EMBO/sd-character-level-ner huggingface dataset:
Tpl2[D270A] mutation impairs phagosome maturationA.Figure 1A. Phagocytic uptake of fluorescently-labelled latex beads by WT and Tpl2[D270A] (Tpl2D270/D270A) BMDMs. Intracellular fluorescence was monitored following uptake of AF488 latex beads by BMDMs, (n = 4 wells). Data information: One representative experiment out of three shown. Error bars and shaded areas represent SEM. **** P < 0.0001. B. Intra-phagosomal proteolysis in WT and Tpl2[D270A] BMDMs was assayed following uptake of DQ Green BSA / AF594 latex beads. As positive assay control, BMDMs were separately pre-treated with 100 μg/ml leupeptin for 1 h to inhibit serine-cysteine proteases (n = 4 wells). Data information: One representative experiment out of three shown. Error bars and shaded areas represent SEM. **** P < 0.0001. Paired Mann-Whitney t-test; All differences relative to WT are ****. C. Cathepsin activity assay in WT and Tpl2[D270A] BMDMs 0.5 h after uptake of latex beads. BMDMs were stained with the Magic Red cathepsin L substrate (red). Average fluorescence intensity of the cathepsin probe per cell was quantified (n = 40-51 cells). E. pH assay in WT and Tpl2[D270A] BMDMs upon 0.5 h after incubation with latex beads. BMDMs were stained with the LysoTracker Red DND-99 dye (red). Average fluorescence intensity of the Lysotracker Red probe per cell was quantified (n = 95-126 cells). Data information: One representative experiment out of three shown. Error bars and shaded areas represent SEM. **** P < 0.0001. Student's unpaired t-test. D. Intra-phagosomal acidification in WT and Tpl2[D270A] BMDMs was monitored following uptake of BCECF-coupled latex beads. BMDMs were pre-treated with 1 μM bafilomycin A1 for 15 min to inhibit V-ATPases (n = 4 wells). Data information: One representative experiment out of three shown. Error bars and shaded areas represent SEM. **** P < 0.0001. Paired Mann-Whitney t-test; All differences relative to WT are ****. F. Intra-phagosomal oxidative burst in WT and Tpl2[D270A] BMDMs was assayed following uptake of OxyBURST Green BSA / AF594 latex beads. BMDMs were pre-treated with 1 μM bafilomycin A1 for 15 min to inhibit ROS production (n = 4 wells) Data information: One representative experiment out of three shown. Error bars and shaded areas represent SEM. **** P < 0.0001. Paired Mann-Whitney t-test; All differences relative to WT are ****. G. Reactive oxygen species assay in WT and Tpl2[D270A] BMDMs upon 0.5 h after incubation with latex beads. BMDMs were stained with the ROS Deep Red dye. Average fluorescence intensity of the ROS Deep Red probe per cell was quantified (n = 80-110 cells) Data information: One representative experiment out of three shown. Error bars and shaded areas represent SEM. **** P < 0.0001. Student's unpaired t-test.
Figure 5(A) Venn diagram representing the overlap between the N-termini sets identified in wild type and nsi mutant lines. N-termini from four replicates of Arabidopsis wild type and two independent nsi knockout lines (nsi-1 and nsi-2) were retrieved and compared. More than 300 N-termini could be retrieved in all samples (B) Venn diagram of quantified NTAed proteins. Half of the retrieved N-termini could be quantified and 173 were common to all samples. (C) Comparison of NTA yield of retrieved N-termini of proteins starting at position 1 or 2. The majority of these proteins, corresponding mostly to cytosolic components, undergoing or not to N-terminal methionine excision, were not affected by inactivation of GNAT2. For statistical analyses nsi-1 and nsi-2 were pooled and compared to the wild type. Two independent technical replicates of four biological replicates for each of the WT, nsi-1 and nsi-2 samples were analysed. Error bars are ± SD. (D) Comparison of NTA yield of retrieved N-termini of proteins starting at positions > 2. Clear alteration of NTA yield was observed in nsi mutant lines in the pool of nuclear-encoded plastid proteins. nsi-1 and nsi-2 samples were treated as in D. Error bars are ± SD (E) Comparison of NTA yields of retrieved N-termini in plastid proteins. Similar variation as in panel D was observed when NTA of only plastid proteins was analyzed. (F) Volcano plot representing NTA analyses of nsi knockout lines (treated together) and wild type. For this analysis, the p-value was calculated using Excel's two-tailed t-test function, for two-sample with equal variance. The most impacted proteins are shown in green. N is related to the number of quantified N-termini. (G) IceLogo representation of the protein N-termini with modified NTA yield vs proteins with unmodified NTA yield. The color symbol associated to each residue is Black is aliphatic, green is small hydrophilic.
Figure 6Phenotypic analysis. Seedlings of the indicated genotypes were grown in LD conditions (16-h light/ 8-h dark) for 5 days, and then transplanted to new medium containing 0.4 μM NAA and kept in continuous white light or white plus UV-B light (1 W/m2) for 7 days. Images are shown in (A); scale bars = 2 mm. Lateral root density (number of lateral roots/length of primary root) (B) and average length of lateral roots (C) of the indicated genotypes were measured. SDs (n > 8) are indicated. Quantitative RT-PCR analysis of auxin signaling gene expression in roots of wild type (Col-0), uvr8, myb73 myb77, and myb73 myb77 uvr8. (D) Results under white light and (E) under white plus UV-B light. Seedlings grown in continuous white light for 12 days were kept in white light or transferred to white light plus UV-B for 1 day with the addition of 0.4 μM NAA, and roots were collected to analyze auxin signaling gene expression. Error bars represent SD of three biological replicates.
Figure 1CryoEM reconstructions of the extended, spontaneously contracted, and urea-contracted tails colored according to the distance to the axis of the reconstruction. The color code bar shown in panel (A) and which is given in Angstroms is the same for all the three panels. The central axial sections of the reconstructions. Note the C-terminal domain of gp104 and other low-density features that do not visualize well in the isosurface representation (panels (A-C)). Letters G, H and I indicate the position of the cross-sections shown in the homonymously labeled panels below. A 69 Å-thick slice of the reconstruction of the extended sheath cryoEM map orthogonal to the sixfold axis. The proximal and distal parts of one of the three fibers that run at 82° to the sixfold axis are indicated with a semitransparent cyan and magenta lines, respectively. Sections of the spontaneously and urea contracted sheath structures that cut through the baseplate/proximal part of the fiber plane, whose position in the reconstructions is indicated with a red line in panels (E and F). The putative direction of the distal part of the fiber that is completely disordered in the reconstructions is indicated with a solar cross symbol.
TFG depletion disrupts early secretory pathway organizationHuman RPE‐1 cells were mock‐transfected (control) or transfected with a TFG siRNA for 60 h.Figure 5A HumanRPE‐1 cells were mock‐transfected (control) or transfected with a TFG siRNA for 60 h. Cells were fixed and stained using α‐Sec16A and α‐Sec31A antibodies and imaged using confocal microscopy. Images shown are projections of 3D data sets (4 μm in z). Both individual and merged images with Sec16A in green and Sec31A in red are shown (at least 15 cells for each condition). Scale bar, 5 μm. Higher magnification views of the indicated regions (boxed) are also shown in the lower or upper right portion of each image. Inset scale bar, 1 μm.B Quantification of the number of Sec31A‐labeled structures in a 4 μm2 region. For both conditions (control or TFG siRNA‐treated), at least 50 distinct regions away from the peri‐nuclear Golgi were examined. Error bars represent mean ± SEM; at least 15 different cells per condition. **P 0.01 compared with control, calculated using a paired t‐test. C Quantification of the number of Sec16A‐labeled structures in a 4 μm2 region. For both conditions (control or TFG siRNA‐treated), at least 50 distinct regions away from the peri‐nuclear Golgi were examined. Error bars represent mean ± SEM; at least 15 different cells per condition. No statistically significant difference was observed, following analysis using a paired t‐test. D Cells stably expressing low levels of GFP‐ERGIC‐53 and mRuby‐Sec23A were fixed and imaged using SR‐SIM (top), or depleted of endogenous TFG prior to fixation and SR‐SIM imaging (bottom). Images shown are representative of at least 15 individual cells analyzed for each condition. Scale bar, 5 μm. Inset scale bar, 1 μm. E HumanRPE‐1 cells were mock‐transfected (control) or transfected with a TFG siRNA for 60 h. Cells were fixed and stained using α‐Sec16A and α‐ERGIC‐53 antibodies and imaged using SR‐SIM. Images shown are projections of 3D data sets (4 μm in z). Both individual and merged images with Sec16A in green and ERGIC‐53 in red are shown (at least 15 cells for each condition). Scale bar, 5 μm. Higher magnification views of the indicated regions (boxed) are also shown in the lower or upper right portion of each image. Arrowheads highlight Sec16A‐labeled structures that do not exhibit juxtaposed ERGIC‐53 staining. Inset scale bar, 1 μm.F Based on structured illumination microscopy, quantification of the number of Sec16A‐labeled structures juxtaposed (within 500 nm) to ERGIC‐53‐labeled structures is shown. For both conditions (control or TFG siRNA‐treated), at least 1,000 unique Sec16A‐labeled structures were examined. Error bars represent mean ± SEM; at least 15 different cells per condition. **P 0.01 compared with control, calculated using a paired t‐test.
Single cell analysis of the two kidney biopsy samples.Figure 2.cell type annotations visualized by UMAP. B) The sample specific UMAP visualization of two samples. Heatmap of canonical markers applied for cell type assignment. Violin plot displaying the expression signals of kidney cell types.
Figure 1qRT-PCR detection of the fold induction of IFNB1 mRNA relative to unstimulated condition in different cell types. Cells were stimulated with ecGAMP (5 μg/ml) for 4 h. qRT-PCR detection of Ifnb1 mRNA abundance in mBMDMs treated with different eCDNs (5 μg/ml) for 4 h. ELISA detection of IFNβ release by mBMDMs treated for 4 h or 24 h with extracellular 2'3'-cGAMP at indicated concentrations. ELISA detection of IFNβ release by mBMDMs treated for 4 h or 24 h with extracellular c-di-AMP at indicated concentrations. qRT-PCR detection of IFNB1 mRNA in THP-1 cells stimulated with indicated eCDNs (5 μg/ml) for 4 h. ELISA detection of IFNβ in supernatants of THP-1 cells stimulated with indicated eCDNs at indicated concentrations for 4 h. qRT-PCR detection of the fold induction of IFNB1 mRNA relative to unstimulated condition in human CD14+ monocytes derived from PBMC stimulated with indicated eCDNs (5 μg/ml) for 4 h and 8 h. Each symbol represents one individual donor. Data are means+SD averaged from 10 healthy donors. ELISA detection of IFNβ in supernatants of human CD14+ monocytes derived from PBMC stimulated with indicated eCDNs (5 μg/ml) for 4 h. Each symbol represents result from one individual donor. Data are means+SD averaged from 10 healthy donors.
Figure 7(A) Antibody-based affinity purification of endogenous FAM210A from purified murine cardiac mitochondria coupled with mass spectrometry analysis. (B) Top list of FAM210A-interacting proteins in IP-MS (immunoprecipitation-mass spectrometry) analysis. PSM: peptide spectrum match. C) Confocal imaging of co-localization of FAM210A-3xFLAG and endogenous EF-Tu by immunostaining. Scale bar: 40 μm. (D) Left panel: IP-IB validates FAM210A-interacting proteins using pre-immune IgG and FAM210A antibodies. Right panel: IP-IB validates FAM210A-interacting proteins in HEK293T cell lysates subjected to IP using pre-immune IgG and anti-FLAG antibodies. A representative image is shown in three biological replicates. (E) Mapping of interacting domains in FAM210A for binding EF-Tu. (F,G) Protein expression of mitochondrial ETC component genes was measured in AC16 cells after following treatments in comparison with control cells: (F) siRNA knockdown of FAM210A. (G) FAM210A overexpression.
In all, 51 hits including SCOC and WAC are validated by deconvolution and additional assays.Figure 2(A) SCPO, STIPO and STAPO for the 51 genes validated by two out of four individual siRNA duplexes reproducing the GFP-LC3 spot phenotype, above a cutoff of greater or less than 20% of control, and passing additional criteria (Supplementary Table 3). Red boxes indicate an increase >1.2; pink boxes an increase between 1.1 and 1.2; white boxes are between 0.9 and 1.1; light blue boxes a decrease between 0.9 and 0.8; dark blue boxes a decrease to 0.8. Hits underlined were taken forward as three out of four4 hits, (§) not chosen because of restricted tissue expression, (*) are three out of four when alternate method of normalisation is used. (B) Table summarising results from further validation of 20 genes. mRNA levels scored as (+) or (−) indicate efficient or poor knockdown, respectively (see Supplementary Figures S1 and S2). Increasers (left) or decreasers (right) assayed for LC3 lipidation were scored (+) for yes, (−) for no, (+/−) subtle change and (nd) not done (Supplementary Figure S3). (C-E) STIPO results after incubation under different autophagy‐inducing conditions following (C) SCOC knockdown (D) SUPT5H knockdown and (E) WAC knockdown. siRNA‐treated cells were incubated for the indicated time in FL: full medium plus leupeptin; ES; EL: ES plus leupeptin; Torin: 250 nM Torin1 in FL; Resv: 128 μM Resveratrol in FL; LiCl: 10 mM LiCl in FM.
SMO/HH regulates Smaug levels and activity.Figure 2(A) Smaug repression assay. The assay is based on the dual expression of a construct encoding a λN-HA-SNAP-Smaug chimeric protein (abbreviated λN-SNAP-Smaug) and a second that is transcribed into a reporter mRNA (called SNAP-GUS-5BoxB) that carries a translational fusion between SNAP and GUS coding regions followed by five Box B hairpins (5BoxB) inserted in the 3'UTR. A plasmid encoding a GFP-SNAP fusion (not shown here) was also used as a transfection and loading control. (B, C) Smaug downregulates reporter expression. Mean values of the relative levels of SNAP-GUS reporter (SNAP-GUS/GFP-SNAP ratio, in arbitrary units) in extracts of transfected Cl8 cells in the absence (black) or presence of λN-SNAP-Smaug (red), λN-SNAP (pale grey) or SNAP-Smaug (dark grey). See Fig EV3A for the representative gel and Fig EV3B which shows that λN-SNAP-Smaug has no effect on a SNAP-GUS construct that lacks the 5BoxB. N=3 (biological replicates). See also Fig EV3A-C. Here and in D-F: SNAP-GUS/GFP-SNAP ratios were set to 1 in cells expressing λN‐SNAP-Smaug. The error bars correspond to the standard deviation of the mean (SD). Statistical analysis was done by a Kruskal-Wallis test by ranks followed by a Dunn test and p values are: black bracket <0.05, red bracket <0.01, green bracket <0.0001. Here and in D-E: 15 ng of pAct.λN-SNAP-smaug plasmid were used per transfection. , E) HH/SMO reduces Smaug levels and its overall negative effect on reporter expression. Mean values of the relative levels of reporter expression (determined as above) (D) or of λN-SNAP-Smaug (ratio λN-SNAP-Smaug/GFP-SNAP) (E) in the absence of SMO constructs (red) or in the presence of SMOWT-HA (green), SMOPKA-SD-HA (blue) or SMO∆958-HA (yellow), without HH (plain boxes) or in presence of HH (striped boxes). N=6 (independent biological replicates), except for "no SMO, no HH" and "SMOWT with HH" conditions where n=10 (independent biological replicates). A representative gel is shown in Fig EV3D. Note that SMOWT-HA or HH alone had reproducible weak effects that were observed in two independent biological triplicates but were not statistically significant. See also Fig EV3E-F. (F) HH/SMO reduces Smaug's intrinsic repressive ability. A similar experiment was conducted with different amounts of λN-SNAP-smaug expressing plasmid (ranging from 0 to 50 ng) as indicated). The mean values of the relative levels of reporter (y-axis) are plotted against the relative levels of λN-SNAP-Smaug (x-axis). SNAP-GUS/GFP-SNAP ratios were set to 1 in cells expressing λN‐SNAP-Smaug in absence of SMOPKA-SD/HH. The colored area represent the extent of the values observed for the relative amounts of reporter expression obtained with (blue) and without (red) SMOPKA-SD/HH, respectively. N=3 (biological triplicates). See Fig EV3H and also a representative gel in Fig EV3G.
Lrig-1 is required for sustained regeneration.Figure 6A Representative WISH images for Lrig-1. B RNAlow neoblast size in intact animals following control or Lrig-1 knockdown (n ≥ 53 technical replicates). C Quantification of H3P immunostaining in intact animals following control or Lrig-1 knockdown (n = 9-10 technical replicates). D Quantification of H3P immunostaining at homeostasis (fixed at 7 days post-RNAi, white region) and at 48 hpa (amputated at 5 days post-RNAi and fixed at 7 days post-RNAi, yellow region) during the first round of regeneration in control or Lrig-1 KD animals (n = 10-17). E Quantification of unpigmented blastema area (as % total tissue area) in regenerating control or Lrig-1 KD animals from 2 dpa to 7 dpa during the first round of regeneration (n = 10 animals per condition per time point). The same animals were used for all timepoints. Representative images from 7 dpa are shown. Scale bars, 500 µm. F Quantification of H3P immunostaining at homeostasis (fixed at 14 days post-RNAi, white region) and at 48 hpa (amputated at 12 days post-RNAi and fixed at 14 days post-RNAi, yellow region) during a second round of regeneration in control or Lrig-1 KD animals (n = 10-18). G Quantification of unpigmented blastema area (as % total tissue area) in regenerating control or Lrig-1 KD animals from 2 dpa to 7 dpa during the second round of regeneration (n = 9-10 animals per condition per time point). The same animals were used for all timepoints. Representative images from 7 dpa are shown. Scale bars, 500 µm.
Figure 4(a-b) Atoh1+ progenitors show stem cell capacity during normal homeostasis and regeneration. At early stages of recovery, d10-12, Atoh1+ cells lineage trace the colonic crypt (N=4 per condition). Data information: Nuclei are counterstained with Dapi (blue); white dashed lines indicate basement membrane. Scale bars =50μm Data are represented as mean ± SD analyzed using two-way ANOVA with Sidak's multiple comparisons test *P≤0.05, ***P≤0.001. (c-d) By d30 after DSS, Atoh1 derived stem cells continue to lineage trace complete colonic crypts (N=4 per condition). Data information: Nuclei are counterstained with Dapi (blue); white dashed lines indicate basement membrane. Scale bars first two panels of c =50μm; scale bars (last panel of c =100μm. Data are represented as mean ± SD analyzed using two-way ANOVA with Sidak's multiple comparisons test *P≤0.05, ***P≤0.001. (e) Illustration of experimental protocol outlining DT treatment of Atoh1CreERT2;ROSA26tdTomato;Lgr5DTR-GFP mice. Whole mount (f) images of Atoh1 lineage labeled crypts. Data information: Nuclei are counterstained with Dapi (blue); white dashed lines indicate basement membrane. Scale bars =50μm section (g) images of Atoh1 lineage labeled crypts. Majority of crypts are labeled after Lgr5+ stem cell ablation and remain label with the appearance of Lgr5+ stem cells (inset, arrowheads). Atoh1 lineage labeled crypts remain by d30. Data information: Nuclei are counterstained with Dapi (blue); white dashed lines indicate basement membrane. scale bars =100μm. (h) Significantly increased labeling of crypt base was observed at d8 in DT treated mice (N=4 per condition). Data are represented as mean ± SD analyzed using Student's t-test with Welch's correction *P≤0.05, ***P≤0.001. (i) DSS and DT treatment of Atoh1CreERT2;ROSA26tdTomato;Lgr5DTR-GFP mice. (j-k) Scattered labeling of Atoh1+ cells in control conditions, while DSS injury lead to Atoh1 lineage labeled crypts lacking Lgr5+ stem cells. Data information: Nuclei are counterstained with Dapi (blue); white dashed lines indicate basement membrane. Scale bars =50μm Data are represented as mean ± SD analyzed using Student's t-test with Welch's correction *P≤0.05, ***P≤0.001.
Fig.Figure 5(A) Representative images show hippocampal vGlut2 and Homer1 immunostaining. White box outlines the region of interest and yellow box shows the regions where confocal images were taken. (B) Confocal images of synaptic immunostaining in CA1 striatum lacunosum-moleculare (str. lac/mol) at P10. Arrows pointing to the enlarged synapse in the insets. (C, D) Quantification of synapse density in CA1 str. lac/mol in iCKO versus control at P10 (C), and CKO versus control at P21 (D). At P10, N(Ctrl) = 4, N(iCKO) = 3, p = 0.02. At P21, N = 8, p = 0.005. (E) Representative images show hippocampal vGlut1 and Homer1 immunostaining. (F) Confocal images of synaptic immunostaining in CA1 striatum radiatum at P10. (G, H) Quantification of synapse density in CA1 striatum radiatum in iCKO versus control at P10 (G), and CKO versus control at P21 (H). At P10, N = 5, p = 0.83. At P21, N = 5, p = 0.89.
Figure 3A) Michaelis-Menten kinetic parameters of purified PP2A-B56α and PP2A-B55α holoenzymes were determined against the indicated phospho-peptides. Mean and standard deviation shown in plots as black bars (n=3 independent experiments) B-D) In vitro dephosphorylation by the PP2A-B55α and PP2A-B56α holoenzymes of panels of phosphorylated peptides as indicated. Mean and standard deviation shown in plots as black bars (n=3 independent experiments)
Figure 5Analysis of migration patterns of WT and Arghap45−/− activated T cells on 2D surface coated with ICAM-1. Each track represents the migratory path of individual WT and Arghap45−/− activated T cells recorded over 466 s in a single field of view, Trajectories were plotted to a common starting point, and > 200 T cells were recorded per plot using time-lapse microscopy at 10 x magnification. Analysis of migration patterns of WT and Arghap45−/− naive T cells on 2D surface coated with ICAM-1 and CCL21. Same conditions as in (A). Individual cell speed in an experiment involving 188 WT (black dots) and 144 Arghap45−/− (red dots) activated T cells, respectively. Each dot corresponds to the mean speed of a given cell. Bars represent the mean and SD. Three replicates are shown in panel D. n B-D ean and SD are hown. *P ≤ 0.033 (*), npaired T test. Percentage of WT (black dots) and Arghap45−/− (red dots) activated T cells remaining adherent after application of a shear stress of 1 dyne/cm2 for 120 s. Three independent experiments were performed involving more than 200 cells and each dot correspond to the mean percentage of remaining adherent cells in one given experiment. ean and SD are shown. npaired T test. Average cell speed of WT (black dots) and Arghap45−/− (red dots) activated T cells. Three independent experiments were performed involving more than 200 cells and each dot correspond to the mean of the speed in one given experiment. Average cell speed was evaluated by dividing the cumulative displacement of cells tracked over 10 seconds by the total time of displacement. ean and SD are shown. npaired T test. Individual cell speed in an experiment involving 283 WT (black dots) and 305 Arghap45−/− (red dots) naïve T cells, respectively. Same conditions as in (B). Four replicates shown in panel H. n ean and SD are shown. **** P ≤ 0.0001, unpaired T test. Percentage of WT (black dots) and Arghap45−/− naive T cells (red dots) remaining adherent cells after application of a shear stress of 1 dyne/cm2 for 120 s. Same conditions as in (C). n -G mean and SD are shown. **** P ≤ 0.0001, unpaired T test. Average cell speed of WT cell (black) and Arghap45−/− naive T cells (red). Same conditions as in (D). unpaired T test.