EMBO/sd-character-level-ner huggingface dataset:
Figure 4A) Spatial distribution of lamin A/C in the nuclei of HSPC (CD38-), myeloid progenitors (CD33+) and lymphoid progenitors (CD19+). Inverted images of equatorial Z plane of DAPI and lamin A/C are shown. Scale bar: 5 μm. B) Line scans of lamin A/C signal along nucleus contour of representative cells are presented for each cell population. The corresponding variations in lamin A/C intensity and nucleus curvature are plotted in blue and in red, respectively. Negative curvature values correspond to local nuclear invaginations. Red asterisk underlines local nucleus invaginations. C) Dispersion of A/C signal along the nucleus periphery is similarly low in CD38- (n=17, 3 donors), CD33+ (n=61, 3 donors) and CD19+ (n=11, 3 donors) indicative of an homogenous distribution of the protein along the nuclear membrane. (Bars correspond to mean ± SD; n.s: non significant; Mann Whitney test). D) Dispersion of lamin B signal along the nucleus periphery is similarly low in CD38- (n=131, 3 donors), and CD19+ (n=74, 3 donors) indicative of a homogenous distribution of the protein along the nuclear membrane. But is significantly increased in CD33+ (n=146, 3 donors). Bars correspond to mean ± SD; ****:p<0.0001; Mann Whitney test. E-F-G) Spatial distribution of lamin B and local measurements of lamin B intensity and curvature of nuclear membrane in CD38- (E), CD33+ (F) and CD19+ (G) cells. Inverted images of the equatorial Z plane of DAPI (left panel) and lamin B (right panel) of a representative nucleus are shown for each population. Scale bar: 5 μm. The variations in lamin B signal intensity and curvature are plotted in blue in red, respectively. Asterisks indicate local invaginations of the nuclear membrane. In the bottom graphs, positive and negative curvatures are shown in grey and red, respectively. The absence of curvature (curvature =0) is highlighted as a dashed line. In CD33+ cells, negative curvature of the nuclear membrane are associated with low lamin B intensities.
Figure 6A. HEK293 cells were co-transfected with CDKL5 (wild type "WT" or kinase-dead "KD" K42R mutant) fused to an NLS, and FLAG-ELOA (wild type "WT" or a S311A mutant "SA"). Anti-FLAG precipitates or cell extracts were probed with the antibodies indicated. One of three independent experiments is shown. B. Same as A. showing a range of pathogenic (red) and benign (blue) CDKL5 variants. (C-E). Wild type (WT), CDKL5­-disrupted (CDKL5Δ/Δ) or siRNA-transfected cells were subjected to indirect immunofluorescence analysis with the indicated antibodies at laser tracks. Quantification of ELOA-pSer311 signal at the laser tracks is shown. Data represent mean ± SD of total pELOA Ser311 intensities in different biological replicates as indicated (n). For simplicity, only intensities greater than zero are shown. Statistical significance was assessed by one-way-ANOVA-test or unpaired t test with Welch's correction. Asterisks **** indicate P-values of <0.0001. Scale bar is 10 μm
How DnaK dependence and GK usage are linked.Figure 8A) Enrichment of APRs not protected by a specific charge in DnaK substrates, as determined by Niwa and colleagues (Niwa et al, 2012), analysed versus PureTANGO score of the APR considered. Statistical significance was determined through hypergeometric testing, with Bonferroni correction for multiple testing. *: p-value < 0.05. P-values and Pearson correlation coefficients (R2) for linear regression are indicated. B) Distribution of the difference in netcharge between orthologs from a Mycoplasma species devoid of DnaK (M. crocodyli, indicated as MYCCM), and its closest DnaK-expressing relative (M. synoviae, indicated as MYCS5). C) GK usage in DnaK-expressing Mycoplasma synoviae orthologs. D) GK usage in DnaK-depleted Mycoplasma crocodyli orthologs.
Figure 7A. Representative images for co-localization between RPA32 (green) and single stranded C-rich telomeric sequences (ss-Telo-C, red FISH probe), in G2/M-synchronized cells. White arrows indicate co-localization events. B. Quantification of images in (A). A total of at least 55 nuclei were scored from three independent experiments. Each dot represents the number of events per nucleus. C. Quantification of images in (A) as the percentage of single stranded C-rich telomeric sequences associated with RPA32 to total single stranded C-rich telomeric sequences A total of at least 55 nuclei were scored from three independent experiments. Data information: All scale bars: 5 μm. Data are shown as mean ± SD, with the red line marking the mean value. Statistical analyses were performed with Student's two-tailed unpaired t-test with Welch's correction for normality of distribution unless specified otherwise. D. Representative images for co-localization between RPA32 or p-RPA32S33 (green) and telomeres (red FISH probe), in the indicated cell lines treated with siLuc or sihnRNPA1 for 48 h. White arrows indicate co-localization events. Data information: All scale bars: 5 μm. Data are shown as mean ± SD, with the red line marking the mean value. Statistical analyses were performed with Student's two-tailed unpaired t-test with Welch's correction for normality of distribution unless specified otherwise. E. Quantification of images in (D). A total of 85-135 nuclei were scored from three independent experiments. Each dot represents the number of events per nucleus. Statistical analyses were performed using one-way ANOVA with post-hoc Games-Howell's test for multiple comparisons. Data information: All scale bars: 5 μm. Data are shown as mean ± SD, with the red line marking the mean value. Statistical analyses were performed with Student's two-tailed unpaired t-test with Welch's correction for normality of distribution unless specified otherwise. F. Representative images for co-localization between RPA32 (green) and telomeres (red FISH probe) in the indicated cell lines treated with either DMSO or 10 μM NU7026 DNA-PK-inhibitor (DNA-PK-i) for 48 h. White arrows indicate co-localization events. Quantifications are shown on the right. A total of 53-84 nuclei were scored from three independent experiments. Statistical analyses were performed using one-way ANOVA with post-hoc Games-Howell's test for multiple comparisons. Data information: All scale bars: 5 μm. Data are shown as mean ± SD, with the red line marking the mean value. Statistical analyses were performed with Student's two-tailed unpaired t-test with Welch's correction for normality of distribution unless specified otherwise.
Figure 9Mec1 is recruited to RPA-ssDNA following DNA damage to promote Rad53-mediated checkpoint signaling that prevents immediate long-range resection of DNA ends. This anti-resection function of Mec1 is important to protect DNA ends and ensure that subsequent resection occurs in a restrained and controllable manner. Since the Dpb11-Rad9 interaction is not dependent on Mec1 (but mostly dependent on CDK phosphorylation), the Dpb11-Rad9 complex is rapidly stabilized at DNA lesions once initial Mec1 signaling and Ddc1 phosphorylation occurs. As Mec1 signaling builds up, it hyper-phosphorylates the Slx4 scaffold, which becomes a strong interactor of Dpb11 and displaces Rad9 to counteract checkpoint signaling and Rad9-mediated resection block. Mec1 signaling therefore switches to an anti-checkpoint and pro-resection mode. Once long-resection occurs, extensive ssDNA accumulates, leading to increased opportunities for strand invasion, but also increased opportunities for non-allelic recombination (rad9∆ cells have increased non-allelic recombination (Fasullo et al, 1998; Nielsen et al, 2013)). In this context, we propose that a new mode of Mec1 signaling triggered by extensive resection stabilizes the STR complex at lesions via interaction with Dpb11 for proper regulation of HR. It is tempting to speculate that the Dpb11-9-1-1 complex acts as a "processivity factor" for the helicase function of Sgs1 to processively displace, and reject, heteroduplexes.
Figure 8A Left: Schematic representation of VHH antibodies produced in E. coli as monomers or homo-trimeric fusions with the human collagen XVIII trimerization module. The VHH trimers match the three-fold rotational symmetry of the SARS-CoV-2 Spike protein. The molecular weight of the indicated modules is shown. Right: As an example, monomeric and trimeric forms of Re7H02 (a Re6B06 class member, Fig 1A) were analyzed by size exclusion chromatography (SEC, Superdex 200 column) coupled with multi-angle light scattering (MALS). The elution profiles (UV absorbance signal), as well as the calculated molecular weights (per slice), are plotted, and the average molecular weight is indicated. B Re6B06 monomer and trimer variants (20 nM) were analyzed by BLI for RBD-binding, with 900 s association and 1500 s dissociation steps. Note the avidity effect of Re6B06 trimerization. C Neutralization of SARS-CoV-2 by the indicated VHH antibodies. The neutralization experiment was performed Note the gain in neutralization efficacy when a VHH is trimerized. Re6B06, for example, is 30 000-fold more potent when symmetry-matched with the virus Spike.
Figure 1A. Sequential digestion workflow. Proteins or protein complexes are crosslinked and digested with trypsin. After splitting the sample into four aliquots, one remains single digested with trypsin (T) while the others are sequentially digested with either AspN (A), chymotrypsin (C) or GluC (G). Samples are enriched by SEC and the three high-MW fractions are analysed by LC-MS, submitted to xiSEARCH and xiFDR analysis. B. Results of the sequential digestion workflow applied to a synthetic 7-protein mix, compared to using trypsin alone in four replicates and parallel digestion with trypsin, AspN, chymotrypsin and GluC. A trypsin four replicate experiment shows a large overlap of the four datasets with little gain. Parallel digestions with trypsin, AspN, chymotrypsin and GluC demonstrates high complementarity but moderate gains over trypsin. Sequential digestion shows low overlap between the four datasets and the largest gain in unique residue pairs. C. Gains of repeated analysis (trypsin only), parallel digestion and sequential digestion for the same data as shown in panel B. D. Crosslinked peptides obtained by sequential digestion of a synthetic 7-protein mix are smaller than their corresponding tryptic peptides.
VP1407 can serve as an effective on/off switch for VpT6SS1 in V.Figure 2A Illustration of the VpT6SS1 (vp1386-vp1420) activation cascade. Genes are represented by arrows indicating the direction of transcription. Genes belonging to the same operon are denoted by the same color. Locus numbers (vpxxxx) are denoted above. Positive regulators are denoted by a red frame. Dashed arrows denote transcriptional activation and are color coded according to the relevant induced operon. Numbers in black circles next to the positive regulators TfoY, VP1407, and VP1391 delineate the progression of the cascade. B Viability counts of V. natriegens prey before (0 h) and after (4 h) co-incubation with wild type V. natriegens attackers harboring the indicated plasmids, on media containing 3% NaCl at 30 °C. Data information: data are shown as the mean ± SD, n = 3 technical replicates; statistical significance between samples at the 4 h timepoint by an unpaired, two-tailed Student's t-test is denoted above and is color coded to match the relevant samples. A significant difference was considered as P < 0.05. C Expression (cells) and secretion (media) of VgrG1 from V. natriegens containing an arabinose-inducible vp1407 in the chromosomal dns locus (VnatReg) and harboring an empty plasmid (pEmpty) or plasmids containing VpT6SS1 that lacks vp1407 (pT6SS1Ind) or both vp1407 and hcp1 (pT6SS1Ind/∆hcp1; T6SS-). Samples were grown in media containing 3% NaCl and either supplemented (+) or not (-) with 0.1% arabinose (Ara) at 30 °C. Loading control (LC) is shown for total protein lysates. D Illustration of the engineered VnatReg (yellow bacteria) containing a plasmid-borne, inducible VpT6SS1 (pT6SS1Ind). In the presence of arabinose (red circles), VP1407 is expressed from the chromosome and the plasmid-borne T6SS is induced, resulting in T6SS-mediated intoxication of adjacent prey bacteria (blue). E Viability counts of V. natriegens prey before (0 h) and after (4 h) co-incubation with VnatReg attackers carrying the indicated plasmids on solid media plates, as described in C. Data information: data are shown as the mean ± SD, n = 3 technical replicates; statistical significance between samples at the 4 h timepoint by an unpaired, two-tailed Student's t-test is denoted above and is color coded to match the relevant samples. A significant difference was considered as P < 0.05.
Figure 1A-C. Epidermal thickness of the back skin was analyzed at the indicated time points in c-JunΔ/Δ Mx1-Cre (A), c-JunΔ/Δ CD11c-Cre (B) and c-JunΔ/Δ K5-Cre-ERT2 mice (C) on Hematoxylin and eosin (H&E) stained sections. (n = 5-21 (A), n = 6-29 (B), n = 5-18 (C); ≥ 2 independent experiments) Data information: Data are shown as mean ± SEM. P‐values were calculated by one-way ANOVA with Tukey (A- C, Statistical significance: ns > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, **** P < 0.0001. See Appendix Table S3 for exact P‐values. H&E stained sections of back skin from indicated mice treated with IMQ for 2, 3 or 5 days. Bright-field images, Magnification: 20x, Scale bar: 100 µm. Layers of epidermis (left) and Trans-epidermal water loss (TEWL) (right) were analyzed in the back skin of indicated mice. (Left: n = 8-14, right: n = 6-14; ≥ 2independent experiments). Data information: Data are shown as mean ± SEM. P‐values were calculated by one-way ANOVA with Tukey Statistical significance: ns > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, **** P < 0.0001. See Appendix Table S3 for exact P‐values. Flow cytometry of total back skin after 2, 3 and 5 days of IMQ treatment. Analyzed were dermal γδ T cells (γδ TCRint+) (F) Graphs show immune cells as % of live, single cells. (n = 10-28 (F), ; ≥ 2 independent experiments). Data information: Data are shown as mean ± SEM. P‐values were calculated by Bonferroni multiple comparison test Statistical significance: ns > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, **** P < 0.0001. See Appendix Table S3 for exact P‐values. Flow cytometry of total back skin after 2, 3 and 5 days of IMQ treatment. Analyzed were Monocytes (CD11b+Ly6Chi) (G), and Neutrophils (CD11b+Ly6G+) (H). Graphs show immune cells as % of live, single cells. n = 10-21 (G), n = 4-25 (H); ≥ 2 independent experiments). Data information: Data are shown as mean ± SEM. P‐values were calculated by Bonferroni multiple comparison test Statistical significance: ns > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, **** P < 0.0001. See Appendix Table S3 for exact P‐values.
Autophagy Inhibition Increases PI(3)P-Positive Early Endosomes.Figure 2The following experiments were performed in glial shNf-1/shTp53 glial cells serum starved 4 hr before assaying. A: Control, sgAtg7, or sgAtg16l1 cells were treated with 2 ng/mL EGF for 15 min. Cells were then processed for staining using anti-EEA1 antibodies and PI(3) P probe (Alexa 488-labelled 2XFYVE domains). To ensure the specificity of the probe, control cells were pre-treated with 5 mM 3'MA for 30 min. Scale bar: 10 μm. B: Quantification of PI(3) P+ label intensity per cell (in A). C: Pearson's colocalisation coefficient between PI(3) P and EEA1 (in A). Data information: Statistical analyses were performed on at least 3 independent experiments, where error bars represent SEM and p values represent a two-tailed Student's t test: NS p>0.05, * p<0.05, ** p<0.01, *** p<0.001. The following experiments were performed in glial shNf-1/shTp53 glial cells serum starved 4 hr before assaying. D: Control and sgAtg7 cells were stimulated 20 ng/mL EGF for 5 or 15 min before immunofluorescence staining against EEA1 and EGFR. Scale bar: 10 μm. E: Pearson's colocalisation coefficient between EEA1 and EGFR (in D). Data information: Statistical analyses were performed on at least 3 independent experiments, where error bars represent SEM and p values represent a two-tailed Student's t test: NS p>0.05, * p<0.05, ** p<0.01, *** p<0.001. The following experiments were performed in glial shNf-1/shTp53 glial cells serum starved 4 hr before assaying. F: Endogenous VPS34 was immunoprecipitated from control and sgAtg7 cells that were treated with 2 ng/mL EGF for 15 min then lysed in CHAPS-containing detergent buffer and binding partners detected by western blotting. G: Densitometry analyses of proteins coimmunoprecipitated with endogenous VPS34 (in F). Data information: Statistical analyses were performed on at least 3 independent experiments, where error bars represent SEM and p values represent a two-tailed Student's t test: NS p>0.05, * p<0.05, ** p<0.01, *** p<0.001.
Figure 1Plot showing the average Z-scores of 210 endolysosomal genes plotted against the negative log10 of the P values from the one-sample t-test of whether average Z-score ≠ 0. Green circles represent significantly upregulated genes (Z > 0 and P < 0.05). Magenta circles represent significantly downregulated genes (Z < 0 and P < 0.05). Grey circles represent genes whose expression is not significantly changed (P > 0.05). All comparisons were between cancers and normal cells from the same tissue. Dashed line shows position on Y-axis that corresponds to P = 0.05. Positions of MCOLN1 (red circle), VAC14 (blue circle), and MTM1 (yellow circle) are indicated. Inset, schematic showing that Mtm1 and Vac14 regulate the levels of PI(3,5) P2, and thereby, influence TRPML1 activity. Unsupervised hierarchical clustering of Pearson's coefficients of pairwise correlation of gene expression reveal 4 indicated clusters. Violin plots of average Z-scores of genes that belong to the four clusters derived from hierarchical clustering. The thick horizontal dashed lines in each sample set represent median; thin dashed lines represent the quartiles; polygons represent density estimates of data and extend to extreme values. Statistical tests employed were 1-sample t-test or Wilcoxon signed rank test to examine significance of differences from a hypothetical value of 0. GSEA conducted on endolysosomal genes ranked on the basis of increasing Z-scores. Positions of the CLEAR target genes are indicated. Bar graph showing the relative expression of the indicated genes in the indicated cell types. Values were normalized to respective 'HN31+HRAS shRNA' average and represent mean±SEM. Data points represent values from biological replicates. Statistical test employed was Student's t-test. Western blots performed on extracts isolated from the indicated cells probed with antibodies against Tfeb and tubulin. Molecular weights are shown on the left. Bar graph shows relative Tfeb levels in the indicated samples. Values were normalized to 'HN31+HRAS shRNA' and represent mean±SEM. Data points represent values from biological replicates. Statistical test employed was Student's t-test. Bar graph showing the relative expression of the indicated genes in the indicated cell types. Values were normalized to respective 'HN31+HRAS shRNA' average and represent mean±SEM. Data points represent values from biological replicates. Statistical test employed was Student's t-test. 'Attribute circle layout' generated from Pearson's coefficients of pairwise correlation of gene expression using CytoScape. Decreasing node size (clockwise from MCOLN1) reflects decreasing closeness centrality; node color represents degree (number of directed edges); edge color represents Pearson's correlations. Bar graph showing the relative expression of MCOLN1 in the indicated cell types. Values were normalized to HT1197 average and represent mean±SEM. Data points represent values from biological replicates. Statistical test employed was Student's t-test.
Synaptic targeting requires the homeodomain TF Dfd in neurons and muscles.Figure 8(A) Representative confocal images of stage 17 embryonic heads highlighting the expression of Dfd (purple), Myosin in muscles (blue) and FasII in axonal projections (green) in control (elav>RFP) animals and in animals with reduced Dfd expression in neurons (elav>DfdRNAi) or in muscles (Mef2>DfdRNAi) or both in both tissues (elav;Mef2>DfdRNAi). The single channels for the projections (FasII staining) of MN2a and/or MN3 to the MHE, MHD or LR muscles of an early stage 17 Drosophila embryo are shown. Asterisks highlight the location of MN2a and MN3, respectively. MN2a is identified by the FasII expressing axonal projection emerging from a Dfd expressing MN, which normally innervates the Dfd expressing MHE muscle (as shown in the elav>RFP control), while MN3 is identified as the MN underneath MN2a, which normally innervates the MHD muscle (as shown in the elav>RFP control). The panel on the right side represents a schematic drawing of the confocal image shown on the left side, summarizing the innervation of the anterior muscles (LR, MHE, MHD) by projections emerging from MN2a (magenta) or MN3 (cyan) in control and perturbation conditions. (B) Quantitative analysis of phenotypes. Each experiment was performed in triplicates, innervation rates were calculated from n=21 for elav>DfdRNAi, n=23 for Mef2>DfdRNAi n=20 for elav;Mef2>DfdRNAi animals. Note, each genetic experiment was performed in parallel to an adequate control experiment using the same driver line crossed to a line that controls expression of either UAS-RFP or UAS-GFPRNAi, p-values between two genetic conditions were calculated by a two-sided Fisher test. *** p < 0.005.
Figure 3A Model fitting to the experimental tFT-Bcd ratio in early n.c. 14 as function of AP position for the different models Black line represents mean measured ratio of tFT-Bcd reporter with mCherry-sfGFP-Bcd in y/w flies. The shadowed area represents ±1s.d. Inset is same as main panel, except for the tFT-sfGFP-fmCherry-Bcd reporter. B Increasing protein age results in increasing tandem reporter ratio. The fluorophore states of the tFT reporter are shown in the inset: (1) immature; (2) only sfGFP matured; (3) only intermediate mCherry matured; (4) sfGFP and intermediate mCherry matured; (5) only mCherry matured; and (6) both mCherry and sfGFP matured. kg, kr1, kr2 denote the maturation rates of sfGFP, intermediate mCherry and fully-matured mCherry respectively. The different curves correspond to sets of values for kg, kr1, kr2 selected from Gaussian distributions with half-time means 27, 40 and 9 minutes and standard deviations 2.7, 4 and 0.9 minutes respectively. C Least-squares fitting of each model in n.c. 14 to: (left) the sfGFP only; (middle) the tFT-Bcd reporter ratio only; and (right) both the sfGFP profile and the tFT-Bcd reporter ratio simultaneously. Each fit is performed with the fluorophore maturation rates drawn from Gaussian distributions D Quality of SDD model fitting to tFT-Bcd ratio for different Bcd diffusion coefficients and lifetimes. For each pair of these parameters, the fluorescence correction ε is left as a free parameter for fitting. The colormap is the log of the fitting function (Methods). The white lines correspond to parameter combinations that produce a gradient with a length constant λ=75μm (solid) and λ=95μm (dashed).
Figure 4(a) Changes in groups (carbohydrates, amino acids and fatty acids) of storage metabolites measured in circulating haemolymph with progressing wasting, shown as log2 changes measured by LC-MS and calculated per larvae relative to RasV12; ctrl at day 6. (b) Volcano plot showing autophagy-dependent changes to amounts of circulating metabolites at day 6. X-axis shows the log2 of fold change of RasV12;atg13 -/-scrib-/- // atg13 -/- vs. RasV12; scrib-/- , y-axis shows -log10 p-value, calculated by t-test. Metabolite names are shown for metabolites with log2(FC) >± 1 and/or -log10(P) < 2. Green points indicate log2(FC) >± 1, blue indicate -log10(P) < 2 and red indicates for above both thresholds. (c) Autophagy-driven wasting releases metabolites into circulation of 113 reliably detected metabolites, those showing significant differences in any of the three comparisons are shown. Color indicates the log2(fold change) difference and the numbers show the p-value of the comparison. The statistical test to define significance was FDR-adjusted t-test p-value < 0.05