Figure 1. CDK4/6 inhibition induces ROS-mediated senescence and autophagy.
(a) IC50 values of palbociclib in ER+ (MCF7, T47D, ZR75-1) and HMEC (MCF10A) cells treated with increasing concentrations of palbociclib (0.01–12 μM). MCF7, T47D and MCF10A cells were treated with DMSO (Cnt) or palbociclib for 6 days, allowed to recover (rel) and subjected to (b) Clonogenic assay, (c) Cell counting and (d) SA-ß galactosidase activity measurement with representative images. (e) Impact of combined siRNA knockdown of CDK4 and CDK6 along with treatment with DMSO, 1 or 5 μM palbociclib for 6 days. (f) Measurement of MDC-positive acidic vesicles, including autophagosomes, by flow cytometry in MCF7 and T47D cells treated siRNA against CDK4/6. (g) Western blot for LC3B I, II and p62 upon treatment with palbociclib for 6 days. (h) Representative TEM microphotograph of cells treated with DMSO (Cnt) or 1 μM palbociclib for 6 days. Red arrows indicate double-membraned autophagosomes. Scale bars, 500 nm. (i) Western blot of LC3B and p62 in MCF7 and T47D cells treated with a combination of 25 μM CQ for 1 h and palbociclib for 6 days. (j,k) Quantification of GFP-LC3 puncta (j) and RFP-GFP-LC3 puncta (k) and representative images in MCF7 cells treated with 1 μM palbociclib and/or 25 μM CQ for 48 h. (l,m) Cellular ROS measurement and quantification (MFI) of ROS levels in MCF7 and T47D cells upon transfection with siRNA against CDK4 and CDK6 (l) or treatment with palbociclib for 6 days (m). (n,o) LC3B and p62 protein levels (n) and MDC +ve cells (o) upon combined treatment with 10 mM NAC or 0.1 mM Trolox and 5 μM palbociclib for 6 days. All data represent mean±s.d. from three independent experiments; P values were calculated in comparison with cells treated with DMSO (Control) unless indicated. NS: P>0.05; *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001.