Fig 2: COPS5-mediated 4OHT-resistance is dependent on NCoR downregulation.(a) Western blot analysis of COPS5 and NCoR in MCF7 cells with transient expression of COPS5 and/or NCoR. (b) Quantitative RT–PCR analysis of ligand-regulated ERa target gene expression in parental and 4OHT-resistant MCF7 clones with expression of NCoR. *Significant difference compared with corresponding untreated basal levels (P<0.01, Student's t-test). (c) Western blot analysis of COPS5, NCoR and ERa expression in the 4OHT-resistant MCF7 cells stably transduced with COPS5 shRNA or rescuing COPS5 cDNA. (d) Quantitative RT–PCR analysis of ERa target gene pS2 in the MCF7 cell lines shown in c in response to 4OHT (500 nM, 48 h). *Significant difference compared with the untreated CTL sample (P<0.01, Student's t-test). (e) Western blot analysis of NCoR and ERa expression on knockdown by stable NCoR shRNAs in parental MCF7 cells. (f) Quantitative RT–PCR analysis of ERa target gene pS2 in the MCF7 cell lines shown in e in response to 4OHT (500 nM, 48 h). *Significant difference compared with the untreated CTL sample (P<0.01, Student's t-test). Data are presented as mean+s.e.m. of three biological replicates. CTL, control.

Fig 3: COPS5 overexpression leads to degradation of NCoR protein through the ubiquitin–proteasome pathway.(a) Cycloheximide (CHX) chase assay in parental MCF7 cells transduced with vector, HA-COPS5-WT or HA-COPS5-D151N. Cells were treated with CHX and then analysed by western blotting. (b) Half-life of NCoR protein was calculated by quantification of the western blots of anti-NCoR and anti-actin (for normalization). *Significant difference compared with other samples (P<0.05, Student's t-test). (c) Western blot analysis of COPS5, NCoR and ERa in MCF7 cells stably expressing empty vector (EV), WT or D151N COPS5 cDNA in the presence or absence of proteasome inhibitor MG132. (d) In vivo ubiquitination assay in HEK293T cells with overexpression of WT or D151N COPS5. Proteins pulled down by Ni-NTA beads and existed in whole cell lysates (WCL) are tested by western blot using antibodies shown on the left. His-Ubi, His6-tagged ubiquitin; Ubi-NCoR, polyubiquitinated NCoR protein. (e) Western blot analysis of NCoR expression on knockdown of COP9 subunits GPS1, CSN2 and CSN6 by specific shRNAs in 4OHT-resistant MCF7 clone #3. Ctl, control. (f) Quantitative RT–PCR of mRNA of ERa target gene pS2 in the MCF7 cell lines shown in e in response to 4OHT (500 nM) for 48 h. Data are presented as mean+s.e.m. of three biological replicates. All of the 4OHT-treated samples are significantly different from corresponding untreated samples (P<0.01, Student's t-test).

Fig 4: CSN5 inhibition results in the inactivation of a subset of CRLs and the stabilization of their substrates.Effect on CRL components and substrates after treatment of HCT116 cells with either 1 µM MLN4924 or 1 µM CSN5i-3. (a) Immunoblottings for Cul1 and its CRL components Rbx and Skp1; (b) for cullins Cul2, Cul3 and Cul4A; (c) for F-box protein Skp2 and the SCFSkp2 substrates p21 and p27; (d) for F-box proteins Fbxo22 and Fbxo30; (e) for F-box protein ßTrCP and the SCFßTrCP substrates Wee1 and pI?Ba; (f) for the adaptor KEAP1 and the Cul3KEAP1 substrate Nrf2; (g) for F-box proteins Fbxw7 and Fbxo3.

Fig 5: CSN5i-3 is a potent inhibitor of CSN5-catalysed cullin deneddylation.(a) Schematic illustration of the CRL cycle and the role of cullin neddylation and deneddylation. Ad, adaptor protein; SRM, interchangeable substrate recognition module; Sub, substrate. (b) Chemical structures of CSN5 inhibitors illustrating the optimization of the high throughput screening (HTS) hit CSN5i-1a to the cell active intermediate CSN5i-2, its R,R-enantiomer CSN5i-2e and to the final compound CSN5i-3. (c) Co-crystal structures of CSN5 with CSN5i-1b and CSN5i-3. The structures revealed the direction for compound extension into the substrate binding cleft of CSN5 (red arrow). (d) Dose–response curve for inhibition of CSN activity by CSN5i-3 as measured in a biochemical enzyme assay (n=2, ±s.d.). (e) Immunoblotting for Cul1 after treatment of HCT116 cells with 1 µM CSN5i-3 and for Cul1 and CSN5 after treatment with CSN5 siRNA.