EMBO/SourceData huggingface dataset:
Figure 4(A) Superposition of the ATPγS-MjMR-DNA complex (green) and apo-MjMR (3AV0, magenta) structures. Alignment was done using their NBDs (1.3 Å rmsd in the positions of 311 Cα atoms). The coiled-coils of the DNA-bound Rad50 molecules are shifted (arrow) to more parallel orientation. The entire structures can be superimposed with a 1.9 Å rmsd in 692 Cα positions.(B, C) Comparison of the local structures between the apo (B) and the DNA-bound MjMR complex (C). Upon DNA binding, helix α6 translates to DNA backbone, resulting in the tighter packing of Leu155 and Leu156 against a hydrophobic pocket formed by lobe I and II. Arrows indicate the direction of the helix movement by DNA binding. See Fig EV5 for a close-up view.
Figure 5(A) Stereo view of the superposition of the ADP-MjRad50 (3AUX) complex (black) onto the ATPγS-MjMR-DNA complex (green and blue) by aligning their lobe II domains. The α1-α2 loop that could collide with parts of the DNA molecule is indicated. (B) Aligning the lobe II domains of two structures in Fig 5A shows that rotation of the lobe I opens the gate for Mre11, which could allow the access of the unwound DNA. Color schemes are same as those in Fig 5A.(C) DNA-unwinding activities of TmMR. Lane 2, no protein; lanes 3 and 4, no ATP; lanes 5 and 6, with ATPγS; lanes 7 and 8, with ATP. The DNA: protein ratio was 1:2 for lanes 3, 5, and 7, and 1:5 for lanes 4, 6, and 8. Plotted data represent the mean of three experiments, with error bars indicating one standard deviation. A graph of the quantified data is shown in the lower panel.(D) Comparison of the ATP-dependent DNA unwinding activities of WT and mutant TmMR proteins. Lane 2, no protein; lane 3 and 4, WT TmMR; lane 5 and 6, Rad50 (Δ54-56) mutant; lane 7 and 8, R87E mutant; lane 9 and 10, K95E mutant; lane 11 and 12, R94E/K95E mutant; lane 13 and 14, K115E mutant. For each TmMR, reaction contains 1:2 or 1:5 ratio of DNA: protein.
Figure 6(A) Endonuclease activities of the TmMR WT and mutant proteins towards a duplex DNA in the absence (left panel) or presence of ATP (right panel). Major products are marked with dots. The 5'-32P-label is indicated by a star. See Appendix Fig S4A and S4B for the quantified values.(B) Quantification of a bubble DNA binding by WT or mutant TmMR in the presence or absence of AMP-PNP. See Appendix Fig S3E and S3F for the original data.(C) Endonuclease activities of the WT and mutant TmMR proteins towards a bubble DNA molecule in the absence (left panel) or presence of ATP (right panel). See Appendix Fig S4C and S4D for the quantified values.(D) The effects of single or multiple point mutations in the DNA-contacting or neighboring residues of S. cerevisiae rad50 on the sensitivity to chemical drugs. Sensitivities of rad50 expressing the mutants towards indicated concentrations of CPT, PHL, and MMS were examined. (E) The effects of Rad50 mutation on recombination stimulated by inverted Alu repeats. See Appendix Table S1 for the quantified values.(E) The effects of Rad50 mutation on recombination stimulated by inverted Alu repeats. See Appendix Table S1 for the quantified values.
Figure 1B) Single point activity assays of USP1WT and deletion mutants on Ub-Rho show signifi­cantly increased activity in mutants where insert L1 and L3 are both deleted.C) Single point activity assays of USP1WT and deletion mutants (± UAF1) on Ub-Rho show the loss in hyper-activation of USP1ΔL1L3 upon addition of UAF1.D) Michaelis-Menten analysis of USP1 deletion mutants against ubiquitin-rhodamine (Ub-Rho) shows that USP1ΔL1L3 has significantly higher activity compared to WT and other mutants (n=2, biological replicates).E) Michaelis-Menten analysis of USP1 deletion mutants (+UAF1) against Ub-Rho shows that all deletions mutants have similar catalytic activity (n=2, biological replicates).F) Comparing activity of USP1WT and USP1ΔL1L3 on a peptide substrate i.e. LRGG-AMC (100µM). USP1ΔL1L3 cleaves the peptide substrate more efficiently compared to USP1WT (n=2, technical replicates).G) Comparing activity of USP1WT and USP1ΔL1L3 (+UAF1) on a peptide substrate i.e. LRGG-AMC (100µM). Addition of UAF1 allows cleavage of LRGG-AMC by both USP1WT and USP1ΔL1L3 (n=2, technical replicates).
Figure 2A) Quantification of gel-based activity assays where the activity of USP1WT and deletion mutants (+UAF1) on PCNA-UbTAMRA is compared. The USP1ΔL1 mutant has reduced activity on PCNA-UbTAMRA compared to the other USP1 variants tested (n=2, biological replicates).C) Quantification of gel-based activity assays where the activity of USP1 PIP mutants (+UAF1) on PCNA-UbTAMRA is compared. Both the PIP mutants have reduced activity on PCNA-Ub compared to USP1WT (n=2, biological replicates).D) FP based binding assays of USP1 mutants (+ UAF1) with PCNA-UbTAMRA show reduced binding of USP1ΔL1 and USP1PIP1 compared to USP1WT (n=2, technical replicates).E) Quantification of gel-based activity assays where the activity of USP1WT+UAF1 on PCNA-UbWT is compared with PCNA-UbPIM1 shows that PCNA-UbWT is cleaved much faster compared to PCNA-UbPIM1 (n=2, technical replicates (example shown here from 1 of 2 biological replicates))F) Comparing activity of USP1WT and USP1PIP1 (+UAF1) on PCNA-UbWT and PCNA-UbPIM1 in gel-based assays shows that the activity of USP1WT is severely reduced by the PIM1 mutation in PCNA, whereas USP1PIP1 activity is not further affected indicating that PIP1 and PIM mutation affect the same interaction (n=2, technical replicates (example shown here from 1 of 2 biological replicates)).
Figure 3A) Schematic representation of loading and purification of PCNA-Ub on nicked circular DNA. The DNA-loaded PCNA-Ub elutes in the void of the SEC column and this sample is collected and used for studying activity of USP1 on DNA-loaded PCNA-Ub.B) Michaelis-Menten analysis of USP1+UAF1 with and without DNA (65bp dsDNA) shows that DNA binding alone has no effect on USP1 activity (n=2, technical replicates (example shown here from 1 of 2 biological replicates)).C) Coomassie stained gel of in vitro activity assay showing increased activity of USP1-UAF1 on DNA-loaded PCNA-Ub compared to PCNA-Ub (-DNA) and PCNA-Ub (+DNA, +RFC and not ATP).D) Quantification of gel-based activity assays showing enhanced activity of USP1+UAF1 on DNA-loaded PCNA-Ub compared to free PCNA-Ub (n=3, technical replicates (example shown here from 1 of 2 biological replicates)).E) Quantification of gel-based activity assays showing enhanced activity of USP1 on DNA-loaded PCNA-Ub compared to free PCNA-Ub (n=3, technical replicates (example shown here from 1 of 2 biological replicates)).
Figure 4A) Gel based quantification of USP1-UAF1 activity on PCNA-Ub (DNA loaded and free) for three different concentration of enzyme and substrate.B) FP based activity assays on free PCNA-Ub (three concentrations of USP1 and PCNA-Ub) and activity of USP1 on increasing concentrations of Ub-Rho.
Figure 5A) Comparison of USP1WT and deletion mutants (+UAF1) for activity on DNA-loaded PCNA-Ub (solid lines) and free PCNA-Ub (dashed lines) shows no increase in USP1ΔL1 activity on the loaded substrate. Left panel: Quantification of gel-based activity assays showing percentage of cleaved PCNA-Ub at the mentioned time points (n=2, biological replicates), Right panel: Quantification of the activation fold observed in USP1WT and mutants on DNA-loaded PCNA-Ub versus free PCNA-Ub (n=2, biological replicates).B) Multiple sequence alignment of USP1 insert L1 which highlights the DNA binding region and the conservation of the positively charged residues across species. Three groups of positively charged residues (***) are mutated separately and tested for binding to double stranded DNA (65bp) using SPR which shows the importance of this region for DNA binding of USP1.C) Comparison of USP1DM1, USP1PIP1 and USP1PIP1+DM1 (+UAF1) activity on DNA-loaded PCNA-Ub (solid lines) and free PCNA-Ub (dashed lines) shows reduced increase in activity of these mutants compared to USP1WT. Left panel: Quantification of gel based activity assays showing percentage of cleaved PCNA-Ub at the mentioned time points (n=2, technical replicates), Right panel: Quantification of the activation fold observed in USP1WT, USP1PIP1, USP1DM1 and USP1PIP1+DM1 on DNA-loaded PCNA-Ub versus free PCNA-Ub (n=2, technical replicates).D) EMSA based DNA binding experiment shows that USP1PIP1 and USP1WT has similar DNA binding capability while USP1DM1 has lost its ability to bind DNA.
Figure 1A. BMDMM were treated as indicated with either 60ng/mL ultra-pure (UP) LPS, 50ng/mL human derived recombinant GM-CSF, 40µM WEHD-FMK for 16 hours or 5µM nigericin for the last hour. Supernatants were then analysed for IL-1β secretion by ELISA. Data information: n≥ 3 biological replicates (every dot represents one biological replicate). For all panels, error bars are SEM. Significance was calculated using one way ANOVA with multiple comparisons or two-way ANOVA to compare across genotypes. p values are shown.B. HoxB8 macrophages were treated with the indicated dilutions of GM-CSF using supernatants from GM-CSF expressing cells. Shown are ELISA analysis of IL-1β secretion in supernatants and western blots indicating IL-1β expression in lysates.. Data information: n≥ 3 biological replicates (every dot represents one biological replicate). For all panels, error bars are SEM. Significance was calculated using one way ANOVA with multiple comparisons or two-way ANOVA to compare across genotypes. p values are shown.C. HoxB8 macrophages of the indicated genotypes were treated as indicated with 60ng/mL UP-LPS, 1% GM-CSF containing supernatant (approximately 2ng/mL GM-CSF), 40µM WEHD.FMK, 0,5µM CRT-0066101 for 16 hours, 5µM nigericin was added for the last hour. Supernatants were analysed by ELISA for IL-1β. Inlay panels show western blots for levels of IL-1β expression in the indicated knockouts. Data information: n≥ 3 biological replicates (every dot represents one biological replicate). For all panels, error bars are SEM. Significance was calculated using one way ANOVA with multiple comparisons or two-way ANOVA to compare across genotypes. p values are shown.D. HoxB8 cells were treated with 60ng/mL of UP-LPS, 1% GM-CSF supernatant, 50mM KCl, and 5µM nigericin as indicated for 16 hours before supernatants were analysed for IL-1β by ELISA. Data information: n≥ 3 biological replicates (every dot represents one biological replicate). For all panels, error bars are SEM. Significance was calculated using one way ANOVA with multiple comparisons or two-way ANOVA to compare across genotypes. p values are shown.
Figure 5A. HoxB8 macrophages were seeded in an 8-well Ibidi chamber and treated as indicated for 12 hours. Cells were fixed and stained for ASC (Red) and with DAPI (blue). For nigericin treatments nigericin was added for only 30 minutes. Example cells containing ASC Specks are shown with white arrows. Scale bars represent 50µM. Data information: Microscopy pictures are representative images from 3 biological replicates.B. HoxB8 cells were treated as indicated with LPS or LPS+GM-CSF. The cross linker DSS was added to the cells as described in methods. Samples were analysed by western blot for ASC oligomerization. Black vertical lines indicate that non-relevant lanes were removed during figure preparation but samples were run on the same gel. Data information: westerns are representative images from 3 biological replicates.C. Wild type and caspase-1-/- HoxB8 macrophages were treated as indicated for 16 hours. Cell lysates were made and analysed by western blot for levels of caspase-1 and gasdermin D cleavage. Data information: westerns are representative images from 3 biological replicates.D. HoxB8 macrophages were treated as indicated with LPS or LPS+GM-CSF for 12 hours. Cells were lysed and NLRP3 was immune precipitated using a magnetic bead coupled antibody. As a negative control, beads without NLRP3 antibody were used. Beads were boiled and analysed for recruitment of NLRP3, and caspase-1. * indicates light chain from the antibody. Molecular weight markers shown in middle of NLRP3 blot were embedded in image using Chemostar imaging software (INTAS) at time of image acquisition. Data information: westerns are representative images from 3 biological replicates.E. HoxB8 macrophages were incubated with combinations of LPS, GM-CSF, nigericin +/- Z-VAD.FMK as indicated. Proteins were extracted and analysed by western blot for caspase-1 processing. Data information: westerns are representative images from 3 biological replicates.F. HoxB8 macrophages were seeded in a 96 well microplate and co-treated with either 10µM or 50µM z-VAD with LPS+GM-CSF for 16 hours. Then 100nM of Sytox Green was added to the cells. The number of sytox green+ cells per image was quantified. Data information: Significance was calculated using one way ANOVA with multiple comparisons. Every dot represents one biological replicate. P values are shown.
Figure 7A. HoxB8 macrophages were treated for 16 hours with LPS +/- GM-CSF as indicated. Proteins were extracted and analysed for levels of IL-1β by western blot.B. HoxB8 macrophages were treated with LPS or LPS+GM-CSF as indicated for 16 hours. Cells were lysed in buffer containing FLAG tagged K63 TUBE and K63 ubiquitylated proteins were precipitated using anti FLAG pull down. Pulled down proteins were analysed for IL-1β and ubiquitin by western blot.C. IL-1β-/- hoxB8 macrophages were infected with lentivirus expressing IL-1β-FLAG-6xHis or the indicated mutants from the IL-1β promoter. Cells were treated as indicated with LPS or LPS+GM-CSF or LPS+Nigericin and levels of secreted IL-1β were analysed by ELISA (top panel). GM-CSF treated cells were also lysed in urea buffer and nickel NTA affinity pull down was performed. Nickel NTA pull down was also performed on supernatants (bottom panel). Pulled down proteins form the His tag pull down were analysed for the presence of IL-1β by western blot. Molecular weight markers shown in middle of blot were embedded in image using Chemostar imaging software (INTAS) at time of image acquisition. Data information: n≥ 3 biological replicates (every dot represents one biological replicate). For all panels, error bars are SEM. Significance was calculated using one way ANOVA with multiple comparisons, or two-way ANOVA to compare across genotypes. p values are shown.D. HoxB8 macrophages were treated as in A with addition of 50µM mitoTEMPO or 10mM NAC as indicated. Proteins were extracted and analysed for IL-1β by western blot.E. Wild type and TNF-/- HoxB8 macrophages were treated as indicated with LPS or LPS+GM-CSF. Proteins were extracted and analysed for IL-1β by western blot. Black vertical lines indicate that non-relevant lanes were removed during figure preparation but samples were run on the same gel.F. HoxB8 macrophages were treated as in A but with the addition of 25µM Punicalagin throughout the 16 hour time course. Proteins were extracted and analysed by western blot (left panel). Supernatants were also analysed by ELISA for secreted IL-1β (right panel). Data information: n≥ 3 biological replicates (every dot represents one biological replicate). For all panels, error bars are SEM. Significance was calculated using one way ANOVA with multiple comparisons, or two-way ANOVA to compare across genotypes. p values are shown.
Figure 8A. Wild type and GsdmD-/- HoxB8 macrophages were treated as indicated for 16 hours. Supernatants were analyzed for IL-1β secretion by ELISA. Data information: n≥ 3 biological replicates (every dot represents one biological replicate). For all panels, error bars are SEM. Significance was calculated using one way ANOVA with multiple comparisons, or two-way ANOVA to compare across genotypes. p values are shown.B. HoxB8 macrophages were seeded in an 8-well Ibidi chamber and treated for 14 hours with LPS and GM-CSF. Cells were fixed and stained for GasderminD (green), IL-1β (red) and DAPI (blue). Images were taken at the Zeiss LSM880 Confocal microscope Data information: shown. Microscopy pictures are representative images from 3 biological replicates. Scale bars in microscopy images represent 10µM.C. HoxB8 macrophages were co-stimulated with three different PEG 3mM for 16 hours as indicated. Supernatants were analyzed for IL-1β secretion by ELISA. Data information: n≥ 3 biological replicates (every dot represents one biological replicate). For all panels, error bars are SEM. Significance was calculated using one way ANOVA with multiple comparisons, or two-way ANOVA to compare across genotypes. p values are shown.D. HoxB8 macrophages were seeded in 96 well microplate and treated as indicated. Before treatment, sytox green was added and cells were imaged in an incucyte incubator microscope every hour for 16 hours. Shown is the sytox green signal normalized to the total cell area. Data information: n≥ 3 biological replicates (every dot represents one biological replicate). For all panels, error bars are SEM. Significance was calculated using one way ANOVA with multiple comparisons, or two-way ANOVA to compare across genotypes. p values are shown.E. Wild type and GsdmD-/- HoxB8 macrophages were treated as indicated for 16 hours. Proteins were extracted and analysed for IL-1β ubiquitination by western blot. Data information: westerns are representative images from 3 biological replicates.
figf1(a) Representative confocal micrographs of mitochondrial morphology in MEFs of the indicated genotype 24 h after transfection with mtYFP. Where indicated, cells were starved for 2.5 h. Scale bar, 20 μm. (b) Morphometric analysis of mitochondrial shape. Experiments were carried out as in a. Data represent mean ± s.e.m. of three independent experiments (n=100 cells per condition in each experiment).(c) Top left, 48 h after transfection with the indicated siRNA, MEFs were lysed and 25 μg samples of proteins were separated by DS-polyacrylamide gel electrophoresis (PAGE) and immunoblotted with the indicated antibodies. Top right and bottom, representative images show mitochondrial morphology of MEFs transfected with the indicated siRNA and after 24 h with mtYFP. After a further 24 h, confocal micrographs were acquired. Scale bar, 20 μm. Uncropped images of blots are shown in Supplementary Fig. S8. (d) Morphometric analysis of mitochondrial shape. Experiments were carried out as in c. Data represent mean ± s.e.m. of five independent experiments (n=100 cells per condition in each experiment).(e) Representative images of mitochondrial fusion over time (indicated in min). MEFs were co-transfected with mtPAGFP and mtRFP and after 24 h, mtPAGFP was photoactivated in a region of interest and cells were imaged by real-time confocal microscopy. Where indicated, MEFs were starved for 2.5 h. Scale bar, 20 μm. See also Supplementary Movies S1 and S2. (f,g) Quantification of mitochondrial fusion in MEFs of the indicated genotype. Experiments were carried out as in e. Data represent mean ± s.e.m. of four independent experiments.(h) Representative electron micrographs of muscle (longitudinal sections; four top micrographs) and liver (four bottom micrographs) from CD1 mice. Where indicated, mice were fasted for 12 h. The outlined regions of micrographs from muscle samples are magnified ×7 in the images below. Scale bars, 2 μm.
figf2(a-d) Levels of mitochondria-shaping proteins during starvation. Protein samples (20 μg) from MEFs of the indicated genotype were separated by SDS-PAGE and immunoblotted with the indicated antibodies. Cells were starved for the indicated times.(e) Association of DRP1 with mitochondria during starvation. Mitochondria were isolated from MEFs starved for the indicated times and 25 μg samples of proteins were separated by SDS-PAGE and immunoblotted with the indicated antibodies. (f) Levels of Ser 637 phosphorylation of DRP1 during starvation. Equal amounts of cell lysates from wild-type MEFs starved for the indicated times were immunoprecipitated with the indicated antibody and the immunoprecipitated proteins were separated by SDS-PAGE and immunoblotted with the indicated antibodies. (g) Quantitative analysis of Ser 637 phosphorylation of DRP1 during starvation. Experiments were carried out as in f. Data are normalized to total levels of DRP1 and represent the mean ± s.e.m. of three independent experiments.(h) MEFs were treated for the indicated times with 100 nM rapamycin or with 25 μM forskolin (FRSK) for 0.5 h, lysed and equal amounts (50 μg) of proteins were separated by SDS-PAGE and immunoblotted using the indicated antibodies.(i) HeLa cells were transfected for 2 days with the indicated siRNA or treated with 25 μM forskolin for 0.5 h and lysed. Equal amounts (50 μg) of proteins were separated by SDS-PAGE and immunoblotted using the indicated antibodies. Uncropped images of all blots in this figure are shown in Supplementary Fig. S8.
figf3(a) Pseudocolour-coded images of EPACI-camps FRET from real-time imaging of wild-type MEFs transfected with EPACI-camps. Where indicated, cells were perfused with the starvation solution for the indicated times (in min) or with 25 μM forskolin (FRSK). Scale bar, 20 μm. Colour scale indicates F1/F0 ratio. See also Supplementary Movie S3. (b) Quantitative analysis of CFP/YFP FRET ratio. Experiments were carried out as in a. Where indicated, cells were perfused with the starvation solution or with 25 μM forskolin. Data represent mean ± s.e.m. of 13 independent experiments.(c) Lysate samples (50 μg) from MEFs of the indicated genotypes were analysed by SDS-PAGE and immunoblotting using the indicated antibodies. Where indicated, MEFs were starved, or treated with 25 μM forskolin. Where indicated, 20 μM H89 was added during starvation. Uncropped images of all blots in this figure are shown in Supplementary Fig. S8.(d) Representative images of the effect of H89 on mitochondrial morphology on starvation. Wild-type and Mfn2−/−MEFs were transfected with mtYFP, and after 24 h confocal micrographs were acquired. Where indicated, cells were starved for 2.5 h and 20 μM H89 was added. Scale bar, 20 μm. (e) Morphometric analysis. Experiments were carried out as in d. Data represent mean ± s.e.m. of five independent experiments (n=100 cells per condition).(f) Starvation-induced mitochondrial elongation depends on Ser 637 of DRP1. Representative confocal micrographs of mitochondrial morphology of wild-type MEFs co-transfected with mtRFP and the indicated plasmids. At 24 h after transfection, where indicated cells were starved for 2.5 h and imaged. Where indicated, 20 μM H89 was present during starvation. Scale bar, 20 μm. (g) Morphometric analysis of mitochondrial shape. Experiments were carried out as in f. Data represent mean ± s.e.m. of five independent experiments (n=50 cells per condition).(h) Representative confocal micrographs of mitochondrial morphology of Drp1−/− MEFs co-transfected with mtRFP and the indicated plasmids. At 24 h after transfection, where indicated cells were starved for 2.5 h and imaged. Where indicated, 20 μM H89 was present during starvation. Scale bar, 20 μm. (i) Morphometric analysis of mitochondrial shape. Experiments were carried out as in h. Data represent mean ± s.e.m. of five independent experiments (n=50 cells per condition).
figf4(a-c) MEFs of the indicated genotype were treated as indicated, counted and 2.7×105 cells were lysed. Lysates were separated by SDS-PAGE and immunoblotted using the indicated antibodies. (d) Ratio between the densitometric levels of cyclophilin D and those of PMP70 in MEFs of the indicated genotype. One representative experiment of five independent repetitions performed as in a-c is shown.(e) MEFs of the indicated genotype starved for 5 h were treated where indicated with 0.5 μM wortmannin (Wortm.). Lysates from 2.7×105 cells were separated by SDS-PAGE and immunoblotted with the indicated antibodies. Uncropped images of all blots in this figure are shown in Supplementary Fig. S8.