Fig. 5

A sequential exposure to palbociclib and PI3K/AKT/mTOR inhibitors, with palbocilcib maintained during all the treatment, induces synergistic effects. MDA-MB-231 (a-c) and HCC38 cells (d-f) were pre-incubated with 0.5 μM palbociclib for 24 h. Then, the cells were treated with increasing concentrations of BYL719, BEZ235 or BKM120 alone or in combination with palbociclib. After 48 h cell proliferation was assessed by CV assay. The effect of the drug combinations was evaluated using the Bliss interaction model. Data are mean values ±SD of three independent experiments (n = 5). g MDA-MB-231 cells were treated with 0.5 μM palbociclib for 48 h, 2.5 μM BYL719, 2.5 μM BKM120 or 50 nM BEZ235 for 24 h or were pre-incubated with palbocilcib for 24 h and then with palbociclib combined with the PI3K/mTOR inhibitors for further 24 h. Then the cells were stained with PI and the distribution of cells in cell cycle phases was determined by flow cytometry. Results are representative of three independent experiments (n = 5). *p < 0.05, **p < 0.01, ***p < 0.001 vs G0/G1 ctrl; ^#p < 0.05 vs G0/G1 Palb; ^§§§p < 0.001 vs G0/G1 BYL719, BEZ235 or BKM120. h The cells were treated as in g and the expression of the indicated proteins was evaluated on cell protein extracts by Western blotting. Results are representative of two independent experiments