Fig. 3: Loss of PIN1 reactivates APC/C^CDH1 and destabilizes mitotic proteins.

a Long-term colony-formation assay of indicated BC wild-type and PIN1 KO cell lines. Cells were grown for about 2 weeks, fixed and stained with crystal violet. b Gene Ontology (GO) enrichment analysis was applied to proteomics of PIN1 KO versus WT MDA-MB-231 cells. Color codes for p-value and symbol size codes for the ratio of proteins related to specific GO term/total number of proteins significantly altered. Data were obtained from Kozono et al. 2018 and analyzed by one-sided hypergeometric test. c IB analysis for indicated proteins derived from WT and PIN1 KO MDA-MB-231 cells synchronized in M phase by nocodazole and then released back into the cell cycle for the indicated time. d Cell-cycle profiles of WT (blue) and PIN1 KO (pink) in (c) as determined by FACS. e DNA contents were measured by FACS in WT and PIN1 KO MDA-MB-231 cells synchronized at the G1/S boundary by double thymidine block and then released back into the cell cycle for 4 h. f Cell cycle phase distribution of WT and PIN1 KO MDA-MB-231 cells from (e). n = 3 independent experiments. Data in graphs are mean ± SD and analyzed by unpaired two-sided t-test. WT vs. PIN1-KO, p = 0.0007 (0 h), p = 0.0006 (4 h). g, Tracking cell division (green square) and cell death (black rhomboid) at the single cell level. Asynchronous cultures of MCF-7 WT and PIN1 KO cells expressing the APC-degron reporter were followed for 72 h for single cell expression of mCherry-Geminin (shades of blue). h Frequency of G0/G1 arrest (ratio of G0/G1 arrested cells to total cells) in WT and PIN1 KO MCF-7 cells stably expressing the APC/C-degron reporter from (g). WT, n = 168 cells; PIN1 KO, n = 191 cells. The error bar indicates 95% confidence interval determined by bootstrapping. i Cycloheximide (CHX) chase assay for indicated proteins derived from WT and PIN1 KO MDA-MB-468 cells treated with 50 µg/ml CHX for the indicated time. The images were representative images from 3 independent experiments (a, c–e, i). Source data are provided as a Source Data file.