EMBO/sd-character-level-ner huggingface dataset:
B) HeLa cells were incubated for 4 h with complete medium in the presence (Hla) or absence (Ctr) of 10 µg/ml α-hemolysin or they were infected for 4 h with the wt strain of S. aureus (wt) or the α-hemolysin deficient mutant (Hla−). Cells were disrupted and whole cell lysates were subjected to pull-down assays using GST-Ral-GDS-RBD-sepharose. The levels of GTP-bound Rap2b were determined as was described in Materials and Methods by Western blot analysis using a rabbit anti-Rap2b and the corresponding HRP-labeled secondary antibody. The bands were subsequently developed with an enhanced chemiluminescence detection kit. The band intensities were quantificated with the Adobe Photoshop program is shown in the lower panel. * p<0.05 (paired Student's t-test). These data are representative of two independent experiments.
Neurons expressing M18Y473D are not impaired at SV docking. Autaptic munc18-1 null hippocampal neurons expressing M18Y473D or M18WT as controls were analysed with electron microscopy on chemically fixed samples ((C) Total number of SVs is not different (M18WT: 142 ± 11 SVs; M18Y473D: 148 ± 15 SVs, multilevel analysis, p = 0.456).(B-E) M18WT : N=6 autaptic neurons, n=137 synapses; M18Y473D: N=8 autaptic neurons, n=116 synapses.
(e) Gamma-H2AX-ChIP signal in the Sµ region is shown as the fraction of immunoprecipitated DNA (% IP) normalized to the total amount of DNA used for the immunoprecipitation (n=3; mean + sd; two-tailed unpaired Student's t-test; ns = not significant).
Quantitative phosphoproteomics dataset showing phosphorylation sites enriched in cells lacking RAD9. Among the most highly enriched sites are Sgs1 T1269, Uls1 T540, Mec1 S1964, and 2 S-N sites in Rfa2. This response is similar in both the presence of 40μg/mL phleomycin (x axis) and 0.02% MMS (y axis). Note downregulation of Rad53 signaling (red dots) due to absence of RAD9.
(A) Protein sequence alignment of the C-terminal region of D. melanogaster, human, and yeast Glycogen synthases. Identical residues are blue; all other residues are red. Conserved in all three species, R593, W609, and S651 are underlined.
Subcutaneous injection of limiting numbers of JNKi-treated MDA231-LM2 cells in NSG mice.
A. Representative time-lapse spinning disk confocal images of n = 3 of live COS7 cells stably expressing ER marker GFP-TMD (green). The cells were loaded with SiR-lysosome to visualize LE/Lys (orange). Zoom insets show a merge of the ER (GFP-TMD) (green) and SiR-lysosome labelled LE/Lys (orange). Zoom insets show a region of interest containing a mobile endosome as denoted by the white arrow. Scale bars: full size 10µm, zoom inset 3µm. Images related to movie EV1. B. Kymograph of the representative image in panel (A).
Whole-mount immunofluorescence of P8 mesenteric lymphatic vessels of Foxp2flox/-;Tie2-Cre and littermate control mice showing loss of organized FOXC2high valve regions but unaltered expression of FOXC2 in Foxp2 deficient vessels. Whole-mount immunofluorescence of lymphatic valves in P7 mesenteries of control and Foxp2flox/-;Tie2-Cre mice showing NFATc1 subcellular localization. Note predominantly nuclear localization of NFATc1 in control valves (left panels), but cytoplasmic localization (middle panels, 3 out of 5 mice) or downregulation of NFATc1 (right panels, 2 out of 5 mice) in the mutant valves. Quantification of protein localization in NFATc1+ lymphatic valves from (D) (n = valves [mice], as indicated).
(D) ThP1 cells were infected with HIV-1 at an MOI of 20, in absence (left) or presence (right) of nevirapine (NVP, 10 µM). Blue: DAPI staining. Green: vRNA (RNA-FISH). Red: vDNA (EdU). (E) Scatter plots show intensities of vDNA and vRNA in detected vRNA clusters.
A) mScarlet was added between the RNA recognition motifs (RRMs) and LC of HRPA-1 and the third exon was replaced with the corresponding human sequence; disease mutation D290V was introduced.
E Percentage of apoptotic cells, determined by measuring Annexin-V and PI positive HCT116 or U87 cells by FACS. The effects were assessed after a 24h or 48h treatment respectively (NEN (N) 1.2 µM; Domperidone (Domp, D) 30 µM, zVAD-FMK (zVAD) 100 µM, Staurosporine 100 µM. Cells were pre-treated for 1h with the zVAD-FMK inhibitor prior to adding the drugs. Data information: Data are presented as mean (SD) (N=3) and were analyzed by one-way ANOVA with Tukey post-hoc test (for (E, upper graph) HCT116 cells) or by Kruskal-Wallis with Dunn´s post-hoc tests (for (E, lower graph) U87 cells). Significance is indicated for multiple comparisons as indicated. * p< 0.05; ** p< 0.01;*** p< 0.001; **** p < 0.0001.
Cell-cycle profiles of HEK293, RPE and U2OS FAM111A-expressing cell lines that were induced with doxycycline for 48 h and analyzed by flow cytometry.
A, Co-IP assay of PIF3-TAP with TOC1-GFP vs. 5X-GFP in N. benthamiana. Protein extracts were immunoprecipitated by IgG resins followed by HRV-3C protease digestion. PIF3 and TOC1 were detected by anti-MYC and anti-GFP, respectively. B, Quantification of protein interaction in A. Results from 3 independent trials were averaged. Error bars indicate SEM, asterisks indicate significant difference (*p<0.05, student's t test).
e, Immunoblot of beclin 1 in post-nuclear supernatant (PNS) and Golgi-enriched fractions in HeLa cells stably transduced with empty vector or GAPR-1-Myc after peptide treatment (20 μM, 2 h).
Average urea production from each group was determined by a commercially available urea assay kit.
(E) Immunostaining of Galectin-3, CathD and CD68 in spinal cord sections from 5-month-old WT, Tmem106b-/-, Grn-/-, and Tmem106b-/-Grn-/- mice. Scale bar = 10 µm.
C-F Viability counts of the indicated prey bacteria before (0 h) and after (4 h) co-incubation with VnatReg attackers harboring the indicated plasmids, on media containing 3% NaCl and 0.1% arabinose at 28 °C. Data information: Data are shown as the mean ± SD, n = 3 technical replicates. Statistical significance between samples at the 4 or 24 h timepoints 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.
(J, K) THP-1 cells stably expressing full-length nucleocapsid or truncated nucleocapsid lacking amino acids 70-160 and 290-360 were stimulated with 10 μM nigericin for 30 min following pretreatment with 1 μg/ml LPS for 3 h. An ELISA assay was performed to determine supernatant IL-1β levels (J) and a cell viability assay was conducted to verify the percentage of viable cells through checking cellular ATP levels (K). For J, K, Control vector used here was empty vector corresponded to the nucleocapsid-containing vector. For J, K, Student's t-test was used and data were shown as means±SD of three technical replicates. *, P<0.05. Results were repeated for three times with similar results.
(A) Cytosolic ATP synthesis in the indicated VSMC types, measured by incorporation of phosphate-32 (32Pi) into ADP. [32Pi]-ATP was separated from 32Pi using the molybdate method, as explained in the Methods section.
A. Representative images and quantification of Drosophila midguts treated with the pH indicator Bromophenol Blue. Moreover, quantification of the three categories of Drosophila of indicated conditions. The three categories can be divided into: homeostasis, a well-defined acidic (yellow colored) copper cell region (CCR) is flanked by basic (blue colored) anterior midgut (AM) and posterior midgut (PM). "Perturbed A", the acidic region is lost and the whole gut is basic; "Perturbed B", the strongly acidic region is lost, and the remainder of the gut also becomes less basic. The numbers of quantified guts from left to right are 90, 90, and 90. Error bars show the SD of three independent experiments.
A. Reciprocal Co-IP of USP44 and USP7. HEK293T cells were transfected with constructs encoding MYC-USP44 and FLAG-USP7. These cells were lysed and pertinent factors were immunoprecipitated using either anti-FLAG or anti-MYC. Pulled-down proteins were observed by immunoblot analysis using either anti-MYC or anti-FLAG antibodies.
(K) Quantitative analysis of area of UF. n=5, one‐way ANOVA, Bonferroni post hoc.
Top 100 genes up-regulated in collecting in comparison to lymphatic capillary LECs. Expression in BECs is shown for comparison. Heatmap color coding shows log2 fold change. Red box indicates LEC-specific/enriched collecting vessel signature genes that include Foxp2 (red arrow).
(A) Determination of differentiation states of skin cutaneous melanoma (SKCM) biopsies from the TCGA cohort (n=458) The vast majority of these tumors exhibited a differentiated profile (89%; melanocytic and transitory states). The others display a dedifferentiated profile (11%; neural crest-like cells and undifferentiated states). Human melanoma 501Mel cell line is classified as differentiated melanoma cells (melanocytic cells), according to the gene expression profile (four categories were defined by (Tsoi et al, 2018); melanocytic, transitory, neural crest-like and undifferentiated cells). 501mel cell line was selected for the CRISPR screens.
(B) Serum CRAMP determination by ELISA (n = 8). Data information: Data in B were representative and were the mean ± SD from three independent experiments. P values were calculated by unpaired two-tailed t-test for comparison of two groups
Comparison of the novel 78-kb PAI reported in this study, with the 89-kb PAI reported in epidemic strains from two outbreaks in China. The key factors for virulence of epidemic strains are indicated in green, including SalKR, NisKR, a Type IV-Like Secretion System and a Tn916 element. The antibiotic-resistance genes are shown in red. Three regions in the 89-kb PAI are absent from the 78-kb PAI. No virulence-related genes were found within these regions.
Whole-mount immunofluorescence of embryonic mesenteries of the indicated developmental stages. Note induction of FOXP2 expression at E17.
T The number of MuSC-related events in FACS experiments is significantly increased in linc-MYH-KO animals (n = 7 KO/7 WT, Student's t test, two-tailed, *p = 0.0126). Data are mean ± S.E.M.
D-E. Representative immunofluorescence of c-Jun (green), CD1a (red), CCL2 (D) or Il23p19 (E) (white) and DAPI in psoriatic lesions. Arrows indicate triple-positive cells and an asterisk highlights a representative one that is shown enlarged in a white-framed inlet. Magnification: 40x. Scale bar: 50 µm. (n = 2 patient samples).
(C) Exemplary TEM image of a WT cell incubated with OA for 1h. A grid is randomly placed on top of the image and the width of perpendicularly oriented ER profiles passing through grid lines are measured. Yellow lines indicate measured sites. (D) ER profile analysis of (C). Bars: average profile width/cell, ± SEM, n= 11-9 cells (149-224 profiles), *p<0.05 (unpaired T-test).
Immunoblot showing LC3-II formation in WT or GCN5 KO HeLa cells in the presence or absence of the lysosome inhibitor Baf.
A. Representative images for fragile telomeres using the CO-FISH assay. Lagging strands (C-probe) and leading strands (G-probe) were marked with green PNA probes and red LNA probes, respectively. White circles and arrows indicate "smeary" or "doublet" telomeric signals, respectively. Quantification of fragile telomeres is shown on the right as the percentage of total telomeres counted (n=932-1073). Comparison of fragile to non-fragile telomere percentage was performed using Χ2-test. Data information: All scale bars: 5 μm. Data are shown as mean ± SD.
(N) Kaplan-Meier analysis for overall survival of patients with high or low expression of ASS1. P < 0.001. P-value for Kaplan-Meier curves was determined using log-rank test.
B Venn diagrams show overlapped genes in signaling pathways response to sorafenib.
De novo identification of DNA consensus motifs enriched in the 1000 IL-2 pDHSs determined by HOMER.
Immunoblot analysis of whole cell lysates from primary healthy control and patient fibroblasts either left untreated or treated with the proteasome inhibitor MG132 (10 μM) for 24 h. Data are representative of two independent experiments.
Percentage of PDGFRαH2BEGFP+ cells in G1 cell cycle phase with age (n=1 E12.5; n=2 P2, P10; n=3 E16.5, E17, P0; n=4 E10.5; n=5 E17.5, E18.5, P50; n=7 P4 biological replicates) fitted using an exponential model.
F) Western blot of LRP6 and TBLR1 expression in WT and miR-424/503-KO basal cell-derived organoids after 14 days in culture with and without Wnt3a (100ng/ml). The arrow shows the correct TBLR1 band.
A BMDMs from WT, Dectin-1−/− or MyD88−/− mice were infected with swollen conidia (MOI = 1), and TNF-α was measured in the SN after overnight incubation. TNF-α levels were significantly reduced in Dectin1−/− and Myd88−/− BMDMs.
(G) Htt-552 in lysosomal membranes (MEMB) and matrices (MTX). Isolated lysosomes were incubated cell lysates containing expressed Htt-552 for 20 min, lysosomes were recovered by centrifugation. Lysosomal membranes and matrix were separated after hypotonic shock and centrifugation, and were processed for Western blot analysis.
A. HeLa cell stably expressing GFP-LGN and transiently expressing RFP-H2B was treated with noscapine to induce chromosome misalignments. Cells were imaged every 5 minutes. The time is relative to NEB. The red arrows indicate cortical regions where the chromosomes are in close proximity to the cell boundaries, accompanied by the displacement of LGN from the cortex.
(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
C. PHSL1-CDC20 ama1∆ cdc28-as2 control cells and cells containing spo13∆ or cdc5-as were treated with 1Na-PP1 at t = 3 h in SPM to inhibit Cdc28-as2. At t = 9 h in SPM, Cdc28-as2 was activated by washing cells with cSPM lacking 1Na-PP1 (washout). cdc5-as cells were washed with cSPM plus CMK to activate Cdc28-as2 and inhibit Cdc5-as. Top, time-lapse series from the imaging of Mpc70-GFP and RFP-tubulin. The weak, nuclear signal originates from Ndt80-GFP. Bottom, quantification of spindle formation and Mpc70 loading to SPBs. D. PHSL1-CDC20 ama1∆ cells containing SPC72, spc72-7, or spc72-7 plus cdc5-as were shifted from 24 to 36°C at t = 4.2 h in SPM (to inactivate Spc72-7, arrow heads) and treated with CMK at t = 6 h in SPM (to inhibit Cdc5-as). Top, time-lapse series from the imaging of SPBs (Cnm67-RFP) and Mpc70-GFP. Bottom, quantification of SPB separation and Mpc70 loading to SPBs. Inhibition of Cdc5-as in spc72-7 cells advances the time of Mpc70 loading by 119 min (95% CI, 76-162; P < 0.0001; Welch's t-test). Data are representative of two independent experiments.
D.- E. Network representation of enriched Gene Ontology (GO) Biological Process (BP) terms of top 50 genes most strongly negatively correlated with STAT3 (GS ≤ - 0.6, adj. p-value ≤ 0.01) in cluster 2 (D, blue) and cluster 3 (E, pink) (MM ≥ 0.8, adj. p-value ≤ 0.01). Node size indicates the percentage of associated genes. Similar colors indicate terms of the same GO group. GS = Gene significance, MM = module membership.
(e) mxl-3 and hlh-30 transcriptional levels in wild-type, hlh-30(tm1978) and mxl-3(ok1947) mutants in the basal and fasted states show that mxl-3 transcription is hlh-30 independent, and hlh-30 transcription is mxl-3 independent. Expression is presented relative to wild-type fed animals as mean ddCt+s.e.m. No significant differences relative to wild type in the same feeding state were observed, n = 3 independent experiments.
Averaged DPOAE responses for clarinet mice at P28, showing significantly reduced responses in Clrn2clarinet/clarinet mutants at all frequencies tested. DPOAE data shown are mean ± s.d. *p<0.02, **p<0.01, one-way ANOVA. Please see Appendix supplemental table 1 for exact P-values.
AHeLa cells, transfected with siRNA targeting Drp1 and GFP‐parkin (green), were treated with 25 μM antimycin A for 90 min following a 30‐min pretreatment with 50 nM bafilomycin A1, then fixed and immunostained against PDH E2/E3 bp (red) and TOM20 (blue). PDH E2/E3 bp‐positive/TOM20‐negative MDVs colocalizing with GFP‐parkin (arrows) or not (circles) are indicated. Scale bar, 30 μm.
Percentage of fetal spheroids decreases during culture (n=3 individual wells from single organoid culture (see material and methods); experiment was repeated in ten independent organoid cultures with similar results).
C. Schematic diagram of the granulocyte-macrophage colony-stimulating factor (GM-CSF)/interleukin (IL)-4-induced mouse monocyte DCs/macrophages differentiation assay. Cells were treated with specific antagonists of distinct EP receptors: ONO-8711, EP1; PF-04418948, EP2; L-798106, EP3; and E7046, EP4.
a: Dose response curve for the ISA D614 and for the mCherry D614 strains obtained by fluorescence or viral RNA measurement in VeroE6 cells from one representative experiment. Data are represented as mean ± SD (indicated by the error bars). Each experiment was performed in technical triplicates (N=3). b: Table of EC50 values obtained for the two different strains from two technical replicates. and their respective mean ± SD. c
(A) WT, Irgm1-/-, Irgm2-/- and Irgm3-/- mice (n = 4 mice/genotype) were injected i.p. with LPS (8 mg/kg). Serum was collected 4 hours post injection (hpi) and concentration of various cytokines determined via a preconfigured Luminex multiplex panel. Relative concentration (fold change relative mean of WT) is shown for the indicated cytokines (absolute cytokine concentrations of same experiment are shown in Fig EV1).
(M) FACS-isolated SCs were cultured for 2 days in proliferation medium followed by 2 days in differentiation medium; the degree of differentiation was assessed by IF staining of MF20+. Scale bar = 100 µm. Quantifications of the numbers of MF20+ area per nucleus are shown on the right (n=3 mice, each). Error bars represent s.d.'s of the mean. Student's t-test (two-sided): *P<0.05, **P<0.01, ***P<0.001.
(F) Model of autophagic clearance of damaged lysosomes.Source data for this figure is available on the online supplementary information page.
(I) Representative images showing the phospho-AMPK immunolabeling (Pseudo-colored) in low-density cortical neurons on the basal condition and on DHPG treatment for 5 minutes in the presence of scrambled siRNA and in the presence of α-SNAP siRNA. MAP2B immunolabeling was used for identifying neurons and intensity was used for normalization. Scale bar 10μm.
Structure-based sequence alignment between H‑BRCT domains of Sir4 (top) and Dbf4 (PDB 3QBZ, bottom). Secondary structural elements present in the Sir4 H‑BRCT crystal structure are displayed above the Sir4 sequence (α, α-helix; β, β-strand; η, 3.10 helix). Dbf4 residues strongly contributing to Rad53 FHA1 binding are highlighted with black (interface I) and blue (interface II) asterisks. The elongated loop connecting helix α3 and strand β4 in Sir4 H‑BRCT is labelled with an orange bar and the Tyr198 (Dbf4) to Lys1064 (Sir4) exchange is highlighted with colored arrows.
A, B Viability counts of V. natriegens prey before (0 h) and after (4 or 24 h) co-incubation with VnatReg attackers harboring the indicated plasmids, on media containing 3% NaCl and 0.1% arabinose (A), or on media containing 0.1% arabinose at 28 °C (B). Data information: Data are shown as the mean ± SD, n = 3 technical replicates. Statistical significance between samples at the 4 or 24 h timepoints 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.
B. Systematic phospho-Y2H interaction mating approach to identify the active kinase(s) modulating phosphorylation-dependent PPI. One yeast strain (L40matα) co-expressing the interacting bait and prey proteins and was mated against the 266 kinases in the array (L40mata) in a pairwise manner. The growth patterns of the diploid yeast strains that express bait, prey and one kinase from a third plasmid, indicate phospho-modulation of protein interactions.
Whole-mount AP staining of the blister roof epidermis from Col17a1-/- mice (right) and littermate controls (left) at P1. Scale bar: 500 μm.
Identified crosslink between murine ACAD9 and ND2 (spectrum in Fig EV1). ND2: Red - crosslinked peptide, green - C-terminal helix, Yellow - crosslinked Lys319. ACAD9: Orange - crosslinked Lys504. Sequences of identified crosslinked peptides from mouse and the homologous sequences from the human proteins are presented (yellow: crosslinked lysine).
H. Cells of wildtype (WT), ΔnlpI, ΔmepS and ΔnlpIΔmepS containing multicopy plasmids with lacZ were grown onto CPRG indicator agar to assay envelope integrity. CPRG (yellow) cannot penetrate intact Gram-negative envelopes. Its conversion by intracellular β-galactosidase to CPR (red) indicates loss of envelope integrity.
I. PIs of k 2 mRNAs and GAPDH co-detected in HUVECs (n≥19 cells; *P<0.05; Wilcoxon test).
Average DHS signal at the 1015 IL-2 regulated inducible DHSs in TB+ IL-7 and TB+ IL-7nil.
Gene expression profile of P240 PR-SET7ΔHepA livers and comparison with transcriptomes of ductal progenitor cellsA Overexpression of stem cell-specific, oncofetal hepatic and proliferation-dependent genes in P240 PR-SET7ΔHepA livers. Volcano plot of normalized RNAseq data obtained with P240 wild-type and PR-SET7ΔHepA livers. Significance cutoff value was set at P = 0.05 in -log10 scale (horizontal dashed line) and fold change cutoffs at −0.6 and 1 in log2 scale (vertical dashed lines).B Volcano plots showing dots corresponding to representative stem cell marker genes (red), oncofetal hepatic genes (blue) and proliferation-dependent genes (black) are indicated.C, D Comparison of differentially expressed genes in P240 PR-SET7ΔHepA livers with those reported in Lgr5-LacZ+ sorted cells or MIC1-1C3+ sorted ductal cells (Huch et al, 2013).
(C-G) ssDNA repair efficiency of the indicated TS mutants as determined from the maximal and final percentages of cells with Rad52 foci during the time course (right panels). Cells were synchronized in G1 and released into S phase in the presence of 0.033% MMS for 1 hour (60+), treated with 2.5% sodium thiosulfate to inactivate the MMS, washed and released into fresh medium for different times. They were also released into medium without MMS for 1 hour to control the formation of spontaneous Rad52-YFP foci (60-). The percentage of cells with foci at each point was normalized to the highest value of the wild type, taken as 100 (left panels). Cell cycle progression was determined by cell sorting. The mean and SEM of 3 (wild type, rad18∆, mms2∆, rad51∆, rad57∆, rad54∆), 4 (pol30-K164R) and 5 (rad5∆) independent experiments are shown. Statistically significant differences according to an unpaired two-tailed Student's t-test are shown, where three asterisks represent p-values <0.001.
(A) Immunostaining of API in the vacuole of SEY6210 (wild-type) cell showing a dispersed pattern.
SCID mice were xenografted subcutaneously with 10 × 106HCC-1599 cells on both sides. One week after transplantation 10 animals/experimental group were treated intraperitoneally with vehicle (DMSO) or two doses of ATRA (7.5 and 15.0 mg/kg) once/day, 5 days a week for a total of 24 days. At the end of this period, treatment was discontinued until sacrifice.The size of the tumors was determined with a caliper and the volume plotted. Each point is the mean ± SE of 20 tumors. *Significantly lower than the corresponding vehicle-treated group (P-value < 0.05, Student's t-test). **Significantly lower than the corresponding vehicle-treated group (P-value < 0.01, Student's t-test).
(F) Total RNA was isolated from regenerating tails at indicated time points post amputation, reversed transcribed into cDNA and analysed for foxm1 expression by qPCR, using ef1α as a reference gene. The graph represents the mean +/- SD of three independent experiments. One-way ANOVA with Dunnett's multiple comparisons test was used. * p=0.0149
A. Verification of L-OHP resistance by CCK8 assay and schematic diagram of Ribo-seq in HCT116/L-OHP and HCT116 cells (n = 3, biological replicates, mean±SEM).