Fig 2: The RNF20/40 complex catalyses the monoubiquitination of Eg5 at lysine 745.(a) Overexpression of the RNF20/40 complex increased the monoubiquitination of Eg5. GFP-RNF20 or/and GFP-RNF40 expression constructs were transfected into MCF-7 cells together with HA-Eg5 and Myc-Ubiquitin expression constructs. Immunoprecipitation assays were performed with antibodies against HA, followed by immunoblotting with antibodies against HA and ubiquitin. (b) RNF20/40 is essential for the monoubiquitination of endogenous Eg5. Cell lysates from MCF-7 cells transfected with control or RNF20/40 siRNAs were subjected to immunoprecipitation with anti-Eg5 followed by immunoblotting with anti-ubiquitin and anti-Eg5. (c) The RING-finger domain of the RNF20/40 complex is essential for catalysing the monoubiquitination of Eg5. MCF-7 cells were co-transfected with the indicated expression constructs and HA-Eg5. Cell lysates were subjected to western blotting with antibodies against the indicated proteins. (d) Identification of lysine residue monoubiquitinated in Eg5. Cellular extracts from MCF-7 cells expressing HA-Eg5 were immunopurified with anti-HA affinity columns and eluted with HA peptides. The eluates were resolved by SDS–PAGE and coomassie blue-stained. The protein band indicating Ub-HA-Eg5 on the gel were retrieved and analysed by LCMS/MS. (e) Monoubiquitination of Eg5 by the RNF20/40 complex in vitro. Recombinant GST-Eg5N, GST-Eg5M or GST-Eg5C was incubated in the presence of E1, E2, Ub and ATP with or without the RNF20/40 complex purified from Sf9 cells, followed by western blotting using antibodies against GST. (f) Eg5 is monoubiquitinated at K745 by the RNF20/40 complex. GFP-RNF20 and GFP-RNF40 expression constructs were co-transfected with FLAG-Eg5 or FLAG-Eg5K745A into MCF-7 cells. Cell lysates were subjected to western blotting with antibodies against the indicated proteins.

Fig 3: RNF20/40-Eg5 axis in breast carcinogenesis.(a) Depletion of Eg5, RNF20/40 and Eg5 inhibition suppressed breast carcinogenesis. MCF-7 cells infected by lentiviruses carrying empty vectors or MCF-7 cells with lentivirus-delivered Eg5, RNF20 or RNF40 knockdown, were transplanted into female athymic nude mice (n=6). Another two groups of mice with tumour xenografts developed from normal MCF-7 cells were mock treated or treated with monastrol via intraperitoneal injection. Tumours were measured weekly using a vernier calliper and the volume was calculated according to the formula: V=π/6 × length × width2. Each bar represents the mean±s.d. for six animal measurements. (b) Immunohistochemical staining of Eg5, RNF20 and RNF40 in breast cancer tissue microarrays. Normal IgG was used as a negative control. Scale bar, 100 μm. The expression of Eg5, RNF20 and RNF40 (H score) in 99 breast carcinoma tissues and 10 normal breast tissues was statistically analysed. (c) Correlation plot of the expression of Eg5, RNF20 and RNF40 in 99 breast carcinoma tissues. Pearson's correlation analysis was performed, and P<0.01 was defined as statistically significant. (d) Kaplan–Meier overall survival analysis of Eg5 mRNA expression in 3,458 breast cancer patients from a public data set (http://kmplot.com/analysis/index.php?p=service&cancer=breast).

Fig 4: Loss-of-function of RNF20/40 leads to cell G2/M arrest and apoptosis via downregulating Eg5.(a) The expression of Eg5 and RNF20/40 in different human cells lines. Total proteins from indicated cells were extracted, and western blotting was performed using indicated antibodies. (b) Depletion of Eg5 and RNF20/40 decreases the viability of MCF-7 cells. MCF-7 cells were treated with indicated siRNAs for MTT assays. (c) Colony formation assay. MCF-7 cells transfected with indicated siRNAs were maintained in culture media for 8 days and stained with crystal violet, and the number of colonies in each condition was counted. (d) Depletion of Eg5, RNF20/40 and inhibition of Eg5 activity induced G2/M arrest. MCF-7 cells treated with indicated siRNAs or 100 µM monastrol were stained with propidium iodide and subjected to flow cytometry. (e) Eg5 overexpression offset RNF20/40 depletion-induced G2/M arrest. MCF-7 cells with RNF20 or RNF40 depletion were transfected with the empty vector or HA-Eg5, and the cell cycle was analysed using flow cytometry. The P value between the indicated treatments is determined by Student's t-test. Western blotting was performed using antibodies against the indicated proteins. (f) Depletion of Eg5, RNF20/40 and inhibition of Eg5 led to cell apoptosis. Eg5, RNF20/40-depleted or monastrol-treated MCF-7 cells were stained with FITC-labelled annexin V and propidium iodide, followed by apoptotic analysis using flow cytometry. (g) Eg5 overexpression offset RNF20/40 depletion-induced apoptosis. The P value between the indicated treatments is determined by Student's t-test. Western blotting was performed using antibodies against the indicated proteins. For all the bar charts in Fig. 6, each bar represents the mean±s.d. for triplicate experiments and the P value were determined by Student's t-test. &P<0.01, #P<0.005, *P<0.001 versus control.

Fig 5: Ubiquitin ligase RNF20/40 interacts with the motor protein Eg5.(a) Immunoaffinity purification of RNF20-containing protein complexes. Cellular extracts from HeLa cells stably expressing FLAG (vector) or FLAG-RNF20 were immunopurified with anti-FLAG affinity columns and eluted with FLAG peptides. The eluates were resolved by SDS–PAGE and silver-stained. The protein bands were retrieved and analysed by mass spectrometry. (b) The RNF20/40 complex interacts with Eg5 in vivo. Immunoprecipitation assays were performed with antibodies against the indicated proteins followed by immunoblotting in HeLa, MCF-7, and PANC-1 cells. (c) Co-fractionation of Eg5 and the RNF20/40 complex by fast protein liquid chromatography. Cellular extracts from MCF-7 cells were fractionated on Superose 6 size exclusion column. The chromatographic profile with the elution positions of calibrating proteins of known molecular mass is shown. The chromatographic fractions were analysed by western blotting with antibodies against indicated proteins. (d) Eg5 interacts directly with RNF20 and RNF40 in vitro. GST pull-down assays were performed with GST-Eg5 and in vitro transcribed/translated RNF20 and RNF40. (e) Identification of the domains responsible for the direct interaction between Eg5 and RNF20 or RNF40. Bacterially purified GST, GST-Eg5N, GST-Eg5M or GST-Eg5C was incubated with in vitro transcribed/translated RNF20 or RNF40, after which GST pull-down assays were performed.