EMBO/sd-character-level-ner huggingface dataset:
Trim21 exposes AdV genomes to cGAS and STING to trigger NLRP3- dependent inflammasome activation (A) AIM2 mRNA levels from PBMCs, CD19+ve B cells, CD14+ve Monocytes or HMDM derived from CD14+ve monocytes.Figure 7AIM2 mRNA levels from PBMCs, CD19+ve B cells, CD14+ve Monocytes or HMDM derived from CD14+ve monocytes. were assessed by qPCR and copy number was determined relative to Actin copy number (n = 5 mean ± s.e.m). HMDM were stimulated with AdV (50 000 pp/cell) and 20 μg/ml h9C12 for 1h, washed 2x with SFM, then whole cell lysates harvested after a further 5h. Viral hexon and transgene (GFP) expression in the cytosol was assessed by western blot. Data is representative of two independent experiments. THP-1s expressing either a control guide RNA or targeting cGAS and STING were generated and stimulated with 1000 U/ml IFNα for 4 h and protein levels assessed by western blot. THP-1s deficient in cGAS and STING were stimulated with AdV (50 000 pp/cell) and 20 μg/ml h9C12, 200 ng/ well HT-DNA, 10 μM Nigericin or 10 ng/ml LPS for 16 h. Data shows combined data (mean ± s.e.m) of 3 experiments with absolute protein values (upper panel) or as % cytokine output of KO cells relative to Ctrl treated cells (lower panel), *P-value ≤ 0.05, ***P-value ≤ 0.001 unpaired, two-tailed t-test). WT THP-1s were treated with 5 μM H151 for 30 mins before stimulation as in (D). Data in upper panel is representative of 3 independent experiments. Data in lower panel shows combined data of these 3 experiments (mean ± s.e.m) showing H151 treated cells relative to media treated cells (*P-value ≤ 0.05, ***P-value ≤ 0.001 unpaired, two-tailed t-test). ASC-GFP THP-1s were treated with 5 μM H151 for 30 mins before stimulation with AdV-mCherry (50 000 pp/cell) + 20 μg/ml h9C12 or 20 mg/ml IVIg or 200 ng/ well HT-DNA for 8h. A representative image (scale bar 50 μm) and quantification of number of cells with ASC specks from one representative experiment of three is shown.
Figure 4(A) Expression of condensin I subunits in insect cells. The wild-type (WT) or IV-5Q mutant CAP-H subunit was co-expressed with the other four subunits (GST-SMC4, SMC2, CAP-D2 and CAP-G) in insect cells. Cell lysates were prepared and subjected to SDS-PAGE, followed by immunoblotting with a mixture of antibodies against SMC2 and SMC4 (left panel) or against CAP-D2, -G, and -H (right panel). (B) Purification of the WT and IV-5Q mutant condensin I complexes. Protein samples purified through glutathione-affinity chromatography were subjected to SDS-PAGE and analyzed by CBB staining (left panel) or immunoblotting with a mixture of antibodies as described above (middle and right panels). (C) Add-back assay using the WT and mutant condensin I complexes. Xenopus extracts depleted of endogenous condensin complexes were supplemented with the purified complexes (from top to bottom; WT, IV-5Q, ∆G, ∆G[IV-5Q]). The supplemented extracts were then incubated with sperm nuclei to assemble mitotic chromosomes. The samples were fixed and labeled with an antibody against mSMC4 (red). DNA was counterstained with DAPI (blue). The data from a single representative experiment out of two repeats are shown. In the experiment shown here, multiple images were collected for condensin-depleted extracts supplemented with the WT (n = 17), IV-5Q (n = 22), ∆G (n = 20) and ∆G(IV-5Q) (n = 25) complexes. The scale bar represents 10 μm.
NK cells gene silenced for SHP-1 and Cbls siRNA via NKp46 antibody-coated NPs exhibit therapeutic efficacy in human B-cell lymphoma engrafted mice.Figure 6(A) Schematic representation of the experimental timeline. (B) Tumor volumes measured daily in SCID/NOD mice treated with 3x106 irradiated YTS KIR2DL1 and NPs containing Cbl-b, c-Cbl and SHP-1 siRNA (n=13), or mice treated with 3x106 irradiated YTS KIR2DL1, and NPs containing N.S siRNA that served as a negative control (n=12). P values were calculated using two-tailed Student's t-test and are indicated by asterisks. * P < 0.05. Data are shown as mean ± SEM. (C) Average growth rates of tumors from the first treatment to the end point from same experimental groups of mice as indicated in B. P value was calculated by two-tailed Student's t-test and is indicated within the graph P =0.001. Data are shown as mean ± SEM. (D) Images of the tumor bearing mice at the end point of the experiment (upper panel) and excised tumors (lower panels). (E) Following 24 hours after the last treatment, extracted cells from tumors of either SHP-1 and Cbls or N.S siRNA loaded NKp46-NPs treated mice were analyzed by flow cytometry. Expression of CD107a was measured by mean fluorescence Intensity (MFI). Relative expression of CD107a MFI was normalized to the sample treated with N.S siRNA loaded NPs. The data represent three independent experiments. Data are shown as mean ± SEMn=3; P value was calculated by one sample t-test and is indicated within the graph P =0.009. (F) Survival analysis in mice treated with NK cells' gene silenced for SHP-1 and Cbls using siRNA-loaded NPs (n=18) or N.S siRNA-loaded NPs (n=18) as demonstrated by Kaplan-Meier survival curve. P values were calculated using the log-rank test and is indicated within the graph P =0.00009.
Figure 5A. Western blots against CYP46A1 and ACTB in extracts of total brain and synaptosomes from wt and NPC1nmf164 mice as a percentage of wt values. Graphs show 3 biological replicates mean ± SEM of CYP46A1 level normalized to ACTB in arbitrary units. B. Quantitative PCR of Cyp46 of total brain and synaptosomes from wt and NPC1nmf164 mice. 3 biological replicates mean ± SEM is shown. C. Mean ± SEM cholesterol levels in EFV-treated or non-treated synaptosomes from wt (n=5 mice, 14-week-old, unpaired Student t test, *p = 0.0233) and NPC1nmf164 (n=4 mice, 14 week-old, unpaired Student t test, *p = 0.0188) mice. EFV values are expressed as percentage of their corresponding non-treated controls in wt and NPC1nmf164 conditions. D. Basal synaptic transmission in EFV-treated and non-treated hippocampal slices from wt and NPC1nmf164 mice expressed as mean ± SEM EPSP slope (n=9 slices from 5 WT mice, n=11 slices EFV-treated from 5 WT mice, n=8 slices from 5 NPC1nmf164 mice and n=13 slices EFV-treated from 5 NPC1nmf164 mice, all mice 14-week-old, 2-way ANOVA, *p = 0.0117). E. LTP in EFV-treated and non-treated hippocampal slices from wt and NPC1nmf164 mice expressed as mean ± SEM percentage of EPSP slope over baseline (n=7 slices from 5 WT mice, n=9 slices EFV-treated from 5 WT mice, n=10 slices from 5 NPC1nmf164 mice and n=11 slices EFV-tretaed from 5 NPC1nmf164 mice, all mice 14-week-old, 2-way ANOVA, ***p < 0.0001). F. Mean ± SEM paired pulse facilitation in EFV-treated and non-treated hippocampal slices from wt and NPC1nmf164 mice (n=11 slices from 5 WT mice, n=9 slices EFV-treated from 5 WT mice, n=10 slices from 5 NPC1nmf164 mice and n=11 slices EFV-tretaed from 5 NPC1nmf164 mice, all mice 14-week-old, 2-way ANOVA, **pNPC1nmf164=0.0027, *pNPC1nmf164+EFV=0.0229).
Figure 6A, OTUB1 overexpression promotes serum-induced MAPK activation in wt KRAS H1993 lung adenocarcinoma cells. B, OTUB1 overexpression does not affect serum-induced MAPK activation in mutant KRAS A549 lung adenocarcinoma cells. A,B, Serum-starved lung cells expressing Flag-OTUB1 or an empty vector (V) were stimulated with 10% serum for the indicated time periods. Whole cell lysates were analyzed by immunoblotting using antibodies against pERK1/2, ERK1/2, Flag and vinculin. C, Representative immunoblots of Flag-tagged OTUB1 overexpression in lung adenocarcinoma cell lines D, E, OTUB1 overexpression affects anchorage-independent growth of lung adenocarcinoma cell lines. Representative images of soft-agar colonies formed by the indicated cells expressing Flag-OTUB1 or empty vector (V). The number of soft agar colonies formed by OTUB1 expressing cells compared to cells expressing an empty vector. Data are presented as mean ± s.e.m. p-values were determined by two-sided t-Test, n=3. D, E, OTUB1 overexpression affects anchorage-independent growth of lung adenocarcinoma cell lines. Representative images of soft-agar colonies formed by the indicated cells expressing Flag-OTUB1 or empty vector (V). The number of soft agar colonies formed by OTUB1 expressing cells compared to cells expressing an empty vector. Data are presented as mean ± s.e.m. p-values were determined by two-sided t-Test, n=3. F,G, Xenograft tumor growth of A549, H1993 or H1437 cells expressing Flag-OTUB1 or an empty vector (V) subcutaneously injected into nude mice. p-values were determined by 2-way ANOVA.
Inhibition of autophagy in MCL‐1‐deficient neurons activates apoptosis.Figure 8(A) Brain morphology of P14 MCL‐1Δ/Δ Beclin‐1+/− mice. Brain sections were stained with cresyl violet and imaged using a × 1 objective. (B-D) Active BAX (6A7; B) and active caspase‐3 (C) were quantified by staining the cortex of 14‐day‐old MCL‐1Δ/Δ and MCL‐1Δ/Δ Beclin-1+/− mice. The number of BAX‐positive LC3‐negative cells was also quantified. (D) Representative images from at least three sections from each animal were counted. The total number of cells was determined by counting the Hoechst‐positive nuclei. Data are expressed as percent of positive cells for least three animals per genotype±s.d. *P0.05. (E) Survival data from MCL‐1Δ/Δ (n=14) and their MCL‐1Δ/Δ Beclin‐1+/− littermates (n=13). (F) Model for the role of MCL‐1 in regulating both autophagy and apoptosis. See text for details.
Figure 5Left panel shows representative images of hCOs from the different iPSC lines at the analyzed time points. Right panel shows the difference of the areas of f-CTRL, b-CTRL, UL1 and UL4 hCOs at different time in culture. Data shown as Z-scores relative to the mean of b-CTRL. Micrograph sections of d30 b-CTRL, UL1 and UL4 hCOs immunostained for proliferation markers KI67 and PH3. The dashed lines represent the apical surface of the cavities/ventricles. Quantification of the total number of proliferating Ki67+ cells/area (µm2) of ventricles like structures and of the number of PH3+ cells facing the ventricle/apical surface length (µm). Data shown as Z-scores relative to the mean of b-CTRL. Nuclei (blue) are stained with DAPI. Micrograph sections of d60 b-CTRL, UL1 and UL4 hCOs immunostained for proliferation markers KI67 and PH3. The dashed lines represent the apical surface of the cavities/ventricles. Quantification of the total number of proliferating KI67+ cells/ area of ventricles like structures and of the number of PH3+ cells facing the ventricle/the apical surface length (µm). Data shown as Z-scores relative to the mean of CTRL. Nuclei (blue) are stained with DAPI. Scheme of the experiment with conditioned medium exchange between b-CTRL and UL4 hCOs and quantification of the Ki67+ cells by FACS analysis on 36 days old dissociated hCOs. Four samples were analyzed as biological replicates.
Figure 3Representative western blot images of phospho-ULK1 (S757), phospho-S6Kp70 (T389), total S6K, GAPDH after iron treatment (FeSO4, 250 µM) for multiple time points. Quantification of mTORC1 activity analyzed through phosphorylation of S6K T389 to total S6k and ULK1 S757 to GAPDH after iron treatment (FeSO4, 250 µM) for multiple time points. Representative epi-immunofluorescent images of L6 cells transfected with myc-RHEB Q46L and immuno-stained against LC3B and myc after iron treatment (FeSO4, 250 µM) for 24 h. Ferrozine-based colorimetric measurement of intracellular iron in wild-type (wt) L6 and RHEB-Q64L L6 cells after iron treatment (50 µM or 250 µM) for 24 h. Representative confocal images of wt L6 and RHEB-Q64L L6 cells pulsed with Lysotracker Deep Red and immuno-stained against LC3B after iron treatment (250 µM, 24 h). Quantification of LC3B-free lysosome numbers in (E). Experiments were repeated three times, and one representative experiment is presented here. *P < 0.05 (multiple unpaired Student's t-test versus Basal in wt and RHEB Q64L cells) Representative TEM images of wt L6 and RHEB-Q64L cells after iron treatment (250 µM, 24 h). Representative western blot images and quantifications of phospho-IRS1 (Y612, indicated by arrowhead) and AKT (T308, indicated by arrowhead) to GAPDH in wt and RHEB-Q64L L6 cells stimulated with insulin (100 nM, 5 min) after iron treatment (250 µM, 24 h). *P < 0.05 (multiple unpaired Student's t-test versus Basal in wt and RHEB Q64L cells)
Cryo-EM density of the interface between RBD and ACE2.Figure 2 Cryo-EM density of the interface between RBD and ACE2.The density, shown as pink meshes, is contoured at 12 σ.
Figure 4Cartoon representation of the 2.1 Å E. albertii EschOTU-Ub crystal structure with labeled termini and active site. Ub is hidden for this initial view of the OTU fold. Structural alignment of the core OTU folds (central β-sheet and two supporting α-helices) from human OTUB1 (green, PDB 4DDG) and EschOTU (orange). Surrounding regions are less-well conserved and shown as semi-transparent. Structural alignment of the core OTU folds (central β-sheet and two supporting α-helices) from CCHFV vOTU (blue, PDB 3PHW) and EschOTU (orange). Surrounding regions are less-well conserved and shown as semi-transparent. Full view of EschOTU (orange) covalently bound to Ub (red) in the S1 site. Primary and secondary interactions with Ub are labeled, as well as the Ub Ile44 hydrophobic patch. An aligned view as in D, showing S1 site interactions between human OTUB1 (green) and Ub (red) (PDB 4DDG). Ub is rotated 21˚ relative to the EschOTU-Ub structure, but maintains similar primary and secondary contacts. An aligned view as in D, showing S1 site interactions between CCHFV vOTU (blue) and Ub (red) (PDB 3PHW). Ub is rotated by 95˚ relative to the EschOTU-Ub structure and displays swapped primary and secondary contacts. Detailed view of the primary and secondary interfaces observed in the EschOTU-Ub structure. EschOTU (orange) and Ub (red) residues participating in the interface are shown with ball and stick representation. Ub-KG(TAMRA) cleavage assay monitoring the effects of structure-guided EschOTU mutations. These data were collected in parallel with those presented in Fig. 1G and the WT dataset is shown again for clarity. Detailed view of the EschOTU (orange) active site region and its coordination of the Ub C-terminus (red). Residues that coordinate Ub or stabilize the active site are shown with ball and stick representation.
Figure 4Faith's phylogenetic diversity (PD) was used to measure the fecal microbial diversity and demonstrates a significant difference between human IGF-1R endothelial overexpressing mice (hIGFREO) mice and wildtype littermates (WT) mice after high fat diet feeding (n=4-5 mice per group). Faith's phylogenetic diversity (PD) was used to measure the fecal microbial diversity and demonstrates a significant difference between human IGF-1R endothelial overexpressing mice (hIGFREO) mice and wildtype littermates (WT) mice after high fat diet feeding (n=4-5 mice per group). Chao-1 analysis was used to measure the fecal microbial diversity and abundance and demonstrates a significant difference between hIGFREO and WT (n=4-5 mice per group). Chao-1 analysis was used to measure the fecal microbial diversity and abundance and demonstrates a significant difference between hIGFREO and WT (n=4-5 mice per group). Partial least squares discriminant analysis (PLS-DA) model and used the variable importance in projection (VIP) score was used to assess the contribution of each genus, shown as a scores plot in (E) VIP score cut off of 1(n=4-5 mice per group). Partial least squares discriminant analysis (PLS-DA) model and used the variable importance in projection (VIP) score was used to assess the contribution of each genus, shown as a loadings plot of PLS-DA of genus abundances in F. VIP score cut off of 1(n=4-5 mice per group).
Figure 3 (A, B) Representative immunoblots (A) and quantification of Tau degradation (B) from 14 DIV primary neurons transduced with mCh or shRab35, treated for 24 h with either DMSO (CON) or cycloheximide (CHX), and probed for pSer396/404-Tau (PHF1), pSer262-Tau, or pSer202-Tau (CP13) and tubulin. shRab35-expressing neurons exhibit markedly decreased pSer262- and p396/404-Tau degradation compared to mCh-expressing controls, while pSer202-Tau degradation is unaffected (n=4 per condition for pSer396/404-Tau and pSer202-Tau, n=3 for pSer262-Tau, unpaired student's t-test, **p=0.0027, *p=0.0120)  (C) Images of PLA signal (green) for Hrs/Tau interaction in N2a cells co-transfected with FLAG-Hrs and either mCh or mCh-Rab35 (red), and probed with antibodies against FLAG and total (DA9) or phospho-Tau species (pSer396/404-Tau, pSer262-Tau, or pSer202-Tau); scale bar: 5 μm. (D) Rab35 overexpression significantly increases PLA puncta for total, pSer396/404-Tau, pSer262-Tau but not pSer202-Tau (n=121-132 cells for total Tau, 178-195 cells for pSer396/404-Tau, 71-98 cells for pSer262-Tau, 25-29 cells for pSer202-Tau; Mann-Whitney test, ****p<0.0001)
Figure 2(A) Morphologies of smyhc1+/+, smyhc1-/+ and smyhc1-/- embryos are grossly normal at 2 days post fertilization (dpf), despite complete paralysis of smyhc1-/- embryos. In contrast, the smyhc1R673H/+ embryos have notochord kinks in larval stages (arrow head), while smyhc1R673H/R673H embryos have much more severe notochord kinks that compress and distort the body axis (arrow heads). The smyhc1R673H/- embryos completely phenocopy smyhc1R673H/R673H embryos. (B) Quantification of smyhc1R673H embryonic phenotypes at 2 dpf.
Figure 5A BAK-6H on liposomes was activated (+cBID, bottom) or not (Control, top) with cBID for 60 mins prior to deuteration for 10 s, 60 s, 600 s, or 3600 s. Incorporation of deuterium was assessed by mass spectrometry and is expressed relative to a non-deuterated sample and a theoretical maximum. HDX-MS reveals that following activation with cBID, maximal deuteration of the BAK N-terminus (up to amino acid 58) occurred within 10 secs. B BAK mutant with an engineered thrombin cleavage site in α1-α2 loop of hBAK∆N22/∆C25/C166S with a 6xHis C-terminal tag for recombinant protein expression or as full-length protein for ectopic expression in MEFs (BAKthrombin). C BAK cleaved within its α1-2 loop by thrombin is able to mediate liposome permeabilisation. Recombinant BAK-6Hthrombin pre-cleaved or not with thrombin (see Fig EV4) was incubated with liposomes at either 20 or 60 nM and liposome permeabilisation induced by cBID was monitored over time. Data is presented as percentage liposome permeabilisation relative to detergent-lysed liposomes, mean +/-SEM of 3 independent experiments. D BAK wild-type, or BAK with an engineered α1-α2 loop thrombin cleavage site were stably expressed in Bax-/-Bak-/- MEFs. Membrane fractions were incubated with thrombin prior to incubation with copper phenanthroline (CuPhe) to induce disulphide-linkage. Samples were run on SDS-PAGE under non-reducing or reducing conditions and immunoblotted for BAK with an antibody recognising the BAK N-terminus upstream of the thrombin cleavage site (aa23-38) or an antibody recognising the BH3 domain (4B5) downstream of the cleavage site. Schematic indicates the nature of the BAK protein detected by each antibody. Disulphide-linkage indicated by a hashed line. Note that the N-terminal portion of thrombin cleaved BAK could not be detected with the N-terminal antibody unless it was intramolecularly disulphide-linked. *cBID (100 nM) was added to activate BAK during thrombin cleavage. Data representative of two independent experiments. E Cleavage of the BAK α1-α2 loop potentiates MOMP. Membrane fractions from Bax-/-Bak-/- MEFs expressing BAK or BAKthrombin were incubated or not with thrombin in the presence or absence of cBID (10 nM) for the indicated times. Membrane (P) and supernatant (S) fractions were separated and immunoblotted for cytochrome c or BAK 4B5.
Atg19 phosphorylation is required for Atg11 binding and Cvt pathway functionDomain structure of Atg19.Figure 1A Domain structure of Atg19. B atg19Δ cells containing HTB‐Atg19 were grown to mid‐log phase. Atg19 was affinity purified and subjected to mass spectrometric phosphorylation mapping. Phosphorylation sites: enlarged; lysine substitutions: gray; Atg11 binding region: green. C atg19Δ cells containing GFP‐Atg19 wild‐type or mutants as indicated were grown to mid‐log phase. Processing of endogenous Ape1 was analyzed by Western blotting and quantified by calculating the ratio of cleaved versus uncleaved Ape1 normalized to the wild‐type. Atg19 expression was assessed by anti‐GFP Western blotting. D atg19Δ GFP‐Atg11 Atg1‐TAP cells containing myc‐Atg19 as indicated were grown to log phase. Atg11 was immunoprecipitated, and bound Atg19 was analyzed by Western blotting. E GST‐fused propeptide of Ape1, Atg19 as indicated, and a His‐tagged fragment of Atg11 (amino acids 685-1178) were purified from Escherichia coli, mixed, and propeptide‐bound proteins were isolated and analyzed by Coomassie staining. Quantification of three individual experiments is shown. Error bars represent standard deviation.
Figure 6A-B, In vitro analyses using primary mouse uterine epithelial cells demonstrated that TNFα induces the expression of annexin V, a marker of PS presentation at the outer membrane of Rb1f/f epithelial cells but does not in Rb1d/d ones, and P4 restores TNFα-primed annexin V expression in Rb1d/d uterine epithelial cells (mean ± SEM, Student's t test). Using three lines of primary mouse epithelial cells obtained from both Rb1f/fand Rb1d/d mice, the in vivo experiments were performed three times. Each of the annexin V-positive ratios was demonstrated. Scale bar = 100μm; green signal, annexin V. C-D, Annexin V assay using primary mouse uterine epithelial cells showed that TNFα administration increases the expression of annexin V in growth-arrested mouse epithelial cells with thymidine treatment, but does not in the control cells (mean ± SEM, Student's t test). Using three lines of primary mouse epithelial cells obtained from both Rb1f/fand Rb1d/d mice, the in vivo experiments were performed three times. Each of the annexin V-positive ratios was demonstrated. Scale bar = 100μm; green signal, annexin V. E, The expression of TNF receptor type 2 (TNFR2) was upregulated in growth-arrested epithelial cells with thymidine and P4 supplementation (mean ± SEM, Student's t test). Using three lines of primary mouse epithelial cells obtained from both Rb1f/fand Rb1d/d groups, qPCR was performed in duplicate.
Figure 7A Concentrations of C5a in the sera from pancreatitis patients, and correlation between ANGPTL4 and C5a in the sera from pancreatitis patients (n = 90). The box plots show a typical display consisting of a median value depicted by the line in the center of the box; an interquartile range (IQR; 25th to the 75th percentile) depicted by the box; and the maximum (Q3+1.5*IQR) and minimum (Q1-1.5*IQR) values depicted by the whisker. Statistical significance of Mann-Whitney U-tests is indicated (*** P < 0.001). Exact P values are shown in Appendix Table S2. The correlation between C5a and ANGPTL4 is depicted with Pearson correlation coefficient (R). Each dot represents the levels of C5a and ANGPTL4 from an individual patient (n = 90). The immunofluorescent expression of C5a and amylase was observed in pancreatitis and adjacent normal regions in patient tissues. Scale bar represents 50 µm B Immunofluorescence staining of C5a in pancreas tissues from ANGPTL4 -/- and WT animals and macrophage depletion/WT models with pancreatitis. Scale bar represents 150 µm. Levels of C5a were measured in serum (n = 7). C After macrophages were activated with LPS (100 ng/ml), the cells were treated with either neutralizing ANGPTL4 or C5a antibody (100 ng/ml) or an isotype control (IgG). ANGPTL4, C5a, TNF-α, and IL-6 levels were measured in culture media (n = 5). D The neutralizing ANGPTL4 antibody (10 mg/kg) was injected twice (IP) into the AP and SAP models. H&E staining of pancreatic tissues and the concentrations of ANGPTL4, C5a, TNF-α, and amylase in the serum (n = 10, each group). Scale bar represents 50 µm E Summary for how ANGPTL4 induces the severity of pancreatitis in acinar cells and macrophages.