Fig. 2: Role of copper binding in CDK1 activation and G2/M progression.

a Intersection analysis of copper-binding proteins (CBPs) from G2-stage HepG2 cells and G2/M progression regulatory proteins compiled from the AmiGO database. Lysates from the cytoplasm or nuclei of HepG2 cells synchronized to the G2 phase were examined using mass spectrometry (MS) after copper-bead pulldown. b The filtered results are charted for copper-beads enriched proteins. c Pulldown assay to verify copper binding to CDK1 within the nuclear and cytoplasmic compartments of HepG2 cells. d In vitro pulldown assay to assess the copper-binding affinity of purified GST-CDK1, using GST-ATOX1 and GST as positive or negative controls, respectively. e Pulldown assay gauging copper binding to GST-CDK1 wild-type (WT) or its copper-binding deficient mutants. f ICP-OES-quantified copper-binding capacities of 10 μg purified recombinant CDK1^WT and CDK1^CBM proteins. (P-values indicated on top). g, h Examination of the effects of copper-binding deficiencies of CDK1 on cell cycle progression using flow cytometry and FUCCI assay after transfecting cells with CDK1 ^WT or CDK1^CBM expression plasmids. The cells were synchronized to the S phase and then treated with 20 μM copper in the CBM groups, followed by flow cytometric analysis (g) or FUCCI-based assays (h). The same treatment condition was used hereafter, if not specifically noted. Scale bars represent 50 μm. i Analysis of H1.4 and p53 phosphorylation in cells with inducible CDK1 knockdown and transfected by HA-CDK1^WT or HA-CDK1^CBM. j Molecular modeling analysis of CDK1 protein structures to visualize the differences in conformation between WT and CBM mutants. k IP assay to examine interactions of transfected CDK1 WT or CBM mutant with endogenous CCNB1. In (f), unpaired two-tailed Student’s t-test was used for statistical analysis. In (f, g), data are representative of three biologically independent experiments. Source data are provided as a Source Data file.