Skip to main content
. 2024 Apr 15;15:3220. doi: 10.1038/s41467-024-47427-w

Fig. 5. Pharmacologic inhibition of PIN1 and CDK4 synergistically and irreversibly reactivates APC/CCDH1 to induce degradation of PIN1 and other mitotic proteins.

Fig. 5

a IB analysis of immunoprecipitates from WT and PIN1-KO MDA-MB-231 cells treated with Palbociclib. b Dot plots of FACS for PIN1-KO or WT MCF-7 cells stably expressing the APC/C-degron reporter and treated with AApin (1.5 μM ATO + 15 μM ATRA) for 3 days. Dots color, reporter levels. c Tracking cell division and cell death of MCF-7 cells in response to Palbociclib (4 μM), Sulfopin (10 μM) or AApin (1.5 μM ATO + 15 μM ATRA). d Frequency of G0/G1 arrest in MCF-7 cells from (c). n > 90 cells. The error bar indicates 95% confidence interval determined by bootstrapping. Detection of endogenous PIN1-CDH1 (e) and CDK4-CDH1 interactions (f) by PLA in the indicated cells treated with combination of 1 μM Palbociclib and 10 μM Sulfopin for 3 days. Nucleus were stained by DAPI. Scale bars, 5 μm. (Right) Quantification of PLA signals. n > 30 cells. Data are analyzed by unpaired two-sided t-test. p values are shown (d–f). g IB analysis for indicated proteins from WT and CDH1-KO MCF-7 cells treated with combination of 1 μM Palbociclib and AApin (ATO (0.5, 1, 1.5, 2 μM) + ATRA (5, 10, 15, 20 μM)) for 3 days. h IB analysis for indicated proteins derived WT and CDH1-KO BT-549 cells treated with 5 μM Sulfopin, 2.5 μM Palbociclib or their combination for 3 days. i CHX assay for indicated proteins from WT and CDH1-KO MDA-MB-231 cells pre-treated with combination of 10 μM Sulfopin and 1 μM Palbociclib for 36 h followed by 50 µg/ml CHX for the indicated time. j IB analysis of ubiquitinated PIN1 from WT and CDH1-KO MDA-MB-231 cells treated with 1 μM Palbociclib and 10 μM Sulfopin for 3 days and 2 μM MG132 for last 12 h and pulled down by Ni-NTA agarose. k, l Schematic diagrams showing the reciprocal inhibition of PIN1-APC/CCDH1 governs cell-cycle entry and exit. The images were representative images from 3 independent experiments (a, b, e–j). Source data are provided as a Source Data file.