Figure 6. Palbociclib induces apoptosis in NCI-H295R cells by targeting the Wnt/β-catenin pathway. (a) Bar graphs showing apoptosis fold change in SW-13 or NCI-H295R cells upon treatment with either palbociclib or ribociclib. Caspase 3/7 activity was used as a read out to measure apoptosis. Results are the mean and standard deviations estimated based on three independent experiments. (b) Active β-catenin, total β-catenin, phospho-Ser9-GSK3β and total GSK3β were detected by western blot upon treatment with either palbociclib or ribociclib in both cell lines. GAPDH was used as a loading control. (c and d) Bar graphs showing the relative amount of phospho-Ser9-GSK3β on total GSK3β (c), or active β−catenin over total β-catenin (d) in both cell lines. Results are the mean and standard deviations based on two independent experiments. (e) Bar graphs showing levels of AXIN2 mRNA upon treatment with either palbociclib or ribociclib, in both cell lines. The values indicate the fold-change of mRNA levels after treatment with palbociclib or ribociclib, compared to mock-treated cells. AXIN2 mRNA levels in each condition were normalized to ACTNB (β-Actin) mRNA levels. qPCR were performed in triplicate. Mean and standard deviation are based on three different experiments. (f) Bar graph showing levels of secreted cortisol in the culture medium of NCI-H295R cells treated with ribociclib or palbociclib for 24 h and 48 h. Values are reported as percentage of concentration assayed with mock-treated cultures. Assay was performed in duplicate on culture media extracted from three different experiments. In (a), (c) and (d) Values are the mean ratio estimated from independent experiments. In (a), (c) and (d) significance was tested with t-test. In (e) and (f), significance was tested with the paired t-test. *P<0.05, **P<0.01.