EMBO/SourceData huggingface dataset:
Figure 4Z-projection of HeLa cells expressing GFP-centrin1 immunostained for p34-Arc (sub-domain Arp2/3) following prometaphase arrest and forced mitotic exit for 5min. N=3 independent experiments. Scale bar = 10µm and for zoom 2µm.Levels of p34-Arc around the centrosome (3um area) were quantified relative to metaphase and increase during forced mitotic exit. STLC arrested = 1 ± 0.01576, n=153, STLC+RO-3306= 1.268 ± 0.01748, n=191, ****p<0.0001, Unpaired t-test with Welch correction. Error bars represent standard deviation.Z-projection of centrosomes isolated from cells expressing GFP-centrin1 during prometaphase arrest and forced exit, which were immunostained with p34-Arc. N=2 independent experiments. Scale bar = 2µm.Quantification of p34-Arc levels around the centrosome in (C), relative to metaphase, showing its increase during forced exit. STLC arrested = 1 ± 0.00779, n=93, STLC+RO-3306= 1.249 ± 0.02446, n=134, ****p<0.0001, Unpaired t-test with Welch correction. Error bars represent standard deviation.Time-lapse of in vitro assay on centrosomes isolated from prometaphase arrested and cells forced to exit mitosis showing centrosomal actin nucleation over time. Scale bar = 10µm Quantification of actin nucleation from isolated centrosomes as in (E) over an area of 2 μm, (white circle), showing an increase in actin nucleation around centrosomes isolated from cells undergoing forced mitotic exit. N=2 isolations, 5 independent experiments, n=16 STLC and n=19 STLC RO.Color switch experiment using green- and red-labelled actin. Red actin was used first, followed by Capping protein, then green-labelled actin. The accumulation of green in the centre indicates that actin is nucleated at the centrosome. Scale bar = 10µm. Quantification of (G) over the time. N=2 isolations, 2 independent experiments, n=19 STLC and n=14 STLC RO. Error bars represent standard deviation.Time-lapse showing cytoplasmic actin filament formation at centrosomes isolated from cells undergoing forced exit when pre-treated with DMSO or CK666. Scale bar = 10µm. Quantification of (I), showing that centrosomal actin nucleation fails when centrosomes isolated from cells undergoing exit are pre-treated with CK666. N=2 isolations, 2 independent experiments, n = 17 DMSO, 26 Ro CK666 24 STLC DMSO, 23 STLC CK666. Error bars represent standard deviation.
Figure 5Phos-tag Western blot of MCF7-Her2-18 cells synchronised into distinct phases of the cell cycle showing the phospho-shift in WASH1 during mitosis.Blue native PAGE Western blot showing shift in the size of WASH1 protein complex isolated from STLC treated prometaphase arrested cells when compared to cells forced to exit mitosis following Ro3306 treatment.
Figure 6Time-lapse of HeLa cells expressing GFP-alpha tubulin and RFP-lifeact treated with control siRNA or siRNA against WASH1 for 48 hours arrested at prometaphase and forced to exit mitosis with Ro3306 addition (t=0). Scale bar = 5µm.Quantification of actin around the centrosome in (A), showing the failure to accumulate actin around the centrosome in siWASH. N = 4 experiments. Error bars represent standard deviation.Western blots of lysates from cells treated with siRNA control and siRNA against WASH1 probed with WASH1 antibody, showing the reduction in WASH levels following treatment of cells with siRNA against WASH. Scale bar = 5µm and 2µm in zoom.Z-projection of HeLa cells expressing GFP-centrin1 treated with control siRNA or siRNA against WASH, arrested at prometaphase or forced to exit mitosis and immunostained with phalloidin. Scale bar-5µm Quantification of actin around the centrosome in (F), showing the failure to accumulated actin around the centrosome during forced exit in cells treated with siRNA against WASH. siControl-STLC= 1 ± 0.0189, n=165, siWASH-STLC=0.9138 ± 0.0298, n=118, siControl-STLC+RO-3306=1.911 ± 0.06221, n=147, siWASH-STLC+RO-3306=1.103 ± 0.0301, n=140. Error bars represent standard deviation. P<0.0001, one-way ANOVA.Time-lapse from in vitro assay with centrosomes isolated from cells undergoing forced exit were pre-treated with either no antibody or anti-WASH1 antibody for 1 hour. Scale bar- 10µm Quantification of in vitro assay from (F), showing the reduction in actin nucleation around centrosomes when they are pre-treated with anti-WASH1. Error bars represent standard deviation. 2 independent experiments.
Figure 1A TRAF6 expression in differentiating CD4+ T cells. Naïve CD4+ T cells were obtained from wild type C57BL/6 mice by FACS and activated with anti-CD3/CD28 (1µg, and 2µg/ml) for the indicated times in the presence of distinct Thelper lineage-directing cytokines or under neutral activation conditions (Th0). After total RNA extraction and cDNA conversion, RT-PCR determined of Traf6 mRNA in differentiating Th0, Th1, Th17 and iTregs was determined.B TRAF6 mRNA expression by human Tregs and non-Treg CD4+ T cell. Human Tregs (CD3+/CD4+/CD8-/CD25HIGH/CD127low/CD39+) and non-Treg CD4+ T cells (CD3+/CD4+/CD8-/CD25-) were obtained from the peripheral blood of healthy donors by FACS after Ficoll-Paque PLUS gradient centrifugation and magnetic bead enrichment of CD4+ T cells. TRAF6 mRNA was measured by qRT-PCR.C, D Evidence of lymphoproliferative disease in Traf6fl/flFoxp3Cre+ mice. (C) Spleens and lymph nodes were recovered from Traf6fl/flFoxp3Cre+ mice and Traf6fl/fl littermates at 8 weeks of age (Scale bars: 5 mm). (D) The cellularity of the lymphoid tissues of Traf6fl/flFoxp3Cre+ mice and their Traf6fl/fl Foxp3Cre- littermates was determined (8 mice/group).E, H Effect of Treg-specific TRAF6-deficiency on baseline T cell activation. The frequencies of effector cells (CD44high/CD62Llow), memory cells (CD44high/CD62Lhigh) and naïve cells (CD44low/CD62Lhigh) in the CD4+ T cell compartments of Traf6fl/fl and Traf6fl/flFoxp3Cre+ mice were determined by flow cytometry (5 mice/group).F, I Effect of Treg-specific TRAF6-deficiency on baseline T cell activation. The frequencies of effector cells (CD44high/CD62Llow), memory cells (CD44high/CD62Lhigh) and naïve cells (CD44low/CD62Lhigh) in the CD8+ T cell compartments of Traf6fl/fl Foxp3Cre- (wild type) and Traf6fl/flFoxp3Cre+ mice were determined by flow cytometry (5 mice/group).G, J Impact of TRAF6-expression on in vitro Treg differentiation. naïve CD4+ T cells were isolated from Traf6fl/flFoxp3Cre+ and Traf6fl/fl mice and differentiated into iTregs. Conditions of sub-optimal TGFβ concentrations (0.5ng/ml, 0.05ng/ml) were tested as well and intracellular FOXP3 was measured after 4 days.
Figure 2A In vivo suppressive function of WT and Traf6fl/flFoxp3Cre+ Tregs. Tregs from the indicated mice (CD45.2+) were isolated by FACS, as were naïve (CD62Lhigh/CD25-) CD4+ responder T cells from congenically distinct (CD45.1+) donor mice. Tregs and Tresponders were mixed at a 1:5 (2x105:10x105) ratio before injection into Rag2-/- mice. 7 days later, spleens were harvested, and the relative frequencies and absolute numbers of each transferred cell populations were determined by flow cytometry.B Implanted B16 melanoma growth in Traf6fl/flFoxp3Cre+ and wild type (Traf6fl/flFoxp3Cre-) mice. 1x105 B16F10 cells were injected subcutaneously (s.c.) into the shaved flanks of the indicated mice (n= 5/group). Tumor volumes were monitored every 3 days.C, D Proinflammatory cytokine production in tumor-bearing mice with and without Treg-specific TRAF6 expression. Cell suspensions of the tumor-draining lymph node and tumor-infiltrating leukocytes (TILs) were recovered after 21 days of tumor growth. Ex vivo stimulation with PMA and ionomycin in the presence of Golgistop for 5 hour preceded intracellular staining for IFNγ and IL-17 and flow cytometry analysis.E, F FOXP3 expression by CD4+ T cells in tumor bearing Traf6fl/flFoxp3Cre+ and wild type (Traf6fl/flFoxp3Cre-) mice. The frequency of FOXP3+ cells in the CD4+ cells of tumor-draining lymph nodes (dLN), peripheral, non-tumor draining lymph nodes (pLN), spleens, and TILs were determined by flow cytometry.
Figure 3A Assessment of K63-type ubiquitination of FOXP3 upon expression of wild type and catalytically deficient TRAF6 mutants. 293T cells were transfected with the given combinations of vectors encoding wild type TRAF6, the enzymatically deficient L74H or C70A TRAF6 mutants, HA-FOXP3, and FLAG-labeled ubiquitin molecules possessing a single lysine residue (K63) that restrain the possible ubiquitin monomer linkages on polyubiquitinated proteins to the K63-type only. Cells were lysed and proteins modified by K63-ubiquitin chains were recovered from lysates by pulling down using a bead-immobilized K63-specific TUBE reagent. The presence of FOXP3 among these modified proteins was determined by immunoblotting as were levels of FOXP3 and TRAF6 in the pre-IP whole cell lysate (WCL).B Co-immunoprecipitation (co-IP) of FOXP3 with TRAF6 and its facilitation of K63-linked ubiquitination. 293T cells expressing the indicated constructs encoding HA-FOXP3 and FLAG-TRAF6 were lysed and incubated with bead immobilized anti-FLAG antibodies. FOXP3 molecules co-IPed in this manner were resolved by SDS PAGE and detected by immunoblotting with anti-HA antibodies.C　293T cells were transfected with the indicated combinations of expression constructs encoding HA-FOXP3, Myc-TRAF6, and FLAG-tagged ubiquitin molecules - either wild type (FLAG-Ub), or a variant with a K-to-R mutation at either lysine residue 48 (K48R) or 63 (K63R) responsible for preventing K48- and K63-type polyubiquitination, respectively. .D Endogenous, reciprocal co-IP of FOXP3 and TRAF6 in murine iTreg lysate. Naïve CD62Lhigh/CD25-/CD4+ T cells were obtained by FACS and activated with anti-CD3/CD28 antibodies (1ug and 2ug/ml, respectively) in the presence of IL-2 and TGFβ (100U/ml and 5ng/ml, respectively) for four days before lysis. Antibodies against TRAF6 (left) or FOXP3 (right) were used to pull down target proteins and their interaction partners, which were resolved under denaturing conditions and visualized by immunoblotting.E Degree of K63 ubiquitination in the cellular FOXP3 pools of Traf6fl/flFoxp3Cre+ and Traf6wt/wtFoxp3Cre+mice. CD4+/YFP+ cells from Traf6fl/flFoxp3Cre+ ("Traf6-/-") or wild type Traf6wt/wtFoxp3Cre+mice ("Traf6+/+") were isolated by FACS, lysed and either total FOXP3 (right) or K63-ubiquitin-modified proteins (left) were immunoprecipitated. Levels of polyubiquitinated FOXP3 were observed in each by probing with antibodies specific for anti-K63-ubiquitin and FOXP3, respectively, and levels of total FOXP3 and actin were also measured by immunoblot analysis of whole cell lysate.
Figure 4A Immunoblot analysis of TRAF6-mediated ubiquitination of wild type, and mutant FOXP3 molecules. 293T cell lines were transfected with a normal HA-tagged Foxp3 construct, another encoding a ubiquitination resistant mutant in which all lysine residues were replaced by arginines (20R), or one of 20 single lysine-containing constructs with only the indicated lysine residue available for modification. These cell lines also received expression vectors encoding TRAF6 and ubiquitin molecules labeled with Myc and FLAG tags, respectively. Negative controls did not receive TRAF6 or expressed FOXP3 alone. Labeled FOXP3 proteins were pulled down from cell lysates (anti-HA) and ubiquitinated species were visualized by immunoblotting for FLAG.B Assessing the impact of K262 mutation on FOXP3 ubiquitination by TRAF6. 293T cells were transfected with combinations of expression constructs encoding Myc-TRAF6, FLAG-ubiquitin, and an HA-tagged FOXP3 molecule that was either normal (HA-FOXP3) or possessed a K-to-R mutation at residue 262 (K262R). FOXP3 proteins were pulled down with anti-HA beads and either ubiquitinated proteins or total FOXP3 proteins were detected by probing for FLAG and HA, respectively.
Figure 5A Impact of K63-ubiquitination loss on FOXP3's gene silencing capacity. Jurkat T cells transfected with a wild type Foxp3 expression vector, one encoding the K262R mutant, or an empty (control) vector also received a duel luciferase reporter construct where luciferase expression was under the control of the Il2 promoter. After activation with PMA and ionomycin for 8 hours, luciferase activity was assayed.B IL-2 production in the presence of wild type and K63-ubiquitination resistant FOXP3. Naïve CD4+ T cells isolated from Traf6fl/flCD4Cre+ or WT mice were activated overnight by anti-CD3/CD28 antibodies, then transduced by retroviruses carrying normal Foxp3, the K262R mutant, or an empty vector. Ires-GFP in the retroviral vector serves as an internal control. GFP+ cells were sorted out 48 hours post-transduction. The sorted cells were cultured for one additional day. Culture supernatants were collected for measuring the levels of IL-2 by ELISA.C Cellular distribution of wild type and K262R FOXP3. Hela cells were transfected with expression vectors encoding either wild type FOXP3 or the K262R mutant. The relative overlap of FOXP3 protein signal and DAPI-stained nuclei was observed by fluorescence microscopy. (blue: DAPI, green: FOXP3, Scale bars: 50 μm).D Immunostaining of FOXP3 in Traf6fl/flCD4Cre+ and wild type (Traf6fl/flCD4Cre-) derived suspensions of lymph node and spleen cells. (blue: DAPI, green: FOXP3, Scale bars: 50 μm).E Quantification of FOXP3 distribution in murine Tregs. The degree of FOXP3-nuclear coIocalization in CD4+/FOXP3+ cells isolated from the lymph nodes and spleens of Traf6fl/flCD4Cre+ and wild type (Traf6fl/flCD4Cre-) mice was determined by Imagestream analysis, and the frequencies of cells with either a low or high probability of nuclear FOXP3 distribution are shown (red: nuclear stain, green:FOXP3, Scale bars: 10 um).
Figure 6The in vitro suppressive potency of wild type- and K262R Foxp3-expressing T cells. Naïve CD4+ T cells were purified from Thy1.1+ BALB/c mice and subjected to retroviral transduction to express either wild type FOXP3 or a K262R mutant resistant to ubiquitination that lysine residue 262. These engineered "Tregs" were then co-cultured with naïve responder CD4+ T cells stained with CFSE at the indicated ratios and activated with anti-CD3/CD28 antibodies. Dilution of CFSE signal by the responder populations (i.e. proliferation) was assessed by flow cytometry.The in vitro suppressive potency of wild type- and K262R Foxp3-expressing T cells. Naïve CD4+ T cells were purified from Thy1.1+ BALB/c mice and subjected to retroviral transduction to express either wild type FOXP3 or a K262R mutant resistant to ubiquitination that lysine residue 262. These engineered "Tregs" were then co-cultured with naïve responder CD4+ T cells stained with CFSE at the indicated ratios and activated with anti-CD3/CD28 antibodies. Dilution of CFSE signal by the responder populations (i.e. proliferation) was assessed by flow cytometry.C In vivo suppression of colitis by wild type- and K262R mutant-Foxp3 expressing T cells. 2x105 naïve CD4+ transductants from A were mixed with 1x106 colitogenic naïve CD4+ T cells from wild type mice and transferred i.v. into Rag2-/- mice. Rag2-/- mice receiving no cell transfers, naïve CD4+ T cells alone, or purified wild type Tregs served as controls (n= 6/group). Recipient mouse body weights were monitored weekly for each group.D, E After 8 weeks, colons were excised, fixed in buffered formalin, and processed for H and E staining (D) and histopathology scoring (E). Scale bars shown in 1 mm).
Figure 1B Western blot analysis of liver lysates (100 µg) shows that intein-mediated protein trans-splicing efficiently reconstitutes full-length eGFP. Single AAV: n=5; AAV-intein: n=5. Arrows indicate both full-length eGFP and excised inteins. C Quantification of eGFP protein bands. Values are reported as mean±SEM. Each dot represents the eGFP protein band quantification from animals injected with either single AAV n=5 or AAV intein n=5.
Figure 2B Western blot analysis of lysates of HEK293 cells 72 hours post-transfection with the various F8 variants. Neg, non-transfected cells.C Chromogenic assay of F8 activity in the medium of transfected cells are reported as International Units/deciliter (IU/dl). Data are presented as mean±SEM. Each dot within the same group corresponds to a biological replicate: Neg n=5; Wild-type n=8; N6 n=8; SQ n=7; V3 n=6. Significant differences between groups were assessed using The Kruskal-Wallis test followed by the posthoc analysis: Nemenyi's All-Pairs Rank Comparison Test. The Kruskal-Wallis test p value = 2.88e-05. **indicates the significant difference between the N6 and the Wild-type groups: p value ≤ 0.01.*indicates the significant difference between the V3 and the Wild-type groups: p value ≤ 0.05.
Figure 3A Western blot (WB) of protein lysates of HEK293 cells 72 hours post-transfection (n=3; biological replicates) with either Npu inteins or Heterologous (N-intein DnaB + C-intein DnaE) split-inteins. I + II, N6 split-intein proteins; I+II (Het.), heterologous split-intein proteins; I, 5'N6 coding sequence (CDS)-N-DnaE protein; I (N-int B), 5'N6 CDS-N-DnaB. II, C-DnaE-3'N6 CDS protein. Excised inteins (∼12kDa) are present only in the down part of the blot when I + II (Npu inteins) are provided. Arrows indicate the full-length N6 protein, single halves and excised inteins.B WB of medium of the transfected cells showing the secreted proteins (n=3; biological replicates). Arrows indicate the full-length N6 protein as well as single halves and excised inteins.C Chromogenic assay performed on the medium of transfected cells to detect F8 activity levels reported as International Units/deciliter (IU/dl). Data are presented as mean ±SEM. Each dot within each group represent a different biological replicate (n): I + II n=3; I+II (Het.) n=3; I (N-int E) n=3; I (N-int B) n=3; II (C-int E) n=3; Neg n=4. Significant differences between groups were assessed using Kruskal-Wallis rank sum test Kruskal−Wallis, p value = 0.013. *indicates the significant difference between the I+II and the I (N-int E) groups: p value ≤ 0.05. ***indicates the significant difference between the I+II and the Neg groups: p value ≤0.001.
Figure 4A Western blot (WB) of protein lysates of HEK293 cells 72 hpt with the AAV-N6 split- intein plasmids and with the codon-optimised set. I + II, N6 split-intein proteins; I, 5' N6 CDS-N-DnaE protein; II, C-DnaE-3'N6 CDS protein. (n=3; biological replicates). Arrows indicate the full-length N6 protein, excised inteins and both single halves. Codop: codon-optimised.B WB of medium from the transfected cells showing increased secretion of the Codop-N6 full-length protein compared to the non-codon-optimised. (n=3; biological replicates). Arrows indicate the full-length N6 protein, excised inteins and both single halves.C Chromogenic assay performed on the medium from transfected cells to measure F8 activity levels reported as International Units/deciliter (IU/dl) (n=3; biological replicates).Data are presented as mean ±SEM. Significant differences between groups were assessed using Kruskal-Wallis test p value = 0.027. *indicates the significant difference between the I+II (N6 split-intein) and the II (C-DnaE-3'N6 CDS) groups: p value ≤ 0.05. **indicates the significant difference between the Codop I+II and the I (5' N6 CDS-N-DnaE) codop groups: p value ≤ 0.01. **indicates the significant difference between the Codop I+II and the II (C-DnaE-3'N6 CDS) codop groups: p value ≤ 0.01.
Figure 5A Alkaline gel Southern blot analysis of AAV DNA. AAV DNA was hybridized to a probe specific for the HLP promoter. Neg, AAV DNA treated with Dnase I; CodopV3, AAV-CodopV3; I, AAV-5'N6-N-intein; II, AAV-C-intein-3' N6.B Chromogenic assay performed on plasma samples to detect F8 activity in AAV-treated mice compared to controls groups. Data are presented as mean ±SEM. The same animals were analysed at the various time-points. Each dot within the same group and within different groups of treatment corresponds to a single animal. The baseline includes all animals (n=20) before the treatment: CodopV3: n=8; N6 intein: n=5; CodopN6 intein: n=5. The statistical difference between groups has been assessed at 4weeks post-injection (wpi) with the One-way Anova p value = 0.056; at 8 wpi with the Kruskal-Wallis test p value = 0.68; at 12wpi with the Kruskal-Wallis test p value = 0.45; at 16wpi with the Kruskal-Wallis test p value = 0.58.C Activated partial thromboplastin time (aPTT) assay performed on plasma samples both at the baseline and at the last time-point of the analysis (16 wpi). Data are presented as mean ±SEM. Each dot within each group of treatment corresponds to different animals: The baseline includes all animals before the treatment n=21; CodopV3: n=8; N6 intein: n=5; CodopN6 intein: n=5. Significant differences between groups were assessed using the Kruskal-Wallis test. **indicates the significant difference between the baseline and the N6 intein groups: p value ≤ 0.01.*indicates the significant difference between the baseline and the CodopN6 intein groups: p value ≤ 0.05.D Tail-clip assay reported as bleeding time in seconds. Significant differences between groups were assessed using One-way Anova test p value is = 0.11.Data are presented as mean±SEM. Each dot corresponds to a different animal. Knockout (K.O.) group: n=4; CodopV3: n=4; N6 intein: n=4; CodopN6 intein: n=E Haemoglobin content measured as Optical Density (OD) at 416 nm after the tail-clip assay. Significant differences between groups were assessed using One-way Anova test. Data are reported as mean±SEM. Each dot corresponds to a single animal. Knockout (K.O.) group: n=4; CodopV3: n=4; N6 intein: n=4; CodopN6 intein: n=6.F Western blot analysis of liver lysates (100 µg) from either CodopN6 intein-treated mice (n=6) or untreated haemophilic mice (n=2). A lysate from HEK293 cells transfected with the N6 full-length plasmid was used as positive control (50 µg). Arrows point at N6 full-length protein (N6), the 5' half of N6 (5'N6) and 3' half of N6 (3'N6).
Figure 6A The amount of anti-F8 antibodies analysed by indirect ELISA is reported in Arbitrary Units/milliliter (AU/ml). Each numbered bar represents a single mouse. CodopV3, AAV-CodopV3 injected group n=8; N6 intein, AAV-N6 intein injected group n=5; CodopN6 intein, AAV-CodopN6 intein injected group n=5. Significant differences were assessed as follow: Paired T-test has been used for CodopV3 n=5: p value ≤ 0.001; Wilcoxon test for CodopV3 n=3: p value ≤ 0.05; Paired T-test has been used for N6 intein n=5: p value ≤ 0.05; Paired T- test has been used for CodopN6 intein n=5 p value ≤ 0.01.B Bethesda assay performed on plasma samples mice injected with AAV-CodopV3; on the plasma of animals injected with AAV-N6 intein n=3 and the plasma of animals injected with AAV-CodopN6 intein n=2; plasma of a wild-type animal was used as a control. The dotted line indicates the threshold above which anti F8-antibodies are considered inhibitors.C Pro-inflammatory cytokines levels followed over-time and reported as percentage of the baseline levels (time point before the injection). Data are represented as mean±SEM. Five animals belonging to the CodopV3 group were analysed. Significant differences were assessed using the Kruskal−Wallis test; for IP-10: * indicates the p value ≤ 0.05 between 0 and 4 wpi; * indicates the p value ≤ 0.05 between 0 and 8 wpi; * indicates the p value ≤ 0.05 between 0 and 16 wpi. (n=20); for eotaxin: * indicates the p value ≤ 0.05.