EMBO/sd-character-level-ner huggingface dataset:
Figure 1Partial embryonic lethality after E11.5. Chi-square, **** p<0.0001. E11.5 n=114 embryos, E14.5 n=70 embryos, Adult n=314 born pups. Hindbrain vessel morphology. IsolectinB4 (IsoB4; white), ERG (red) and EdU incorporation (green). EdU signal was masked with ERG signal. Scale bar, 50 µm. Hindbrain vessel density. IsolectinB4 vessel area normalized to wildtype (WT) mean; one dot is two fields/mouse; error bars: SD; unpaired T test, * p <0.05. n=18-20. Hindbrain EC proliferation. EdU incorporation in ERG-positive ECs (double-positive cells, yellow in B). One dot is two fields/mouse; error bars: SD; unpaired T test, *p < 0.05. n=7-12. Reduced weight gain in the adult. Error bars: SD; 2-way ANOVA p=0.0009; Sidak's multiple comparison test, **p <0.01. n=13-20 mice. P6 retina vessel morphology. IsolectinB4 (IsoB4) in black; the outer 20% of the vascularized area in pink. Scale bar, 250 µm. Vessel parameters in the P6 developing retina. G. Radial outgrowth (distance between optic nerve and vascular front). Vascularized area. I. Vessel area in the outer 20% of the retinal vasculature; pink in F (vessel area normalized to vascularized area). Each dot represents one retina/mouse; n=3-4. EdU incorporation in ERG-positive ECs normalized to total ERG-positive ECs. Each dot represents one retina/mouse; error bars: SD. n=8-9.
Figure 4A Lysates from WT and SNX10 KO Caco-2 cells treated with or without 100 μg/mL OMVs for 24 h were analyzed by immunoblots with the indicated antibodies. B Lysates from Caco-2 cells transfected with SNX10-Flag were subjected to pull-down assay with anti-Flag antibody conjugated agarose, followed by immunoblots with the indicated antibodies. C Representative images of co-staining of SNX10-Flag and caspase-5 in Caco-2 cells expressing SNX10-Flag. Scale bar: 20 μm. D WT and SNX10 KO Caco-2 cells were treated with or without OMVs (100 μg/mL) for 24 h and then subjected to IP with anti-caspase-5 antibody. E Representative images of co-staining of Lyn and caspase-5 in WT and SNX10 KO Caco-2 cells treated with or without OMVs (100 μg/mL) for 24 h. Scale bar: 20 μm. F The co-localization of Lyn and caspase-5 was quantified by ImageJ software. n = 3 independent experiments, n = 18 fields analysed. Data are means ± SD. One-way ANOVA followed by Bonferroni post hoc test was used for statistical analyzes. ***p < 0.001. G Cytoplasmic and nuclear proteins were extracted from Caco-2 cells transfected with control siRNA, CASP4 siRNA or CASP5 siRNA and the indicated proteins were detected by immunoblots. H Lysates of Flag-tagged full-length SNX10 or its different truncated mutants transfected Caco-2 cells were subjected to immunoprecipitation. I Caco-2 cells were transfected with Flag-tagged full-length SNX10 or its indicated truncated mutant, followed by treatment with or without OMVs (100 μg/mL) for 24h. Lysates were extracted and subjected to immunoblots with the indicated antibodies.
Figure 5A ELISA analysis of VEGF concentration in intestinal tissues of mice treated with ERK inhibitor or anti-VEGF antibody. SCH772984, ERK inhibitor; Bevacizumab, anti-VEGF antibody. For each group, n = 4. n, biologically independent samples (mice). Data information: All data are shown as mean ± SD. P values are determined by one-way ANOVA. B Evaluation of intestinal permeability in animals treated with SCH772984 or Bevacizumab by Evans Blue dye extravasation assay. For each group, n = 6. n, biologically independent samples (mice). Data information: All data are shown as mean ± SD. P values are determined by one-way ANOVA. C, D Representative H&E images (C) and the degrees of inflammation (D) of intestinal tissues from animals treated with SCH772984 or Bevacizumab. The inflammatory infiltrates were indicated by a yellow arrow. The edema area was indicated by a red star. Scale bar, 100 µm. For each group, n = 4. n, biologically independent samples (mice). Data information: All data are shown as mean ± SD. P values are determined by one-way ANOVA. E Model for the SARS-CoV-2 spike RBD-mediated vascular hyperpermeability and intestinal inflammation. ① Binding of spike RBD and receptors on enterocytes; ② Activation of the Ras-Raf-MEK-ERK pathway; ③VEGF over-production; ④VEGF-triggered phosphorylation of Y731 and internalization of VE-cadherin; ⑤Increase of endothelial permeability accompanied by interstitial edema and inflammation in the intestinal tissue.
a) The ternary NDP52–GAL8–LC3C complex (green line), binary NDP52–GAL8 complex (blue line) and NDP52 complex alone (red line) by SEC-MALS.f1(a) The ternary NDP52-GAL8-LC3C complex (green line), binary NDP52-GAL8 complex (blue line) and NDP52 complex alone (red line) by SEC-MALS. The dotted line represents measured MW and the vertical bars (1 and 2) indicate the gap in MW. The SEC-MALS result of NDP52 alone (red line) shows that the oligomeric state is ambiguous between dimer and trimer. However, the NDP52-GAL8 complex shows a MW difference of 70 kDa compared with NDP52 alone, which confirmed that two molecules of monomeric GAL8 (MW: ~33.5 kDa; Supplementary Fig. S1b) bound to homodimeric NDP52. Furthermore, the ternary NDP52-GAL8-LC3C complex showed a MW difference of ~30 kDa compared with the binary NDP52-GAL8 complex, which also confirmed that two additional molecules of monomeric LC3C bound to the binary complex. (b) Size-exclusion chromatography data of NDP52-GAL8-LC3C complex. The first peak (marked by an orange bar) represents the ternary complex, as shown by the SDS-polyacrylamide gel electrophoresis (SDS-PAGE) results marked by an orange bar (lane 1, 2 and 3). The second merged peak (marked by green and yellow bar) and corresponding SDS-PAGE results show the remaining free GAL8 (lane 4, 5, 6 and 7) and LC3C (lane 5, 6, 7 and 8).
CEP55 overexpression provides survival advantage during perturbed mitosis (A) Representative cytogram of control and CEP55 knockdown MDA-MB-231 cells showing cell cycle profiles following treatment with and without the B12536 (5 nM).Figure 3(A) Representative cytogram of control and CEP55 knockdown MDA-MB-231 cells showing cell cycle profiles following treatment with and without the B12536 (5 nM). Percentage of polyploidy (>4N) following B12536 (5 nM) in control and CEP55 knockdown MDA-MB-231 cells. Graph represents the mean±SEM of three independent experiments. Percentage of polyploidy (>4N) following nocodazole (0.5 µM) in control and CEP55 knockdown MDA-MB-231 cells. Graph represents the mean±SEM of three independent experiments. Apoptotic fraction (Sub-G1 population) determined by propidium iodide staining of cells treated with PLK1i Graph represents the mean±SEM of three independent experiments. Apoptotic fraction (Sub-G1 population) determined by propidium iodide staining of cells treated with Nocodazole Graph represents the mean±SEM of three independent experiments. (F) Percentage of sub-G1 fraction following B12536 (10 nM) or nocodazole (0.5 µM) in control and CEP55 knockdown Hs578T cells. Graph represents the mean±SEM of two independent experiments. (G) Representative cytogram of empty vector (EV) and CEP55-overexpressing MCF10A cells showing cell cycle profile following treatment with and without the B12536 (10 nM). (H) Percentage of polyploidy following treatment with and without B12536 (10 nM) in empty vector (EV) and CEP55-overepressing MCF10A cells. Graph represents the mean±SEM of three independent experiments. (I) Corresponding sub-G1 population determined using propidium iodide staining of cells treated with B12536 as described in panel H. Graph represents the mean±SEM of three independent experiments.
Figure 6Functional complementation of the mepM deletion in BW25113(ycbB, relA') ΔmepM was performed with the pHV30 vector or recombinant plasmids encoding each of the eight endopeptidases under the control of the PrhaBAD promoter and of the TIS1 translation initiation signal. Induction of endopeptidase (ED) genes was performed with 0.2% L-rhamnose in the presence or absence of 8 µg/ml ceftriaxone. BHI agar plates contained 40 µM IPTG and 1% L-arabinose for induction of ycbB and relA', respectively.
Figure 7A. Diagram showing the procedure used for siRNAs mediated knockdown of Bclaf1 in mice and mTNF treatment. siRNAs against Bclaf1 and control siRNAs (siCtrl) mixed with in vivo-jetPEI (Polyplus) were injected into C57BL/6 mice. The mice were then treated with mTNF for two hours before sacrifice. The small intestines were excised and processed for immunohistochemical staining for Bclaf1 (B) and Hematoxylin-and-eosin staining (C) Bclaf1 knockdown efficiency is detected by immunohistochemical analysis (B). Hematoxylin-and-eosin staining showing the morphological change (C). Villus height and crypt depth were measured (D). Data are shown as mean ± SD. n=5 mice for each group. ns, not significant; **p<0.01; ***p<0.001; ****p<0.0001. One-way ANOVA test. Scale bars: 50 μm in B, 100 μm in C. siRNAs against Bclaf1 and control siRNAs (siCtrl) mixed with in vivo-jetPEI (Polyplus) were injected into C57BL/6 mice. The mice were then treated with mTNF for two hours before sacrifice. The small intestines were excised and processed for immunohistochemical staining for cleaved caspase 3 (E), phospholated RIPK3 (F) CC3+ and p-RIPK3+ positive cells in five fields per intestine were quantified. Data are shown as mean ± SD. n=5 mice for each group. ns, not significant; **p<0.01; ***p<0.001; ****p<0.0001. One-way ANOVA test. Scale bars: 50 μm siRNAs against Bclaf1 and control siRNAs (siCtrl) mixed with in vivo-jetPEI (Polyplus) were injected into C57BL/6 mice. The mice were then treated with mTNF for two hours before sacrifice. The small intestines were excised and processed for western blotting analysis (G). HIEC-6 cells were transfected siCtrl or siBclaf1-1 and -2. Then the cells were treated with TNF for 24 hours were subjected to Annexin V/7AAD staining followed by flow cytometry analysis (H) Data are shown as mean ± SD. n=3 biological replicates. ****p<0.0001. One-way ANOVA test. HIEC-6 cells were transfected siCtrl or siBclaf1-1 and -2. Then the cells were treated with TNF for 24 hours were subjected for protein extractions and western blotting analysis (I and J) HIEC-6 cells were transfected siCtrl or siBclaf1-1 and -2. Then the cells were treated with TNF for indicated hours followed by total RNA extraction and RT-PCR analysis (K). Data are shown as mean ± SD. n=3 biological replicates. ****p<0.0001. One-way ANOVA test.
Figure 4The mepM gene was expressed under the control of TIS1 or TIS2 (translation initiation) and of PrhaBAD (the L-rhamnose-inducible promoter of vector pHV30). Growth of BW25113(ycbB, relA') ΔmepM harboring the pHV30 derivatives was tested on BHI agar supplemented with ceftriaxone 8 µg/ml, IPTG 40 µM (induction of ycbB) and L-arabinose 1% (induction of relA'). (A) For TIS2, growth around paper disks containing 1 or 2 mg of L-rhamnose indicated that induction of the expression of mepM was required for ceftriaxone resistance. In contrast, a higher level of translation from TIS1 was sufficient for ceftriaxone resistance in the absence of the inducer. (B) The experiment was repeated with bacteria pre-exposed to L-rhamnose that were harvested at the vicinity of the disk containing 2 mg of L-rhamnose. Expression of ceftriaxone resistance remained dependent on the presence of L-rhamnose indicating that the requirement for MepM is not transient. The diameter of the growth zones is larger in panel (B) than in panel (A) as expected from the fact that bacteria in the inoculum used in (B) had been grown in the presence of the inducer and already contained MepM. In (A) growth is only possible after diffusion of L-rhamnose in the medium prior to the action of ceftriaxone. At a distance from the disk, diffusion was not sufficiently rapid to observe resistance.
a) COS7 cells were transfected with Bif-1–HcRed together with GFP–Atg5 or GFP–LC3.figf4(a) COS7 cells were transfected with Bif-1-HcRed together with GFP-Atg5 or GFP-LC3. Twenty hours after transfection, the cells were cultured in EBSS (middle and bottom panels) or complete medium (top panel) for 2 h and subjected to confocal microscopy analysis. Magnified images are shown as insets. Representative Bif-1-Atg5 or Bif-1-LC-3 double-positive foci are indicated by arrows. (b) COS7 cells transfected with Bif-1-GFP were cultured in EBSS for 2 h and the localization of Bif-1-GFP was examined by immunogold electron microscopy using anti-GFP antibodies. Arrows indicate representative gold particles that are detected on autophagosomal membranes. The scale bars represent 10 μm in the main images and 1 μm in the insets in a, and 1 μm in b.
Figure 2(A) Explanted tumors at Day 12 after repetitive injections of LPS as depicted in the scheme at the bottom of the panel. (B) Detection of blood vessels by immunohistochemistry using anti-CD31 antibody (brown staining) in frozen sections of tumors from mice treated or not with with LPS as in the scheme in (A). Nuclei are stained with hematoxylin. Two representative sections are shown, magnification 20X. Left panel scale bars, 500m; right panel scale bars 100 m. Quantification of vessel mean dimensions and area coverage by vessels is also shown. Three sections from three tumors have been analyzed. Error bars depict SEM. Statistical significance was determined with a two-tail t test. NT, untreated.
Fig.Figure 4RAW264.7 WT, LRRK2 KO or Rab8A macrophages pre-treated with 1 μM GSK2578215A (GSK inh) were treated with 1 mM LLOMe for 30 min. CHMP4B vesicle numbers were analysed by immunofluorescence and high content imaging. Scale bar = 20 μm. Right panels show the quantification of number of CHMP4B positive vesicles per cell. Mean ± SEM of three independent biological experiments. ns=non-significant, *p≤0.05, **p≤0.01 by One-way ANOVA followed by Sidak's multiple comparisons test. RAW264.7 WT, LRRK2 KO or Rab8A macrophages pre-treated with 1 μM GSK2578215A (GSK inh) were treated with 1 mM LLOMe for 30 min. Galectin-3 vesicle numbers were analysed by immunofluorescence and high content imaging. Scale bar = 20 μm. Right panels show the quantification of number of Galectin-3 positive vesicles per cell. Mean ± SEM of three independent biological experiments. ns=non-significant, *p≤0.05, **p≤0.01 by One-way ANOVA followed by Sidak's multiple comparisons test. Live cell imaging of LysoTracker positive spots in WT, LRRK2 KO macrophages treated with 1 mM LLOMe, followed by lysosomal recovery after LLOMe wash-out. One representative experiment out of three shown. Differences between slopes in the LLOMe treatment window were estimated using linear regression. Live cell imaging of LysoTracker positive spots in WT, Rab8A KO macrophages treated with 1 mM LLOMe, followed by lysosomal recovery after LLOMe wash-out. One representative experiment out of three shown. Differences between slopes in the LLOMe treatment window were estimated using linear regression.
Figure 7  Figure 7. Working model for Tel1 function at replication forks in CPT. CPT-induced Top1 trapping on DNA results in the accumulation of DNA supercoiling ahead of an incoming replication fork. In the presence of this torsional stress Mrc1 promotes fork reversal. Reversed forks can be targeted by nucleases, mainly MRX-Sae2, which nucleolitically process the regressed arm. Tel1 counteracts the action of MRX-Sae2 either directly or through a still unknown factor (X), thus preventing unscheduled fork degradation. Reversed forks stabilization by Tel1 can support the completion of DNA replication after the repair of CPT-induced damage by either reversed fork reactivation or fusion with an incoming fork. Expanded view Figure and Table legends
Positive- and negative regulation of innate immune responses by serum GM3 species in an acyl-chain dependent manner.Figure 4(A) Profiling of bioactivities of serum GM3 species in LPS-mediated monocyte activation (schematic). (B, C) GM3-mediated enhancement and inhibition of proinflammatory cytokine production from LPS-stimulated monocytes (LPS: 0.06, 0.13, 0.25 ng/mL). TNF-α (B) and IL-6 (C) production in culture supernatant was measured by ELISA. (D, E) Co-stimulation of monocytes by LPS plus GM3 species or complex ganglioside species (1.5, 3.0, 4.5 µM). TNF-α (D) and IL-6 (E) production were shown in heat maps. (F) Co-stimulation of monocytes by LPS plus GM3 species or precursor GSL species. TNF-α, IL-6, and IL-12/23 production were shown in heat maps. (G) Inhibitory effect of LCFA and unsaturated VLCFA-GM3 on LVCFA-GM3 species. IL-6 production in culture supernatant was measured by ELISA.
Figure 2(A) Seedlings were acclimated for 16 h in MS containing ABA and then imaged 30 min after being swapped to MS containing ABA/NAA (control), MS or MS containing flg22. Images are representative maximum intensity projection of 10 Z-stacks per condition. Experiments were repeated 3 times independently with similar results. Scale bar: 10 µm. (B) Quantification of GFP foci per 0.0025 mm2, for samples treated as described in A. Values are presented as mean ± standard deviation of the mean and are based on 3 independent experiments, with 3 individuals per condition. Bars marked with an asterisk (*) are statistically significant (P<0.05) according to the T -test. (C) GFP-ATG8a cleavage immunoblot for given treatments. The ratio of free GFP to loading control, normalized to the 16h pre-treated sample (set to 1) is provided below each band. (D) Seedlings were acclimated for 16 h in MS containing NAA and then imaged 30 min after being swapped to MS containing ABA/NAA (control), MS or MS containing 6-BA. Images are representative maximum intensity projection of 10 Z-stacks per condition. Experiments were repeated 3 times independently with similar results. Scale bar: 10 µm. (E) Quantification of GFP foci per 0.0025 mm2, for samples treated as described in (D). Values are presented as mean ± standard deviation of the mean and are based on 3 independent experiments, with 3 individuals per condition. Bars marked with an asterisk (*) are statistically significant (P<0.05) according to the T -test. (F) GFP-ATG8a cleavage immunoblot for given treatments. The ratio of free GFP to loading control, normalized to the 16h pre-treated sample (set to 1) is provided below each band.
Figure 4A) Representative CLEM images of mitochondria in HCT AKO cells (control) and HCT AKO cells expressing GFP-BAX or tBID-GFP. Black arrows indicate MOM disruptions. B-D) Effect of GFP -BAX and tBID-GFP on HCT AKO mitochondrial structure. (B) Number of disruptions at the MOM per mitochondrial area, each dot represents an individual cell. (C) Mean area per mitochondrion at the condition tested, each dot represents an individual mitochondrion. (D) Analysis of cristae morphology, each dot represents an individual crista. Cristae shape calculated as length/width. Data represent n>50 mitochondria from 3-5 independent cells. In B-D), boxes represent 96% confidence interval, the average is represented by the line inside the box and whiskers correspond to S.D. The statistical significance was assessed by unpaired Student's t-test. *** p<0,001 and ** p<0,025 with respect to HCT AKO or untransfected condition. E) Representative confocal and STED images of GFP-BAX and tBID-GFP expressed in HCT AKO. Scale bar 1 µm for BAX images, 5 µm for tBID images. F) Zoomed-in structures of GFP-BAX supramolecular assemblies with the shape of lines (*double lines), arcs and rings in HCT AKO cells. Scale bar 100 nm.
Figure 4A, B PBMC of a healthy donor were incubated with the indicated concentrations of bsAbs, and after three days proliferation (A) were measured using a 3H-thymidine uptake- and a Legendplex assay, respectively. Mean values and standard deviations from triplicate samples. B PBMC of a healthy donor were incubated with the indicated concentrations of bsAbs, and after three days cytokine release (B) were measured using a 3H-thymidine uptake- and a Legendplex assay, respectively. Mean values and standard deviations from triplicate samples.
Figure 3(A) The relative frequency of Gly in the final 50 residues of functional human proteins with different subcellular localizations. The number on each graph denotes the left-tailed p-value of the -1 residue. CT-inside and CT-outside represent membrane proteins with their C-termini facing the cytosol or extracellular space, respectively. (B) Comparison of stabilities between wild-type and diGly degron-tagged membrane proteins. The C-terminal topology of tested proteins is indicated at top. (C) GPS reporter cells expressing indicated membrane proteins with a C-terminal diGly degron were treated with or without KLHDC2 overexpression or knockdown and then analyzed. (D) Stability analysis of C-terminal diGly degron-tagged membrane proteins with or without KLHDC2 overexpression and CB-5083 treatment (to inhibit p97/VCP). (E) Abundance analysis of GFP-diGly degron fusion proteins with or without N-terminal tagging with the MTSs of PDHA1, COX8A or GLUL, and with (+) or without (-) blockage of C-degron pathways by dominant-negative Cul2 (DNCul2). (F) Abundance comparison of MTS-GFP-degron fusion proteins in cells treated with shRNAs against various BC-box proteins. The MTS and C-degrons used are labeled at top and left, respectively. The MTS and C-degron sequences are provided in the Materials & Methods. (G) Confocal images of U2OS cells expressing MTSGLUL-GFP proteins with or without a C-terminal diGly degron. MitoTracker staining shows mitochondria. Scale bar = 20 µm. (H) Confocal images of U2OS cells expressing MTSGLUL-GFP-diGly degron fusion proteins co-stained with MitoTracker, and with or without DNCul2 treatment. Scale bar = 20 µm.
XIAP mediates IL8 secretion in melanoma by involving TAB1 and RIPK2 A Western blot analysis of cell lysates of BLM and BLM-XIAPKO cells (input) and XIAP-IP.Figure 2A Western blot analysis of cell lysates of BLM and BLM-XIAPKO cells (input) and XIAP-IP. Data are representative of two experiments. B Confocal microscopic analysis of transfected HCT116 cells with GFP-XIAP for 16 h and stained for TAB1 or RIPK2 (red). Nuclei were stained with DAPI (blue). C Cell-free ubiquitylation assay of recombinant GST-RIPK2 by Flag-XIAP after addition of respective E1/E2 and recombinant ubiquitin. Poly-ubiquitylated RIPK2 and XIAP were detected by Western blotting. D Western blot analysis of cell lysates of BLM, BLM-XIAPKO, SK-Mel28, SK-Mel28-XIAPKO, B16, and B16-XIAPKO cells (input) and RIPK2-IP. Poly-ubiquitin is detected using anti-ubiquitin antibody. E IL8 measurement in the supernatant of transfected BLM (left panel) or SK-Mel28 (right panel) cells with siScr (control), siXIAP, siTAB1 or siRIPK2 after 48 h. Western blot analysis of the respective cell lysates of BLM cells (left bottom) or SK-Mel28 cells (right bottom) transfected with the indicated siRNA. F IL8 measurement in the supernatant of BLM or SK-Mel28 treated with 2 µM d89 (BIR2 antagonist) or DMSO as a negative control for 24 h. G IL8 measurement in the supernatant of BLM or SK-Mel28 treated with 20 µM birinapant (Bir), 2 µM BV6 (both BIR3 antagonist) or DMSO as a negative control for 24 h.
Figure 2A Deubiquitylating enzymes (DUBs) and their ubiquitin substrates. Less efficiently cleaved substrates are indicated in brackets. B UBA-pull down from WT MDFs treated with TSI for 3 hrs were subjected to the DUBs shown in (A). Beads eluates were analysed by Western blot and probed with antibodies as indicated. Representative of three independent experiments. C 1.4 mL cleared cell lysate from 4 x 106 TSI-treated WT MDFs was split into three parts of the indicated volume, followed by UBA-pull down and DUB incubation. Bead eluates were analysed by Western blot and probed with the indicated antibodies. Representative of three independent experiments.