Fig 2: Cohesin is the primary USP13 interactor in the nucleus.A, the USP13 epitope tagging vector was designed to modify the first coding exon (exon 1), adding a 1xFLAG-SBP (F-SBP) dual epitope tag immediately after the initiating methionine. Subsequent Cre-mediated recombination removed the FLOXed IRES-NeoR gene in intron 1. B, Western blot with USP13 antibodies was performed on extracts from parental HCT116 cells and two independently derived heterozygous USP13 epitope-tagged derivatives. This confirmed the presence of the expected equimolar doublet, comprising untagged USP13 protein from the untargeted allele, and a slightly larger epitope-tagged USP13 protein from the epitope-tagged, targeted allele. C, mass spectrometry was performed on dual-affinity purifications from USP13 epitope-tagged cells (and parental control cells). All proteins present in affinity purifications from epitope-tagged cells but absent in affinity purifications from untagged parental cells are shown. The Uniprot accession number, number of unique peptides, ion area, and percent coverage are shown. Ion area is considered a qualitative measure of relative protein abundance. Known subunits of the cohesin complex are in red.

Fig 3: Pharmacological inhibition of USP13 sensitises HPV + cervical cancer cells to BH3 mimetics.A Representative western blot of HeLa and SiHa cells after treatment with increasing doses of Spautin-1 or DMSO control for 48 h. Lysates were analysed for the expression of USP13 and Mcl-1. GAPDH was used as a loading control. B RT-qPCR analysis of MCL1 mRNA expression in HeLa and SiHa cells after treatment with Spautin-1 (10 µM) or DMSO control for 48 h. mRNA expression was normalized against U6 mRNA levels. C HeLa and SiHa cells were treated with Spautin-1 (10 µM) or DMSO control for 48 h. Cells were additionally treated with 10 µM MG132 for 6 h. Lysates were analysed for the expression of USP13 and Mcl-1. GAPDH was used as a loading control. D Representative western blot of HeLa and SiHa cells after transfection of FLAG-USP13 with or without Spautin-1 (10 µM) or DMSO control. Lysates were analysed for the expression of USP13 and Mcl-1. GAPDH was used as a loading control. E Cell viability assay (MTT) of HeLa and SiHa cells treated with increasing doses of ABT-263, with or without Spautin-1 (10 μM), for 24 h. F Synergistic activities of Spautin-1 and ABT-263 in HeLa and SiHa cells. Left: heatmaps of growth inhibition; right: Bliss synergy map. G Flow cytometric analysis of Annexin V assay in HeLa and SiHa cells after treatment with DMSO, Spautin-1 (10 μM), ABT-263 (5 μM) or both for 24 h. H Colony formation assay of HeLa and SiHa cells after treatment with DMSO, Spautin-1 (10 μM), ABT-263 (5 μM) or both for 24 h. Error bars represent the mean ± standard deviation of a minimum of three biological repeats. *p < 0.05, **p < 0.01, ***p < 0.001 (Student’s t-test).

Fig 4: Restoration of Mcl-1 expression partially rescues the proliferation defect in USP13 depleted cervical cancer cells.A Representative western blot of HeLa and SiHa cells after transfection of a pool of four specific USP13 siRNA, or scramble siRNA, for 72 h. After 24 h, cells were transfected with hMcl-1 or control vector. Lysates were analysed for the expression of USP13, Mcl-1 and GAPDH was used as a loading control. B Growth curve analysis of HeLa and SiHa cells after transfection of a pool of four specific USP13 siRNA, or scramble siRNA, for 72 h. After 24 h, cells were transfected with hMcl-1 or control vector. C Colony formation assay (anchorage dependent growth) of HeLa and SiHa cells after transfection of a pool of four specific USP13 siRNA, or scramble siRNA, for 72 h. After 24 h, cells were transfected with hMcl-1 or control vector. Bars are the means ± standard deviation from at least three biological repeats. *p < 0.05; **p < 0.01; ***p < 0.001 (Student’s t-test).

Fig 5: USP13 interacts with soluble cohesin complexes preferentially during DNA replication.A, whole cell lysates (W), chromatin lysates (C), and soluble lysates (S) were prepared from 293T cells and Western blot was performed with antibodies to USP13, STAG2, tubulin (soluble protein), and histone H3 (chromatin protein). B, cohesin complexes were immunoprecipitated with SMC3 antibodies from the protein lysates described in A, and Western blot was performed with the antibodies indicated. C–D, 293T cells were treated for 20 h with dimethyl sulfoxide (DMSO) vehicle alone (DM); hydroxyurea (HU) to arrest cells in S phase; RO-3306 (RO) to arrest cells in G2; and nocodazole (NOC) to arrest cells in mitosis. C, flow cytometry was performed to document the cell cycle arrests. D, SMC3 immunoprecipitations were then performed on whole cell lysates, followed by Western blot with USP13 antibodies. Quantification of USP13 bands in SMC3 immunoprecipitates using ImageJ is shown.