EMBO/SourceData huggingface dataset:
Figure 1(B) Western blot of RNAi efficiency and specificity for oligos used in (C-G). HeLa Kyoto cells were incubated for 72 h with control oligos or RNAi-oligos depleting CUL4A, CUL4B or DDB1. Specific downregulation of the indicated proteins was analyzed by immunoblotting. GAPDH controls for equal loading. (C) Quantification of western blot bands and the ratio between neddylated vs unneddylated CUL4 with the paralog downregulated is calculated. Graphs displays mean with SEM of N=3 biological replicates. **P = 0.001.(E) Flow cytometry analysis of Hela cells treated with the indicated RNAi for 72h and immune-stained for γH2A.X, DNA staining with propidium iodide. One of three biological replicates is represented.(G) Live-cell imaging of mitosis in RNAi depleted HeLa Kyoto cells. Brightfield images were taken every 3 min, and mitotic duration was quantified by measuring the time from cell rounding to telophase. Mean with SEM is depicted from N=3 biological experiments analyzing a total of 30 single cells. nsP= 0.9995; **P=0.0040; **P= 0.0044. Red symbols mark incomplete mitotic events during the movie. White arrows mark cells undergoing mitosis. Scale bar: 10 μm.
Figure 2(A) CUL4A and CUL4B were analyzed by immunoblotting of extracts prepared from HeLa Kyoto cells arrested in S-phase (S= thymidine), G2 (R0-3306), or mitosis (M= nocodazole or taxol). One of three biological replicates is shown. H3 pS10 (phosphorylated histone H3 at serine 10) was used as a mitotic marker. Same samples but separate blot for H3pS10 staining due to size differences.(C) Mitotic population of unperturbed cells was harvested by shake-off. Three subsequent harvests are depicted (R1-R3). The interphase population are cells that remained attached to the culture dish.(G) In vitro kinase assays with purified CDK1-cyclinB and Sf9-purified CUL4B and mutant proteins in the presence of [γ-32P]ATP. The autoradiogram (top panel) shows CUL4B phosphorylation, while coomassie staining (lower panel) controls equal loading of CUL4B and mutants (N=3 technical replicates for WT, P50L; NP1; N=2 for NP1-A49T and NP1-A49T-P50L). Note that the A49T mutation in the NP1 mutant restores phosphorylation of the T49 residue, but only in the presence of the adjacent proline at position 50.
Figure 3(A) Western blot analysis to monitor CUL4A or CUL4B levels in extracts prepared from HeLaFLP control cells or HeLaFLP cells deleted for CUL4B by CRISPR/Cas9 (∆CUL4BFLP) but stably expressing no CUL4B (-), CUL4B or the indicated NP1 or P50L mutants from the doxycycline-inducible promoter. GAPDH was included to control equal loading. One of three biological replicates is shown.(D) The expression levels of endogenous HA-tagged WT and the indicated CUL4B mutants were compared by immunoblotting with HA.II antibody. GAPDH controls for equal loading(F) In vitro E3 ligase activity assays using the indicated reconstituted CRL4BCRBN complexes and the neo-substrate CKlα. At time 0, lenalidomide was added to start the reaction and CKlα ubiquitination was followed at the times indicated (in minutes) by immunoblotting with CKlα antibody. N≥3 technical replicates for CUL4B, CUL4A, P50L, K895R and L864fs*13; N=1 for NP1. Note that the in vitro E3 ligase activity of CRL4B and the P50L patient mutation is comparable.
Figure 4(C) To control RNAi efficiency and specificity, HeLa Kyoto cells were incubated for 72 h with control oligos (control) or RNAi-oligos depleting CUL4B, LIS1 or WDR1. Specific downregulation of the indicated proteins was analyzed by immunoblotting. GAPDH controls for equal loading. One of three biological replicates is shown.(E) HA-immunoprecipitation (IP) of extracts (input) prepared from unedited (-) or HA-CUL4B endogenously expressing HeLa Kyoto cell lines arrested in S-phase (thymidine) or mitosis (nocodazole) were probed with antibodies against LIS1, WDR1 and DDB1. The antibody recognizing phospho-H3(S19) controls mitotic arrest and specificity of the immunoprecipitation.(F, G) HeLa Kyoto endogenously expressing HA-CUL4B or the indicated NP1 or P50L mutants were arrested in mitosis with nocodazole. Extracts (input) and HA-immunoprecipitants were analyzed for the presence of HA-CUL4B for control and bound DDB1 and LIS1 (F) or DDB1 and WDR1 (G), respectively. One of 3 biological replicates is shown.
Figure 5(A) Coomassie-stained SDS-PAGE of in vitro pull-down assays of FLAG-tagged LIS1 and WDR1, and His-DDB1, co-expressed in baculoviral Sf9 cells, F is FLAG and H is His. One of 3 biological replicates is shown. Note that DDB1 specifically interacts with LIS1 and WDR1.(C, D) Western blot of in vitro pulldown assays of Strep-tagged CUL4B and FLAG-tagged LIS1 (C) or WDR1 (D) co-expressed in Sf9 cells in the presence (+) or absence (-) of DDB1. One of 3 biological replicates is shown. Note that LIS1 and WDR1 specifically bind CUL4B, but this interaction is enhanced by the presence of DDB1.(G) FP assay investigating binding of CRL4B and CRL4A to Alexa-labeled WDR1Fluorescence polarization assay comparing the binding of CRL4B and λ-PP-treated CRL4B to Alexa-labeled WDR1. The mean with SD of N=3 technical replicates is depicted. (L) Quantification of in vitro binding assays (Strep-pull down) of Strep-LIS1 with purified CRL4B, or the NP1 phospho-mutant (CRL4BNP1) (N=2 technical replicates), or λ-PP treated CRL4B (CRL4Bλ-PP) (N=1).
Figure 6(C) Extracts (input) prepared from S-phase (S; thymidine) or mitosis (M; nocodazole)-arrested HeLa Kyoto cells (-) and endogenously expressing HA-tagged WT CUL4B, or the NP1 or P50L mutants were immunoprecipitated with HA.II-antibodies (IP). Bound ACTR3 or DDB1 was detected using specific antibodies. One of three biological replicates is shown. Note that in contrast to DDB1, ACTR3 binds CUL4B in a phosphorylation-dependent manner.(D) HA-immunoprecipitants (IP) of extracts (input) prepared from unedited (-) controls or endogenously expressing HA-CUL4B HeLa Kyoto cells arrested in mitosis with nocodazole were probed with antibodies against DDB1, DCTN1 (dynactin) and actin. One of two biological replicates is depicted.(F) Live cell imaging of HeLa cells expressing H2B-mcherry and lifeAct-GFP, treated for 72 h with control oligos (siControl) or RNAi-oligos depleting CUL4B, LIS1 or WDR1. Representative cells are shown in time intervals of 6 min during metaphase with identical contrast settings. The unanchored spindle in CUL4B- and LIS1 deleted cells is marked with a white arrow. Scale bar: 10 μm.
Figure 7hESCs were differentiated to generate human forebrain specific regionalised organoids. Sections were stained as indicated with antibodies against SOX2 or CTIP2 to visualize neural stem cell and neuronal cell populations, respectively. (A) Example images of day 35 organoids. The stained sections are also shown as merged images at different maginifications, with scale bars of 200 μm and 50 μm, respectively. (B) The number of cortical units per mm2 total area was quantified in unedited and ΔCUL4B brain organoids at day 35. Ventricles were counted and the number divided by the total organoid area on 20 sections from one biological replicate.
Figure 1C57BL/6J mice were intraarticularly infected with MRSA (4 × 106 CFU; n = 3-4 per group) and left untreated for 24 hours. H. The amount of infiltrating immune cells in synovial fluid was quantified and detected using MRSA expressive of green fluorescent protein (GFP) within immune cells (Scale bar: 100 μm). MRSA bioburden in synovial fluid was quantified in colony-forming units (CFU). Data information: Error bars show means ± SD with individual data points. Two-tailed unpaired t-test analysis was conducted to determine statistical significance (*p<0.05 or **p <0.01; N.D. = not detected).C57BL/6J mice were intraarticularly infected with MRSA (4 × 106 CFU; n = 3-4 per group) and left untreated for 24 hours. K. Paraffin-embedded tissues were sectioned and used to measure inflammation score and synovial hyperplasia. GLUT1 and MCT4 expression was detected, and the percentage of positively staining cells were determined (Scale bar: 1,000 μm). Data information: Error bars show means ± SD with individual data points. Two-tailed unpaired t-test analysis was conducted to determine statistical significance (*p<0.05 or **p <0.01; N.D. = not detected).M. Human cartilage and synovial tissue were infected with MRSA (4 × 106 CFU) for 24 hours. The co-expression and -localization levels of GLUT1 and MCT4 in human cartilage tissue was measured and confirmed by line profile analysis (Scale bar: 100 μm). Expression of GLUT1, MCT4, IL-1β, and MMP3 was measured in human synovial tissue (Scale bar: 1,000 μm).
Figure 2B. Physiological changes were identified and representative images were generated.C. Blood was collected and complete blood counts (CBC) were measured. Data information: In vivo experiments were repeated in at least two independent experiments. Error bars show means ± SD with individual data points. One-way ANOVA with Tukey's post hoc analysis was conducted to determine statistical significance (*p<0.05 or **p<0.01; N.D. = not detected; N.S. = not significant).D and E. Infiltrating immune cell counts within synovial fluid and MRSA synovial fluid bioburden were quantified. MRSA bioburden in synovial tissue was quantified. F. Paraffin-embedded tissues were sectioned and measured at 7 and 14 days with respect to inflammation score, synovial hyperplasia, synovial cellularity, OARSI score, proteoglycan depletion, osteophyte formation, bone erosion, and bone formation (Scale bar: 1,000 μm). G and H. Lactate level in synovial fluid was measured at 14 days and expression of GLUT1, MCT4, cleaved-IL-1β, and NLRP3 in synovial tissue was measured with GAPDH as a loading control. Data information: In vivo experiments were repeated in at least two independent experiments. Error bars show means ± SD with individual data points. One-way ANOVA with Tukey's post hoc analysis was conducted to determine statistical significance (*p<0.05 or **p<0.01; N.D. = not detected; N.S. = not significant).
Figure 3B. Infiltrating immune cell counts within synovial fluid were quantified. Data information: In vivo experiments were repeated in at least two independent experiments. Error bars show means ± SD with individual data points. One-way ANOVA with Tukey's post hoc analysis was conducted to determine statistical significance (*p<0.05 or **p<0.01; N.D. = not detected).C. Serum and synovial fluid lactate levels were measured and the relative luminescence intensity is shown. Data information: In vivo experiments were repeated in at least two independent experiments. Error bars show means ± SD with individual data points. One-way ANOVA with Tukey's post hoc analysis was conducted to determine statistical significance (*p<0.05 or **p<0.01; N.D. = not detected).D. Expression of GLUT1 and MMP3 in synovial tissue was measured using GAPDH as a loading control.E. Physiological changes were identified, and representative images were generated.F. Blood was collected, and complete blood counts (CBC) were measured. G. Paraffin-embedded tissues were sectioned and measured with respect to inflammation score, synovial hyperplasia, OARSI score, proteoglycan depletion, osteophyte formation, bone erosion, and bone formation (Scale bar: 1,000 μm). The expression of GLUT1 was detected and the percentage of positively staining cells was determined. Data information: In vivo experiments were repeated in at least two independent experiments. Error bars show means ± SD with individual data points. One-way ANOVA with Tukey's post hoc analysis was conducted to determine statistical significance (*p<0.05 or **p<0.01; N.D. = not detected).
Figure 5C57BL/6J mice were intraarticularly infected with MRSA (4 × 106 CFU; n = 4-5 per group) for 24 hours. Synovial fluid cells were collected and (5 × 105) seeded on antibiotic-free DMEM medium with or without DMF (300 µg/ml) for 6 and 24 hours. B. MRSA expressive of green fluorescent protein (GFP) was detected and measured (Scale bar: 100 μm). Data information: Error bars show means ± SD with individual data points. Two-tailed unpaired t-test analysis was conducted to determine statistical significance (*p < 0.05 or ** p < 0.01 or *** p < 0.001 or **** p < 0.0001; N.D. = not detected; N.S. = not significant).G. Inhibition zones in disk fusion assays using vancomycin (1,000 µg) and DMF (75 to 300 µg) were measured and presented in a histogram. Data information: Error bars show means ± SD with individual data points. Two-tailed unpaired t-test analysis was conducted to determine statistical significance (*p < 0.05 or ** p < 0.01 or *** p < 0.001 or **** p < 0.0001; N.D. = not detected; N.S. = not significant).J. RAW264.7 cells were stained with CellTracker and infected with MRSA (4 × 106 CFU) for 2 hours. The cells were then washed to remove extra-cellular MRSA. Medium containing DMF (700 µg/ml), vancomycin (100 µg/ml), and DMF (700 µg/ml) with vancomycin (100 µg/ml) was added and GFP intensity was measured at 1-hour intervals for a total of 12 hours. Representative images of differential fluorescence with CellTracker and GFP at 4-hour intervals for a total of 12 hours. Intracellular and extracellular MRSA were semi-quantitatively measured at 12 hours. Data information: Error bars show means ± SD with individual data points. Two-tailed unpaired t-test analysis was conducted to determine statistical significance (*p < 0.05 or ** p < 0.01 or *** p < 0.001 or **** p < 0.0001; N.D. = not detected; N.S. = not significant).
Figure 6C57BL/6J mice were subcutaneously treated with vancomycin (30 mg/kg) and rifampin (20 mg/kg) for 3 days following MRSA (8 × 106 CFU) infection, followed by systemic (14 mg/kg) or local (70 μg/joint) DMF treatment at 3 days interval for a total of 3 times (n = 6 per group). D. Paraffin-embedded tissues were sectioned and measured with respect to inflammation score, synovial hyperplasia, synovial cellularity, proteoglycan depletion, osteophyte formation, bone erosion, and bone formation (Scale bar: 2,000 or 500 μm). Bone resorption by osteoclasts was measured by tartrate-resistant acid phosphatase (TRAP) staining (Scale bar: 2,000 μm). Lameness score was also measured. E. Illustrated summary of DMF's effect on MRSA-induced septic arthritis. Data information: In vivo experiments were repeated in at least two independent experiments. Error bars show means ± SD with individual data points. One-way ANOVA with Tukey's post hoc analysis was conducted to determine statistical significance (*p < 0.05 or ** p < 0.01 or *** p < 0.001 or **** p < 0.0001; N.D. = not detected).
Figure 1C) A representative selection of 2D classes of UBR5. UBR - UBR box,
Figure 3B) Sucrose gradient sedimentation profiles of GFPAKIRIN2 in the presence or absence of UBR5. Gels were imaged using fluorescent imaging (488nm) to visualise the recombinant GFP tag, and stain-free imaging to visualise all proteins. Gels of assays are a representative of at least three experimental replicates. Reactions in B were performed at 37°C. The final concentration of E3 enzyme in B : 0.3µMC) Ubiquitination of GFPAKIRIN2. D) Ubiquitination of GFPUbAKIRIN2. E) Ubiquitination activity of wild-type and mutant UBR5ΔUBA on GFPUbAKIRIN2 substrate. Gels were imaged using fluorescent imaging (488nm) to visualise the recombinant GFP tag, and stain-free imaging to visualise all proteins. Gels were subsequently stained using Coomassie staining to visualise ubiquitin levels. Gels of assays are a representative of at least three experimental replicates. Reactions C were performed at 37°C. Reactions in D performed at 25°C. The final concentration of E3 enzyme in C, D: 0.3µMF) Sucrose gradient sedimentation profiles of GFPUbAKIRIN2 in the absence (upper panel) or in the presence of UBR5wt (middle panel), and UBR5ΔUBA (lower panel). Gels were imaged using fluorescent imaging (488nm) to visualise the recombinant GFP tag, and stain-free imaging to visualise all proteins. Gels of assays are a representative of at least three experimental replicates.G) Ubiquitination activity of wild-type and oligomerisation-defective mutants on GFPUbAKIRIN2. Gels were imaged using fluorescent imaging (488nm) to visualise the recombinant GFP tag, and stain-free imaging to visualise all proteins. Gels were subsequently stained using Coomassie staining to visualise ubiquitin levels. Gels of assays are a representative of at least three experimental replicates. Reactions in G were performed at 25°C. The final concentration of E3 enzyme , in G: 0.2µM.
Figure 4C) A selection of 2D classes from Fig 1C. Arrows point to the position of the HECT domain. Scale bar: 100Å. Reactions in C were performed at 25°CF) Ubiquitination of GFPUbAKIRIN2 by UBR5 and UBR5ΔPL and G) isolated UBR5HECT. Gels were imaged as described in Figure 3. Gels of assays are a representative of at least two experimental replicates. All reactions were performed using a final concentration of 0.3µM E3.
Figure 1B) Representative CD3 immunohistochemistry staining of a muscle biopsy from a patient with inclusion body myositis (6-IBM).D) UMAP feature plots displaying the normalized expression of CD4, CD8A and CD8B genes. Data information: Each dot represents one cell.
Figure 2B) UMAP feature plots displaying the normalized expression of CD4, CD8A and CD8B genes. Data information: Each dot represents one cell.
Figure 3C) Violin plots displaying the normalized expression level of selected genes per cluster in muscle T cells (n=6 IIM, n=1 undefined myopathy). Data information: CM: central memory; EM: effector memory; TRM: tissue resident memory; Tregs: regulatory T cells. IIM: Idiopathic Inflammatory Myopathies.D) Violin plots displaying the normalized expression level of selected genes per patient in the four TRM clusters (n=6 IIM, n=1 undefined myopathy). IMNM: Immune-Mediated Necrotizing Myopathy; DM: DermatoMyositis; ASyS: AntiSYnthetase Syndrome; IBM: Inclusion Body MyositisE) Representative immunofluorescence stainings of HOBIT (red), CD3 (green), dystrophin (purple) and Hoechst 33342 (blue) performed on muscle tissue for patient 6 (IBM), patient 3 (DM) and 4 (ASyS). Images were acquired using a LSM 880 confocal without Airyscan microscope (63x oil objective). Scale bar=20μm. The white arrows show HOBIT+ T cells. DM: DermatoMyositis; ASyS: AntiSYnthetase Syndrome; IBM: Inclusion Body MyositisF) Isotype mouse and rabbit IgG1 immunofluorescence stainings. IBM: Inclusion Body MyositisG) Quantification of HOBIT+ T cells in muscle biopsies of patient 6 (IBM), patient 16 (IBM), patient 3 (DM) and 4 (ASyS). The number of counted CD3-positive T cells is indicated under each bar as n. ASyS: AntiSYnthetase Syndrome; IBM: Inclusion Body Myositis
Figure 4A) Stacked bar plots showing the frequency of T-cell clones per patient in the muscle (left) and peripheral blood (PB) (right), including non-expanded (unique, lightest shade) and expanded clones (colored from light to dark shades, from less expanded to more expanded clones)). IMNM: Immune-Mediated Necrotizing Myopathy; DM: DermatoMyositis; ASyS: AntiSYnthetase Syndrome IBM: Inclusion Body Myositis; IIM: Idiopathic Inflammatory Myopathies.
Figure 6B) Feature plots of IFI44L expression levels in each patient at early diagnosis in muscle T cells (upper panel) and peripheral blood memory T cells (lower panel). Data information: IMNM: Immune-Mediated Necrotizing Myopathy; DM: DermatoMyositis ASyS: AntiSYnthetase Syndrome; IBM: Inclusion Body Myositis;
Figure 1(B, C) HCT116, PKcs-/-, and Lig4-/- cells were subjected to Agilent Seahorse XFe24 analyzer to measure oxidative phosphorylation (B) via the oxygen consumption rate (OCR) and glycolysis (C) via the extracellular acidification rate (ECAR).(J, K) M059K/J cells were also subjected to the OCR and ECAR analyses.
Figure 2(B) HeLa S3 total cell lysate, nuclear and cytosolic and mitochondrial fractions were analyzed by western blot to show sub-cellular localization of DNA-PKcs and ATM.(C, D) 100 μg of purified HeLa S3 mitochondria were subjected to limited protease K (at the indicated amount) digestion in a final 50 μl reaction volume at 37C for 30 minutes (C) or sodium carbonate (100 mM) verse sodium chloride (133 mM) extraction followed by western blot (D).
Figure 3(A) HeLa cells expressing Flag-tagged ANT1-3 were subjected to Co-IP using anti-Flag antibody, then analyzed by western blot as indicated.(E) HeLa cells expressing Flag-tagged VDAC1-3 were subjected to Co-IP using anti-Flag antibody and analyzed by western blot as indicated.(F) PLA analysis show that the interaction between DNA-PKcs and Flag-ANT2 was disrupted by knockdown of VDAC2, but not VDAC1 or VDAC3. Quantification shown on the right. ***, P < 0.001. Data information: The bar graphs were generated from three independent biological replicates analyses; data are represented as mean ± SD. Statistical significance was established using a two-way analysis of variance (ANOVA, *, P < 0.05; **, P < 0.01; ***, P < 0.001).
Figure 4(A, B) HCT116, DNA-PKcs-/-, and Ligase 4-/- cells were subjected to ADP-ATP exchange assay using MgGreen 5K+ fluorescent dye. The exchange was stimulated by 5mM ADP and halted by ANTs inhibitor carboxyatractyloside (cATR). Normalized MgGreen 5K+ fluorescence was first measured to indicate changes in free magnesium concentration (A). The decreased free magnesium concentration was converted into efflux of ATP ([ATP]e, mM) after the addition of ADP (B). Bar graph on the right shows the normalized MgGreen 5K+ fluorescence and efflux of ATP concentration after ADP until the reaction was stopped by cATR treatment.(F) shGFP and shATM pair of HeLa (Right) or MCF7 (Left) cells were subjected to an ADP-ATP exchange assay using MgGreen 5K+ fluorescent dye.
Figure 5(A) Western blot results show attenuation of ANT2 protein level in DNA-PKcs-deficient and depleted cells.(L) Treatment with VDAC2 siRNA in Hela cells attenuated ADP-ATP exchange. Bar graph on the right shows the efflux of ATP concentration after adding ADP.
Figure 6(B) PLA analysis show that the interaction between DNA-PKcs and Flag-ANT2 was disrupted by 10 Gy irradiation, but prevented by pre-treatment with 5 mM NAC. Quantification shown at the right. Data information: The bar graphs were generated from three independent biological replicates analyses; data are represented as mean ± SD. Statistical significance was established using a two-way analysis of variance (ANOVA, *, P < 0.05; **, P < 0.01; ***, P < 0.001).(C) Co-IP with endogenous DNA-PKcs show that the interaction between DNA-PKcs and Flag-ANT2 was temporarily disrupted by 10 Gy irradiation, whereas VDAC2 remained interaction with DNA-PKcs.
Figure 7(A, B) Paired shGFP and shATM HeLa (A) and MCF7 cells (B) were subjected to ADP-ATP exchange assay using MgGreen 5K+ fluorescent dye. The decreased free magnesium concentration was converted into efflux of ATP ([ATP]e, mM) after the addition of ADP. The bar graphs were generated from three independent biological replicates analyses;, D) PLA analysis show that the interaction between DNA-PKcs and Flag-ANT2 in HeLa (C) and MCF7 (D) was temporarily disrupted by treatment of 10Gy IR. Knockdown of ATM disrupted the dissociation. Quantification shown on the right. The bar graphs were generated from three independent biological replicates analyses(E) HeLa cells expressing Flag-tagged VDAC2 and V5-tagged ANT2 were subjected to Co-IP using anti-Flag antibody and analyzed by western blot as indicated. ANT2, but not VDAC2, temporarily dissociated from DNA-PKcs after treatment of 10Gy IR. ATM knockdown and inhibition (AZD1390, 10 nM) disrupted the dissociation.
Figure 8(A-G) Non-transformed cell lines including (A, B) human retinal pigment epithelial-1 (RPE-1), (C, D) primary human skin fibroblasts (HSF), (E) hTERT immortalized human skin fibroblasts 48BR, (F) primary mouse embryonic fibroblasts (MEF) RPE-1, and (G) A-T patient derived skin fibroblasts GM02052 were subjected to ADP-ATP exchange assay at various time points (up to 16 hrs) in response to 100 μM of H2O2 or 10 Gy IR as indicated. ATM inhibitor (AZD1390, 10 nM) was included in parallel analyses. The grafts display sham (white), bottom (red), and recovery (blue) of ADP-ATP exchange activities after treatments. The bar graphs were generated from three independent biological replicates analyses; data are represented as mean ± SD.