1. High proteasomal activity is involved in the palbociclib‐induced G1 arrest
High proteasomal activity is involved in the palbociclib‐induced G1 arrest AMCF7 cells were treated with 1 μM palbociclib for 0, 6, or 30 h to examine levels of endogenous proteins in response to palbociclib‐induced protein degradation. Additionally, a second set of cells were additionally treated with 10 μM MG‐132 for the last 1 h before sample collection to inhibit the proteasome. HMGCR and CDK4 protein levels are reduced by palbociclib treatment.BProtein levels of CDK4 were quantified from the Western blot samples (n = 4).CTop, Workflow schematic of cell cycle synchronization and analysis of cell cycle progression. Bottom, Representative DNA histograms of each sample at the time of analysis from MCF7 cells. Palbociclib prevents progression beyond the G1 phase, which can be overridden by addition of bortezomib.D, EQuantification of cell cycle status after G1 cell cycle synchronization and 15 h with 1 μM palbociclib with or without bortezomib treatment in MCF7 cells (D) (n = 4) and T47D cells (E) (n = 3).FProteasome activity levels, as measured by Me4BodipyFL‐Ahx3Leu3VS probe, in wild‐type (WT) and palbociclib‐resistant (Resistant) MCF7 and T47D cells (n = 4).GSame as (F), but data are from MCF7 and T47D cells after 6‐h treatment with 1 μM palbociclib. The data have been normalized to each control to enable comparison of the relative proteasome activation caused by palbociclib (n = 4).Data information: In panels (B, D–G), data are presented as means ± SD; each n represents an individual biological replicate. P‐values were determined by ANOVA and two‐tailed Student's t‐test with Holm–Sidak post hoc test; ns depicts not significant (P > 0.05); *: P < 0.05; **: P < 0.01; ***: P < 0.001.Source data are available online for this figure.
Teemu P Miettinen et al. EMBO J. 2018;embj
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