Fig 2: Mechanisms supporting APC/CCdh1 activity under ER stress conditions.(A) HeLa cells were treated with DMSO, 0.5 µg/ml of TM alone, or 0.5 µg/ml of TM plus 5 µM of MG-132 for 16 h. Immunoprecipitates of endogenous Cdc27 were immunoblotted for endogenous Cdh1. Immunoprecipitation using IgG served as negative control. ** indicates Cdh1-specific top band; *indicates non-specific bottom band. (B) HeLa cells were treated with DMSO or 1 µg/ml of TM for 16 h. CDK2 or CDK1 antibodies were used to immunoprecipitate endogenous CDK2 or CDK1 complexes, respectively. Immunoprecipitates were then used in in vitro kinase assays using histone H1 as substrate. Left, autoradiography of 32P-histone H1 phosphorylated by CDK2 complexes. Coomassie stains input of histone H1 in the reactions. Intensity of the autoradioactive bands were quantified by Image J, normalized to histone H1 input, and presented as arbitrary units (A.U.). Immunoblot shows comparable levels of endogenous CDK2 and the indicated proteins before and after TM treatment. Right, autoradiography of 32P-histone H1 phosphorylated by CDK1 complexes, analyzed as indicated for CDK2. (C) Left, total cell lysates from HeLa cells treated with DMSO or 1 µg/ml of TM for 16 h were immunoblotted for the indicated endogenous proteins. Right, quantification of endogenous Emi1 mRNA levels in HeLa cells treated with DMSO or 1 µg/ml of TM for 16 h, measured by SYBR-green qRT-PCR.

Fig 3: APC/CCDH1 inactivation remains reversible until Rb inactivation in cells lacking CDK4/6 activity.a Cells born into DMSO or 1 µM CDK4/6 inhibitor were treated with 10 µM EdU for 15 min, and then assayed for EdU and phospho-Rb(S807/S811) (5000 cells plotted, 1 of n = 3 biological replicates). b Mitogen-released cells treated with DMSO or CDK4/6 inhibitor were measured for percent cells with high phospho-Rb(S807/S811) signal, high APC/C degron, and high EdU signal (after 15 min of 10 µM EdU treatment). Mean ± SEM from three biological replicates. c Emi1 ensures irreversible APC/CCDH1 inactivation. d–h Cells born into DMSO or CDK4/6 inhibitor and have inactivated APC/CCDH1 (with the exception of (f), where it is just cells born into DMSO or CDK4/6 inhibitor) were treated with 3 µM CDK1/2i (d, e) or an extra 100 mM salt (f–h). Dashed lines denote 25th and 75th percentile and bottom row panels are plotted in log2 scale for better visualization. APC/C degron detected via signal over background. Gray boxes denote conditions where cells reactivated APC/CCDH1. n = 2 biological replicates; n > 30 cells per condition. In g, h, to enrich for Rb inactivated cells in the 5 h post APC/CCDH1 degron detection conditions, only cells with cyclin E/A-CDK >0.8 right before treatment were analyzed (threshold determined from Fig. 3e).

Fig 4: EMI1 depletion causes rereplication and genomic stress in iSLK.219 cells. (A) Latent iSLK.219 cells were treated with the indicated siRNA (10 nM) for 24 h and 48 h (left) while LECs were treated with the indicated siRNA (30 nM) for 48 h (right). Cell cycle profiles were obtained after PI staining by flow cytometry. (B) iSLK.219 cells were treated as in (A) and 2 h prior to fixation the cells were maintained in complete medium containing EdU (10 µM). Graph shows quantification of the EdU-positive cells at the indicated time points (n = 3 biological replicates; bars show SD; ***, P < 0.001). (C) iSLK.219 cells were treated for 48 h with the indicated siRNA (10 nM) and afterward stained with 53BP1 or pCHK1-S317 antibodies. Nuclei were counterstained with Hoechst 33342 (scale bar = 20 µm). (D) iSLK.219 cells were treated as in (A) and subjected to immunoblot analysis for the indicated protein targets.