EMBO/SourceData huggingface dataset:
Figure 8A-C. Graph depicting a direct correlation between MT-flux and mitotic spindle where mean spindle length is plotted over mean MT-flux rates for all indicated conditions (A), mitotic conditions containing stable end-on attachments (B), and NDC80 depleted conditions lacking stable end-on attachments (C). Solid and dotted lines represent the linear regression and 95% confidence interval, respectively. Pearson correlation coefficient (r), coefficient of determination (r2) and corresponding p values are indicated. The error bars over x and y axis represent the standard deviation of MT-flux and spindle length, respectively.D. Representative images from spinning disk confocal live-cell time series of U2OS PA-GFP-α-tubulin/mCherry-α-tubulin cells under indicated treatments, with SiR-DNA-labelled DNA. Microtubules are shown in red and DNA in cyan in the merged image. Scale bar: 10 µm.E. Quantification of spindle lengths at metaphase following indicated treatments. Graph represents the spindle length of individual cells with mean ± SD. N (number of cells, number of independent experiments): siControl (49, 5), siMCAK (34,3), siKIF4A + siKIF15 + STLC (46, 6), siKIF4A + siKIF15 + STLC + siMCAK (43, 3), siKIF4A + siKIF15 + STLC + siKIF2A (22, 3), siCLASPs (64, 6), siCLASPs + siMCAK (33, 3), siCLASPs + siKIF2A (32, 3).
figf1(A, B) Two-dimensional tryptic mapping of YFP-p27, YFP-p27T198A and a mixture of both (A). Phosphoamino-acid analysis of spots a and b (B). The migration of phosphoserine (S), phosphothreonine (T) and phosphotyrosine (Y) is marked with circles in B. The asterisk indicates the origin of migration.(C-E) MCF-7 transfected with YFP-p27 or YFP-p27T198A mutant (C, D) or cotransfected with YFP-p27 and an empty RFP vector in a 5:1 (YFP-p27: RFP) ratio (E) were treated with (+) or without (−) MG132 (10 μM) for 12 h before western blot analysis (C) and fluorescence microscopy (D, E).(C-E) MCF-7 transfected with YFP-p27 or YFP-p27T198A mutant (C, D) or cotransfected with YFP-p27 and an empty RFP vector in a 5:1 (YFP-p27: RFP) ratio (E) were treated with (+) or without (−) MG132 (10 μM) for 12 h before western blot analysis (C) and fluorescence microscopy (D, E).(F) Western blots of protein extracts from MCF-7 transfected with YFP-p27, YFP-p27T198A or YFP-p27T198D mutants following incubation with cycloheximide (CHX) for the indicated times. Uncropped images of the blots are shown in the Supplementary Information, Fig. S5.
figf2(a) Crystal violet staining of MCF-7 cell transfected with YFP (C1), YFP-p27, YFP-p27T198A or YFP-p27T198D and selected with G418 for three weeks. Data are presented as mean ± s.d. (n = 3).(b) Flow cytometry analysis of MCF-7 cells transfected with YFP (C1) or YFP-p27, YFP-p27T198A and YFP-p27T198D for 48 h. Data are presented as mean ± s.d. (n = 3).(c) GFP-LC3 U2OS cells 48 h post-transfection with RFP, RFP-p27, RFP-p27T198A or RFP-p27T198D. Data represent results of three independent experiments, from which a total of ∼200 cells were counted (s.d., 10∼15%). The scale bars represent 50 μm in c.
figf3(a, b) MCF-7 cells were cultured for 24 h following siRNA with or without FBS before western blot analysis (a) and cell-viability assays (b).(c) MCF-7 cells were transfected with vector alone (C1), YFP-p27 (WT), or the siRNA-resistant constructs WT-R and T198A-R 24 h before RNA interference (RNAi). PARP cleavage was determined by western blotting and densitometric scanning. Data represent three independent experiments (mean ± s.d.).(d) Western blots of lysates of MCF-7 cells transfected with siRNA before serum-starvation or treatment with LY294002 (LY, 10 μM) or rapamycin (Rap, 100 nM) for 48 h with or without methylpyruvate (MP, 10 mM).(e) MCF-7 cells treated with or without LY294002 for 48 h following RNAi. p27 siRNA pool-1, siRNA1, 2 and 3; arrows, membrane blebbing; triangles, apoptotic bodies. The scale bar represents 50 μm.(f) Western blots of protein extracts from MCF-7 cultured with (+) or without (−) glucose for 24 h following RNAi. Fr, fragment; percentage PARP cleavage = 100 × PARPFr / (PARP + PARPFr).
figf4(a) Western blots of lysates from the indicated cell lines.(b) Pearson correlation analysis of p27 and LKB1 protein levels quantified by reverse phase protein array assays in 74 randomly selected primary breast tumors (MDACC).(c) Western blots of protein lysates from Hela cells transfected with either pCDNA3 or wild-type LKB1 cultured with or without glucose for 24 h.(d-f) Western blots of protein extracts from MEFs treated with AICAR (d and e),(d-f) Western blots of protein extracts from MEFs treated with AICAR (d and e)(d-f) Western blots of protein extractsfrom 3T3L1 cells cultured with or without glucose for 30 min (f).(g) Western blot analysis of 3T3L1 cells 72 h post-transfection RNAi with or without glucose starvation for 1 h before analysis. Densitometric quantification of western blots is shown. Uncropped images of the blots are shown in the Supplementary Information, Fig. S5.
figf5(A) FACS analysis of TUNEL positive p27+/+ and p27−/− cells treated with AICAR for 20 h. Data represent mean ± s.d. of three independent experiments.(B, C) Electron microscopy of p27+/+ (a, c and e) and p27−/− (b, d and f) MEFs treated with (c and d), or without (a and b) AICAR (1 mM), or without glucose (e and f) for 14 h. Magnified images of the boxed areas were shown on the left (a′, b′, c′, d′, e′ and f′) with arrowheads indicating autophagic vacuoles including autolysosomes (black arrowheads). The scale bars represent 10 μm in a-f and 0.5 μm in a′-f′. N, nucleus. Autophagic vacuoles were counted for three randomly selected cells from each electron microscopy section and data represent mean ± s.d. (C).(B, C) Electron microscopy of p27+/+ (a, c and e) and p27−/− (b, d and f) MEFs treated with (c and d), or without (a and b) AICAR (1 mM), or without glucose (e and f) for 14 h. Magnified images of the boxed areas were shown on the left (a′, b′, c′, d′, e′ and f′) with arrowheads indicating autophagic vacuoles including autolysosomes (black arrowheads). The scale bars represent 10 μm in a-f and 0.5 μm in a′-f′. N, nucleus. Autophagic vacuoles were counted for three randomly selected cells from each electron microscopy section and data represent mean ± s.d. (C).
Figure 1A Immortalized Brca1F/−MEFs were infected with retroviruses expressing the indicated shRNAs and/or Cre recombinase, followed by selection with puromycin for 72 h. Cell extracts were prepared 48 h later and analysed by Western blotting as indicated. SMC1 and tubulin were used as loading controls. *non‐specific band.B Cells treated as in (A) were fixed 48 h after selection and stained with an anti‐53BP1 antibody (green). Telomeres were visualized with a Cy3‐conjugated (CCCTAA)3‐PNA probe (red). Yellow arrowheads point to 53BP1 foci that co‐localize with telomeres.C Quantification of TIFs in cells treated as in (B). A minimum of 200 nuclei were scored for each sample. Error bars represent SD of two independent experiments. P‐values were calculated using an unpaired two‐tailed t‐test. *P ≤ 0.05; NS, P > 0.05.
Figure 2A Immortalized Brca1F/−MEFs were infected with retroviruses expressing the indicated shRNAs and/or Cre recombinase, followed by selection with puromycin for 72 h. Cells were arrested in mitosis with colcemid and mitotic chromosomes were processed for CO‐FISH analysis. Metaphase chromosome spreads were stained with Cy3‐conjugated leading strand telomeric PNA probe (red) and FITC‐conjugated lagging strand telomeric PNA probe (green). DNA was counter‐stained with DAPI (blue).B The frequency of chromosome‐type telomeric fusions in cells treated as in (A) was quantified as a percentage of total number of chromosomes (illustrated in Supplementary Fig S2A). A minimum of 2,000 chromosomes were scored for each treatment. Error bars represent SD of at least two independent experiments. P‐values were calculated using an unpaired two‐tailed t‐test. *P ≤ 0.05; **P ≤ 0.01; NS, P > 0.05.
Figure 3A Immortalized Brca1F/− MEFs were infected with retroviruses expressing the indicated shRNAs and/or Cre recombinase, followed by selection with puromycin for 72 h. Mitotic chromosomes isolated 48 h later were fixed and stained with a Cy3‐conjugated (CCCTAA)3‐PNA probe. The frequency of end‐to‐end chromosome‐type fusions is represented as a percentage of fusions observed after TRF2 depletion. A minimum of 2,000 chromosomes were scored for each sample. Error bars represent SD of three independent experiments. P‐values were calculated using an unpaired two‐tailed t‐test. NS, P > 0.05.B Immortalized Brca1F/C61G MEFs were infected with retroviruses expressing the indicated shRNAs and/or Cre recombinase, followed by selection with puromycin for 72 h. The frequency of end‐to‐end chromosome‐type fusions was analysed as in (A).C-F MEFs of the indicated genotypes were infected with retroviruses expressing TRF2 and/or CtIP shRNAs, followed by selection with puromycin for 72 h. Cell extracts were prepared 48 h later and analysed by Western blotting as indicated. GAPDH was used as a loading control. *non‐specific band.C-F MEFs of the indicated genotypes were infected with retroviruses expressing TRF2 and/or CtIP shRNAs, followed by selection with puromycin for 72 h. Cells treated as in (C) and (E) were arrested in mitosis with colcemid, and mitotic chromosomes isolated 48 h later were fixed and stained with a Cy3‐conjugated (CCCTAA)3‐PNA probe (D, F). The frequency of end‐to‐end chromosome‐type fusions is represented as a percentage of fusions observed after TRF2 depletion. Error bars represent SD of two independent experiments. P‐values were calculated using an unpaired two‐tailed t‐test. *P ≤ 0.05.C-F MEFs of the indicated genotypes were infected with retroviruses expressing TRF2 and/or CtIP shRNAs, followed by selection with puromycin for 72 h. Cell extracts were prepared 48 h later and analysed by Western blotting as indicated. GAPDH was used as a loading control. *non‐specific band.C-F MEFs of the indicated genotypes were infected with retroviruses expressing TRF2 and/or CtIP shRNAs, followed by selection with puromycin for 72 h. Cells treated as in (C) and (E) were arrested in mitosis with colcemid, and mitotic chromosomes isolated 48 h later were fixed and stained with a Cy3‐conjugated (CCCTAA)3‐PNA probe (D, F). The frequency of end‐to‐end chromosome‐type fusions is represented as a percentage of fusions observed after TRF2 depletion. Error bars represent SD of two independent experiments. P‐values were calculated using an unpaired two‐tailed t‐test. *P ≤ 0.05.
Figure 4A Immortalized MEFs were infected with retroviruses expressing the indicated shRNAs, followed by selection with puromycin for 72 h. Cell extracts were prepared 48 h later and analysed by Western blotting as indicated. SMC1 was used as a loading control. *non‐specific band.B Quantification of the frequency of end‐to‐end chromosome‐type fusions of cells treated as in (A) represented as a percentage of fusions observed after TRF2 depletion. A minimum of 2,000 chromosomes were scored for each sample. Error bars represent SD of three independent experiments. The P‐value was calculated using an unpaired two‐tailed t‐test. NS, P > 0.05.Immortalized Brca1F/− MEFs were infected with retroviruses expressing the indicated shRNAs and/or Cre recombinase. Mitotic chromosomes isolated 48 h later were fixed and stained with a Cy3‐conjugated (CCCTAA)3‐PNA probe. The frequency of end‐to‐end chromosome‐type fusions is represented as a percentage of fusions observed after TRF2 depletion. A minimum of 2,000 chromosomes were scored for each sample. Error bars represent SD of three independent experiments. P‐values were calculated using an unpaired two‐tailed t‐test. NS, P > 0.05.D Immortalized Brca1F/−MEFs were infected with retroviruses expressing TRF2 shRNAs and/or Cre recombinase, together with a lentivirus expressing PARP1 shRNA, followed by selection with puromycin for 72 h. Cell extracts were prepared 48 h later and analysed by Western blotting as indicated. SMC1 was used as a loading control. *non‐specific band.E Quantification of the frequency of end‐to‐end chromosome‐type fusions of cells treated as in (D) represented as a percentage of fusions observed after TRF2 depletion. A minimum of 1,500 chromosomes were scored for each sample. Error bars represent SD of three independent experiments. The P‐value was calculated using an unpaired two‐tailed t‐test. NS, P > 0.05.
Figure 5A Immortalized MEFs were infected with retroviruses expressing the indicated shRNAs, followed by selection with puromycin for 72 h. Cell extracts were prepared 48 h later and analysed by Western blotting as indicated. SMC1 and GAPDH were used as loading controls. *non‐specific band.B MboI‐ and AluI‐digested DNA from cells treated as in (A) was resolved by pulsed‐field gel electrophoresis and probed with end‐labelled (AACCCT)4 probe. Representative pulsed‐field gel samples run under native and denatured conditions are shown.C Quantification of the 3′ overhang in cells treated as in (B). For each sample, the ss/total DNA ratios were expressed relative to the GFP shRNA‐treated control. Error bars represent SD of two independent experiments.
Figure 6A-C MEFs of the indicated genotypes were infected with retroviruses expressing the indicated shRNAs, followed by selection with puromycin for 72 h. Mitotic chromosomes isolated 48 h later were fixed and stained with a Cy3‐conjugated (CCCTAA)3‐PNA probe. The frequency of end‐to‐end chromosome‐type fusions is represented as a percentage of fusions observed after TRF2 depletion in each experiment. A minimum of 1,200 chromosomes were scored for each sample. Error bars represent SD of two independent experiments. P‐values were calculated using an unpaired two‐tailed t‐test. *P ≤ 0.05; NS, P > 0.05. Each graph represents a separate set of experiments.A-C MEFs of the indicated genotypes were infected with retroviruses expressing the indicated shRNAs, followed by selection with puromycin for 72 h. Mitotic chromosomes isolated 48 h later were fixed and stained with a Cy3‐conjugated (CCCTAA)3‐PNA probe. The frequency of end‐to‐end chromosome‐type fusions is represented as a percentage of fusions observed after TRF2 depletion in each experiment. A minimum of 1,200 chromosomes were scored for each sample. Error bars represent SD of two independent experiments. P‐values were calculated using an unpaired two‐tailed t‐test. *P ≤ 0.05; NS, P > 0.05. Each graph represents a separate set of experiments.A-C MEFs of the indicated genotypes were infected with retroviruses expressing the indicated shRNAs, followed by selection with puromycin for 72 h. Mitotic chromosomes isolated 48 h later were fixed and stained with a Cy3‐conjugated (CCCTAA)3‐PNA probe. The frequency of end‐to‐end chromosome‐type fusions is represented as a percentage of fusions observed after TRF2 depletion in each experiment. A minimum of 1,200 chromosomes were scored for each sample. Error bars represent SD of two independent experiments. P‐values were calculated using an unpaired two‐tailed t‐test. *P ≤ 0.05; NS, P > 0.05. Each graph represents a separate set of experiments.
Figure 1Binding of recombinant (r)CEACAM1 (CC1)-HIS (10 μg/mL) to (A) a panel of Gram-positive bacteria, namely Enterococcus faecalis, Enterococcus faecium, Group A Streptococcus (GAS), Group B Streptococcus (GBS), Group C Streptococcus (GCS), Group G Streptococcus (GGS), Streptococcus pneumoniae, Staphylococcus aureus, (B) an expanded collection of GBS isolates, with serotype and carriage of bac gene shown. rCC1 binding to live bacteria was quantified by a secondary anti-His-FITC monoclonal antibody (mAb). Mean and standard deviation (SD) values are reported for n = 3 independent experiments. In (B), GBS strains from left to right are A909, BS39, 515, BS22, H36B, BS29, 18RS21, BM110, COH1, SBL3066, NCTC10/84, SB35, SB10, SB20, BS26.Western blot analysis of rCC1-HIS (30 μg/mL) binding to purified β protein, but not to control GBS surface proteins α and Rib. Panel on the left show staining of proteins after separation by SDS-PAGE.
Figure 2Inhibition of β-IgSF binding to DB.CC1 by anti-CC1-N mAb (clone CC1/3/5-Sab), but not by anti-CC1-A1B1 mAb (clone B3-17). β-IgSF-biotin was coupled to SPE. Mean and SD values are reported for n = 6 replicates.
Figure 4Adhesion of WT, ∆bac and ∆bac + pLZ.bac complemented A909 strains to human CC1- or CC5-expressing CHO transfectants, or empty vector control cells. Mean and SD from n = 5 independent replicates is displayed.
Figure 6Binding of rCC1-N to dynabeads (DB) coated with β-IgI3 wild type (WT) or mutants. Mutation of β-IgI3 residues 42, 46 and 53 lead to significant changes in CC1-N binding capacity.Binding of β-IgI3 coupled to Streptavidin-PE to DB coated with rCC1-N wildtype or variants. Mutation of CC1-N residues 29, 44, 89, 91 and 95 lead to significant changes in β-IgI3 binding capacity.
Figure 7Binding of recombinant (r)CC1-HIS to dynabeads (DB) coated with β-IgI3, R28-IgI3 or control. Mean and SD is shown for n = 6 independent replicates.Binding of β-IgI3, R28-IgI3 or human serum albumin (HSA) coupled to PE-conjugated streptavidin (SPE), to DB coated with CC1 (DB.CC1). Mean and SD is shown for n = 6 independent replicates.
Figure 1(A) Quantitative analysis of Rnf114 mRNA by realtime-PCR in various mouse tissues and organs shows that Rnf114 was predominately expressed in oocytes.(B) Immunohistochemistry analysis of RNF114 in mouse ovary shows that RNF114 was mainly expressed in the oocytes (Scale bar = 100um).(C) Knockdown efficiency of Rnf114 in the 2-cell stage embryos derived from Rnf114 siRNA #1 or siRNA #2 injected zygotes was verified by realtime-PCR and western blotting. NC indicates negative control injected with non-silencing siRNA. Three independent experiment replicates were performed for realtime-PCR, error bars represent s.e.m.; **:P<0.01, ***:P<0.01 in unpaired two-tailed t-test.(D) RNF114 depletion impaired the early embryonic development. Rnf114 siRNA #1 or siRNA #2 injected zygotes were cultured and quantified (siRNA1#, n = 120; siRNA2#, n = 128; three independent experiments); NC indicates negative control (n = 193; six independent experiments). The error bars represent s.d., *:P<0.05, **:P<0.01, ***:P<0.001 in unpaired two-tailed t-test.(E) Representative images to show early embryonic development defects. The embryos arrested at two-cell stage are indicated by arrows (Scale bar = 100um).
Figure 2(A) In vitro ubiquitination assay. Purified His-RNF114 was subjected to ubiquitination assays with the indicated components and the samples were analyzed by immunoblotting with anti-FLAG (Ub) antibody. Lanes 1-4 indicate negative controls. The results show that recombinant RNF114 efficiently catalyzed the formation of polyubiquitin chains.(B, C) Purified wild-type and RING mutated His-RNF114 were stained by Coomassie Blue (B). Purified wild-type and RING mutated His-RNF114 were subjected to ubiquitination assays together with E1, Ubc4, FLAG-Ub, and ATP. The results show that mutant RNF114 lacked ubiquitination activity (C).(D) Screening for proteins ubiquitinated by RNF114 on a proteome array. Putative substrates of RNF114 were identified on Ubiquitin Ligase Substrate Identification on ProtoArray® Human Protein Microarrays v5.0. Ubiquitination reactions were performed on quintuplicate arrays using the indicated components, the five sub-array are respectively negative controls, E1+E2 (no E3), WT RNF114 (two arrays, duplicate), and mutated RNF114.(E) A heat map comparing hits between E1+E2, E1+E2+WT RNF114, and E1+E2+mutated RNF114 reactions. Candidate substrates of RNF114 selected for further characterization are indicated in red font; HIP1, indicated by blue font, was considered to be a false positive because it was also detected in the mutated RNF114 reaction. Black font indicates proteins with ubiquitin-binding domains such as UIM, UBA or CUE or proteins without clearly reported functions, which were excluded from further investigation.
Figure 3(A) RNF114 substrate validation. HEK293 cells were co-transfected with constructs expressing FLAG-tagged substrates, wild-type or mutated His-RNF114 and EGFP, and western blot was performed with anti-FLAG and anti-His antibodies. The results show that the protein levels of CD74, TAB1, OPTN, TNIP1, APPL1, PSAT1, and RALGPS1 were reduced in the presence of wild-type RNF114 compared to co-transfecting with mutant RNF114.(B) RNF114-dependent in vitro ubiquitination of substrates. Substrate ubiquitination assays were performed with purified RNF114, the reaction products were immunoblotted using anti-His antibody to detect the ubiquitin (upper panel) or anti-FLAG antibody detecting the substrate protein (middle panel), and the lower panel showed the merged signals. The results show that clear polyubiquitin chains were formed in the systems containing recombinant CD74, TAB1, TNIP1, PSAT1 or RALGPS1.
Figure 4(A) Mouse zygotes were microinjected with sterile ddH2O (control group) or Ralgps1, Cd74, Tnip1, Psat1, or Tab1 mRNA. During 96-100 h culturing, the percentages of embryos at various stages were counted under microscopy. The results show that overexpression of Tab1 impaired the early embryonic development. Three independent experiment replicates were performed, error bars represent s.d.; *:P<0.05, ***:P<0.001 in unpaired two-tailed t-test.(B) TAB1 reduced with mouse early embryonic development, while abnormally accumulated at 2-cell stage once treated with Rnf114 siRNAs. TAB1 was detected by western blotting with anti-TAB1 antibody. The relative level of TAB1 protein after Rnf114 siRNAs treatment were presented as mean±s.e.m. (*:P<0.05 in unpaired two-tailed t-test, four independent experiment replicates).(C) Detection of endogenous TAB1 ubiquitination at 1-cell or 2-cell stages. Endogenous TAB1 proteins were immunoprecipated by anti-TAB1 antibody at either 1-cell or 2-cell stages, the immunoprecipates were detected by either anti-TAB1 antibody or anti-ubiquitin antibody. The results show that more Ub chains on TAB1 were detected in the two cell stage embryos than in the zygotes, when the TAB1 proteins were at comparable level.(D) RNF114-dependent TAB1 degradation during early embryo development. (a-c) Representive time lapse embryo images were shown for each treatment. Scale bar = 20um. (a'-c') Quantification of the GFP fluorescent signals for respective treatments in (a-c). n=14, 9 or 12 for the NC, Rnf114 siRNA1# or Rnf114 siRNA2# treatment group respectively. Gray traces: Tab1-GFP fluorescent signals were normalized to the first time point; Black trace: average signal of all embryos for each treatment.(E) Tab1 knockdown partly rescued the early embryo development defect observed with Rnf114 knockdown. Both Rnf114 and Tab1 siRNAs were injected into the GV oocytes, then the oocytes matured, and were fertilized by IVF, and the fertilized eggs were transferred into KSOM media, cultured for 48-50 h. The percentages of embryos arrested at 2-cell stage were counted. Three independent experiment replicates were performed, error bars represent s.d.; **:P<0.01 in unpaired two-tailed t-test.