^ER-TAM, BJ-hTert-HRASV12^ER-TAM-PPM1D-T1 and -T2 cells were treated or not with 4OHT for 5 days. Whole cell lysates were probed with indicated antibodies by immunoblotting. F Cells treated as in (E) were analysed by flow cytometry. Where indicated, cells were incubated in the presence of Z-VAD-FMK. Plotted is the fraction of cleaved caspase 3 positive cells. Bars indicate SD, n = 3. Statistical significance was calculated by Student’s t test. G Parental BJ-hTert-HRASV12^ER-TAM, parental BJ-hTert-HRASV12^ER-TAM-PPM1D-T2, and BJ-hTert-HRASV12^ER-TAM-PPM1D-T2-60 cells that survived 2-month continuous induction with 4OHT were cultured in semisolid media for 10 weeks.