EMBO/sd-character-level-ner huggingface dataset:
HOPS role in importin α mediated p53 nuclear import and PCAF mediated p53 acetylation.Figure 7A HOPS expression and p53 nuclear accumulation. HOPS was overexpressed in RKO and cells were fixed and stained with antibody against p53 (green). Nuclei were stained with DAPI (blue). Samples were analysed with fluorescent microscope and merged images are shown (right panel). Scale bars, 10µm. Cells were scored for p53 localization. The results are shown as percentage of nuclear versus cytoplasmic p53 localization in untransfected and HOPS transfected cells (left panel). Data information: all the experiments detailed above were performed three times, and representative panels are shown. In A data are presented as mean ±SD. B Biochemical analysis of HOPS intracellular localization. RKO cells were transfected or not with HOPS and fractionated into nuclear and cytoplasmic component. Aliquots relative to the same cell number per fraction were immunoblotted using anti-p53 and anti-HOPS antibodies. GAPDH and Lamin B1 are purity controls of cytoplasmic and nuclear fraction respectively. Data information: all the experiments detailed above were performed three times, and representative panels are shown. HOPS and p53 binding to importin α. Untransfected or HOPS transfected RKO cells were treated with etoposide and each sample was evaluated by immunoprecipitation with anti-p53 and analysed by immunoblotting using anti-importin α antibody. Arrowhead indicates the importin α specific signal. Data information: all the experiments detailed above were performed three times, and representative panels are shown. D PCAF-mediated p53 acetylation. p53 and PCAF Flag-tagged were co-expressed in H1299 cells with or without HOPS and treated with trichostatin A and nicotinamide. p53 acetylation levels were evaluated by immunoprecipitation with anti-p53 antibody followed by immunoblotting with anti-acetylated-Lysine (AcK), anti-p53, anti-Flag, anti-HOPS and anti-α-tubulin antibodies. Data information: all the experiments detailed above were performed three times, and representative panels are shown.
Figure 5A, B I-V responses in oocytes (subtracted of the water injected controls) exposed to 40 mM Cl at pH5.8 (A) and pH7.4 (B). The currents were measured while the oocyte plasma membrane was clamped at -60 mv to between 20 and -140 mv for 120 ms with -20 mv increments for each Cl- concentration. The Cl- -elicited current values in D were obtained by subtracting the currents in water injected oocytes from the MtNPF6.5 injected oocytes at each Cl- concentration Data information: Value = mean ± SEM, biological replicates: n = 4 C, D I-V responses in the (C) water and (D) MtNPF6.5 (subtracted of the water injected controls) injected oocytes exposed to different concentrations of Cl- at pH5.8. The currents were measured while the oocyte plasma membrane was clamped at -60 mv to between 20 and -140 mv for 120 ms with -20 mv increments for each Cl- concentration. The Cl- -elicited current values in D were obtained by subtracting the currents in water injected oocytes from the MtNPF6.5 injected oocytes at each Cl- concentration. Data information: Value = mean ± SEM, biological replicates: n = 6
Figure 1A-C 10x106 ZsGreen- and luciferase-expressing U-2932, RC-K8 and RIVA cells were intravenously injected into six-week-old male (M) and female (F) MISTRG mice and monitored weekly using IVIS for at least four and up to six weeks. The color scales on the right indicate the radiance, i.e. the sum of the photons per second from each pixel inside the ROI/number of pixels (photons/sec/cm2/sr). 10x106 ZsGreen- and luciferase-expressing U-2932, RC-K8 and RIVA cells were intravenously injected into six-week-old male (M) and female (F) MISTRG mice and monitored weekly using IVIS for at least four and up to six weeks. D The frequency of involvement of the indicated tissues is shown for one cohort of mice and is representative of two independently analyzed cohorts per cell line. E Gating strategy for the identification of U-2932 cells residing in spleen and bone marrow, among all leukocytes in these organs. Cells were stained for human and mouse CD45. Very few hCD45+ U-2932 cells were detectable in the spleen, whereas their frequencies were much higher in the bone marrow (BM). In BM, >90% of U-2932 cells were positive for ZsGreen. The green curve shows U-2932 cells recovered from BM, overlayed with unlabelled U-2932 cells grown in vitro (grey curve). F, G Frequencies of human lymphoma cells, as judged by hCD45 expression, among all (human and mouse, h+m) CD45+ leukocytes in the spleen (S), femoral bone marrow (BM) and jaw bone marrow (JB) at the study endpoint of the mice shown in D (four weeks post injection for U-2932, five weeks p.i. for RIVA and six weeks p.i. for RC-K8).
Figure 7A. Once the parasite attaches to the surface of a host cell, F-actin strongly accumulates at the posterior pole and at the apical end. During penetration, the junctional complex is formed that contributes to the attachment and stabilisation of the parasite to the host cell in the TJ. F-actin at the TJ provides traction force and stability for nuclear entry, while posterior F-actin provides contraction force to allow nuclear entry. At the same time microtubules might facilitate nuclear entry by pulling. We propose that the nucleus is squeezed through the TJ by a pushing-pulling mechanism controlled by actin and potentially microtubules. B. In some cases (for example due to more permissive host cells or upon modulation of F-actin dynamics), a F-actin ring at the junction is not required/formed and the nucleus can enter the host cell by the action of posterior accumulated F-actin.
Atg12 functions as a binding module for Atg3.Figure 2(A) In vitro pull‐down assay using recombinant plant Atg homologues.CBB, Coomassie Brilliant Blue staining; GST, S; HA, haemagglutinin; PE, phosphatidylethanolamine; WT, wild‐type. (B) Mapping of the mutated residues on the Atg12-Atg5‐Atg16N complex structure. Atg5 and Atg16N are shown with a ribbon model, whereas Atg12 is shown with both surface and ribbon models. The side chains of Tyr 147, Tyr 149 and Phe 154 of Atg12 are shown with a stick model and coloured grey (Tyr 147) or blue (Tyr 149 and Phe 154). Corresponding AtAtg12b residues are indicated in parentheses. Atg12 Phe 185 and Gly 186 and the side chain of Atg5 Lys 149 are shown with a stick model. (C) Proposed model of Atg8-PE conjugate formation mediated by the Atg12-Atg5‐Atg16 complex. CBB, Coomassie Brilliant Blue staining; GST, glutathione S‐transferase; HA, haemagglutinin; PE, phosphatidylethanolamine; WT, wild‐type.
Figure 6TFL457 interference of RIP and calpain-processing in excitotoxicity. Cultures were preincubated with peptides (25 µM, 30 min) or left untreated before NMDA addition (2 h). Different exposures are shown to facilitate visualization of FL, f42/39 or f32. Quantitation of normalized f32 and f42/39 levels. Means ± SEM are shown relative to cultures treated with NMDA and no peptide. Effect of TFL457 on neuronal viability after calpain inhibition. After 30 min preincubation with calpeptin (Calp, 10 µM) and calpain inhibitor III (CiIII, 10 µM), cultures were treated with CPPs and NMDA (4 h) as before. Means ± SEM relative to those obtained in untreated cultures are given. Effect of TFL457 on neuronal viability after metalloproteinase inhibition. Cultures preincubated with GM6001 (10 µM, 30 min) were treated with CPPs and NMDA as before. Analysis was performed as above (without GM6001, ***p = 0.0007; with GM6001, ***p = 0.0006; n = 4). Effect of NMDA on total and cell-surface TrkB-FL levels. Cultures were incubated with NMDA (100 µM) or BDNF (100 ng/ml) for 1 h. Membrane proteins were labeled and purified and compared to corresponding total lysates. TrkB-FL levels are expressed relative to untreated cells. Effect of TFL457 on the decrease of TrkB-FL surface levels induced by NMDA. Analysis was performed as before in cells incubated with CPPs (25 μM, 30 min) and NMDA-treated (1 h). Results obtained in excitotoxic cultures are expressed relative to cells treated with the same peptide but no NMDA. Experimental design to study the effect of endocytosis inhibition on TrkB-FL downregulation in ischemia. Mice were i.p. injected with vehicle or dynasore followed by Rose Bengal. Vessel occlusion and brain damage were induced by cold-light irradiation. Animals were sacrificed early after damage initiation. Effect of endocytosis inhibition on TrkB-FL downregulation. The infarcted area of the ipsilateral hemisphere (I) was compared to the corresponding region of the contralateral area (C). Results from representative mice injected with dynasore or vehicle are shown. Different exposures are presented to facilitate visualization of dynasore effects on TrkB-FL and f32. Quantitation of TrkB-FL and TrkB-T1 in the infarcted area. Normalized protein levels are expressed relative to those of the corresponding contralateral region.
Figure 1Schematic representation of monocistronic and bicistronic AAV constructs enabling neuron-specific expression of i) Venus and ii) HA-tagged APPsα (+ Venus). ITR: inverted terminal repeat, Synapsin: promotor, T2A: Thoseaasigna virus 2A site, SP: signal peptide, HA: influenza hemagglutinin tag. Scheme of the hippocampus with coordinates of the two injection sites (black stars). Overview of the hippocampus of an AAV-APPsα injected cDKO mouse. HA-tag staining (red) reveals APPsα expression within the CA1, CA3 and DG of the hippocampus. Magnification shows the boxed CA1 region. Scale bars: 500 μm (left), 100 µm (right). DG: Dentate Gyrus, CA: Cornus Ammonis. Double immunostaining in CA1 region. APPsα (HA-tag, red) is exclusively expressed in neurons (NeuN, blue, D, E), but not in astrocytes (GFAP, blue, F) or microglia (Iba1, blue, G). Scale bars: 10 μm (D, F, G), 5 µm (E). Western blot analysis of APP expression in hippocampus of LM control (N=5) and cDKO mice (N=5) injected with AAV-Venus or AAV-APPsα. Age of mice: 5-6 months.
Figure 3A Caspase activation was measured by fluorescent cell analysis in wild-type MEF cells treated with S-HAD (10 µM, 24 h) or cisplatin (CP, 30 µM, 16 h). n = 3. Error bars indicate SD with * and ** indicate p <0.05 and p<0.01, respectively using Student's T test. B PARP cleavage was not induced by S-HAD treatment. PARP cleavage was monitored by Western blot analysis in Hela cells treated with S-HAD or cisplatin (CP) as described in (A). The blot was stripped and reprobed with ß-actin antibodies as a loading control. Molecular weight markers (kDa) are indicated on the left. C Wild type and Ccnc-/- MEF cultures were treated with S-HAD and cisplatin (CP) as just described. Cells were imaged and examined for a "rounding up" phenotype diagnostic for death. D Cyclin C is required for normal Bax oligomerization in response to cisplatin. Whole cell extracts prepared from DSP crosslinked wild type and CCNC-/- MEF cells exposed to cisplatin (30 µM, 16 h) were probed for total Bax. Bax multimers are indicated. Molecular weight markers (kDa) are indicated on the left. E S-HAD does not induce Bax oligomerization. Experiments described in D were repeated with cultures treated with S-HAD (10 µM, 24 h). Bax multimers are indicated. Molecular weight markers (kDa) are indicated on the left. F S-HAD treatment sensitizes Hela cells to cisplatin. Hela cultures were treated with cisplatin (30 µM), S-HAD (10 µM) and N-acetyl cysteine (NAC, 1 mM) as indicated. Fluorescent cell analysis was used to quantitate the apoptotic (Annexin V positive) and necrotic (PI positive) population percentages, respectively. n = 3 independent cultures. Error bars indicate SD. Asterisk indicates p <0.05 (Student's T-test).
Figure 4(A) Distribution of GLT-1agold particles in astrocytic processes contacting asymmetric synapses (perisynaptic astrocytic processes: PAP) in SI of 5 WT and 5 KI (2 male, 3 female) mice (P34-35). In cortical PAP of KI mice the density (particles/µm2) of total and membrane-associated gold particles (arrowheads) are reduced compared to WT mice, whereas the density of cytoplasmicgold particles (arrows) is comparable to that in WT (cf Table I for numerical values and statistics). (B) Distribution of GLT-1agold particles in excitatory axon terminals forming asymmetric synaptic contact (AxT) in SI of P34-35 WT and KI mice. In cortical AxT of KI and WT mice, GLT-1a density is comparable (arrowheads: membrane-associated gold particles; arrows: cytoplasmicgold particles) (cf Table I).
Figure 1Associations between CSF T-tau, Ng and NFL (panels A-C). Black squares: CN (n=109), red circles: MCI (n=187), blue triangles: AD (n=93). Associations are shown for Spearman correlations. Aβ42 and T-tau were measured using the the INNOBIA AlzBio3 kit (Fujirebio, Ghent, Belgium), Ng was measured using an in-house immunoassay for Ng (Portelius et al, 2015) and NFL was measured using the NF-light® ELISA kit (Uman Diagnostics, Umeå, Sweden). Associations between CSF T-tau, Ng and NFL and between these biomarkers and CSF A42 (panels D-F). Black squares: CN (n=109), red circles: MCI (n=187), blue triangles: AD (n=93). Associations are shown for Spearman correlations. Aβ42 and T-tau were measured using the the INNOBIA AlzBio3 kit (Fujirebio, Ghent, Belgium), Ng was measured using an in-house immunoassay for Ng (Portelius et al, 2015) and NFL was measured using the NF-light® ELISA kit (Uman Diagnostics, Umeå, Sweden).
Processing of GRI‐peptide by METACASPASE‐9 is required for induction of elevated ion leakageRecombinant AtMC9 (rAtMC9) cleaved GRI25–168 in vitro.Figure 4A Recombinant AtMC9 (rAtMC9) cleaved GRI25-168in vitro. Bacterially produced MBP‐GRI25-168 (from 0 to 2 pmol left to right) was incubated with 1 pmol rAtMC9 or inactive rAtMC9mut (rAtMC9C147AC29A), and cleavage products were analyzed by Western blot with anti‐MBP and anti‐AtMC9 antibodies. Arrowheads in the anti‐MBP blot (from top to bottom) indicate MBP‐GRI25-168 (63.5 kDa), MBPMCS (53.5 kDa), rAtMC9‐cleaved MBP‐GRI25-168 (46 kDa). Arrowheads in the anti‐AtMC9 blot indicate: 1: N‐terminal domain + p20 + p10 subunits of AtMC9, 2: p20 + p10 subunits of AtMC9, 3: N‐terminal domain + p20 subunit of AtMC9, 4: p20 subunit of AtMC9. B Infiltration of wild‐type, prk5‐1 and atmc9‐1 leaves with 37 nM GRIp31-96, GRIp65-84, GRIp68-78 or GST. GRIp65-84 and GRIp68-78 but not GRIp31-96 were able to induce elevated ion leakage in the atmc9‐1 mutant. C Schematic representation of the GRI and the cleavage sites for rAtMC9. The cleavage product detected in Western blot analysis with anti‐MBP antibody is shown as black bar. Mass spectrometric analysis of MBP‐GRI25-168 cleavage with rAtMC9 provided evidence for cleavage after SKTR and KANK; further analysis of GRIp31-96 cleavage by rAtMC9 provided evidence for cleavage after SK, SKTR and after KKIKK. The position of the resulting 11‐aa‐long peptide (68LLVSHYKKIKK78) is indicated by a white inset. D Infiltration of Col‐0leaves with 37 nM of GRIp65-84, GRIp68-78, Y‐GRIp68-78, GRIp31-96 or GST. Y‐GRIp68-78 showed similar activity in cell death induction compared to the other GRI‐derived peptides. E 125I‐labeled Y‐GRIp68-78 (0.46 nM) bound specifically to Col‐0membrane fractions (light gray bars), the binding was significantly reduced in prk5‐1 and prk5‐2 plants. Excess of non‐radioactive Y‐GRIp65-84 (10 μM) reduced binding to background levels (dark gray bars; all bars show the average of four samples, triangles show individual data points). F Excess of GRIp68-78 and GRIp65-84 (10 μM) but not of GRIp31-96 or other peptides (GRIp31-51, GRIp47-68, GRIp80-96) competed the binding of 0.46 nM 125I‐Y‐GRIp68-78 to membrane extracts from Col‐0 (all bars show average of four samples, triangles show individual data points). G Saturation binding curve for 125I‐Y‐GRIp68-78 to Col‐0membrane extracts. Specific binding was calculated by subtracting non‐specific binding from the total binding. The affinity of 125I‐Y‐GRIp68-78 to the receptor (Kd = 1.9 nM) was calculated by non‐linear regression analysis. Scatchard plot is shown in Supplementary Fig SI7.
HCIPs within the autophagy network are shown for 32 primary baits (filled squares) and 33 secondary baits (open squares).figf1
Figure 4A. Second harmonic generation (SHG) and unfiltered multiphoton (MP) imaging of collagen fibres surrounding TEBs (dashed line) in 6-7 weeks old wt and Sharpincpdm mouse mammary glands (n = 6 - 9 mice, 5 - 13 TEBs analysed per mouse; scale bars represent 100 µm). Magnified images of collagen fibres are also shown (scale bars represent 50 µm). B. Frequency of clear collagen bundles around TEBs per mouse (Mean ± SEM, n = 6 - 9 mice). C. AFM indentation analysis of stromal stiffness in 6-7 weeks old wt and Sharpincpdm mouse mammary gland tissue sections (n = 3 mice; 3 - 6 sample sections per mouse and 192 indentation curves per sample).
Pyroptosis allows VHHASC to access the cytosol and target ASC specks.Figure 5(A) Wide-field fluorescence microscopy imaging of live PMA-differentiated and nigericin- (10 µM, top) or PFO-activated (25 ng ml-1, bottom) THP-1 expressing human ASC-GFP. Cells were incubated with VHHASC (200 µg ml-1) alone or in the presence of the caspase-1 inhibitor VX-765 (50 µM). Nuclei were stained with DRAQ5 (blue). Cells were imaged live with a CellDiscoverer 7 microscope. For each condition, a total of 8 positions within 2 wells (2x4 images/well) were imaged. (B) Graphic representation of the percent of specking cells over time. Maximal intensity projections from Z-stacks were generated for each image set before the number of cells and specks per field were calculated using CellProfiler. (A) represents images from one experiment out of three independent experiments of which percentage of specking cells data is represented in (B) with each line representing one field-of-view. (C) GFP+ specks detected in culture supernatants by flow cytometry of ASC-GFP oligomeric state (DSS cross-linked) of extracellular specks recovered from supernatants of cells treated similarly to (A-B). Data in (C) is from two independent experiments (each represented by a different symbol) GFP+ specks detected in culture supernatants by (D) immunoblotting analysis of ASC-GFP oligomeric state (DSS cross-linked) of extracellular specks recovered from supernatants of cells treated similarly to (A-B). (D) is from one out of at least two independent experiments. (E) Mouse BMDMs expressing ASC-mCitrin and primed with LPS (200 ng ml-1) for 3h were pre-treated with VHHmASC (200 µg ml-1) alone or in the presence of the caspase-1 inhibitor VX-765 (50 µM) for 30 min. Cells were then treated with a mixture of LFn-MxiH and PA (Mx/PA, 100 ng ml-1/1 µg ml-1) or PFO (250 ng ml-1) and imaged as in (A) for 3h. Data is a graphic representation of specking cells over time calculated as in (B).
a) Kinetics of the appearance of H-2Db–gp276+ T cells in the peripheral blood of Atg7fl/fl and Atg7fl/flGzmb-Cre mice at various times (horizontal axis) after infection with LCMV clone 13.figf8(a) Kinetics of the appearance of H-2Db-gp276+ T cells in the peripheral blood of Atg7fl/fl and Atg7fl/flGzmb-Cre mice at various times (horizontal axis) after infection with LCMV clone 13. (b) Total IFN-γ-secreting cells in spleens obtained from mice at day 8 or day 15 after infection as in a, assessed after 5 h of stimulation ex vivo with various peptides (horizontal axes). (c) Viral titers in serum of mice at various times (horizontal axis) after infection with LCMV clone 13 as in a. Each symbol represents an individual mouse; small horizontal lines indicate the mean (±s.e.m.). *P ≤ 0.05, **P ≤ 0.005 and ***P ≤ 0.0005 (unpaired t-test). Data are representative of two independent experiments with three to five mice per group (error bars, s.e.m.).
Figure 1A. Pedigree of family 1. Individuals II:3 and III:2 were analyzed by whole exome sequencing. Damaging variants in SHOX and CYP26C1 co-segregate with LWD. B. Pedigrees of family 2 and 3.
Figure 9Following BAK1 depletion, possibly by pathogen effector actions, MAMP-triggered outputs including induction of PROPEPs are weakened. However, effector-dependent PROPEP induction compensates for low amounts of MAMP-induced PROPEPs in the absence of BAK1. Cell death spread following dysfunction of BAK1/BON-mediated control probably facilitates release of cytosolic PROPEPs into the extracellular space. Moreover, in the absence of BAK1/BON1 complexes, PEPR signaling is strengthened by the stabilization of the receptor and by the potentiation of SA-related defenses toward anti-biotrophic resistance.
MICU2 with different numbers of Ca2+ ions bound.Figure 3A Structural alignment of Ca2+-free and partial Ca2+-bound MICU1-MICU2 heterodimers, as well as critical substructures with conformational changes indicated by numbered dashed circles. Middle and right, close-up view of the MICU2 EF-hand 1 (middle, circle 1) and MICU1 EF-hand pair 2 (right, circle 2) conformational changes upon Ca2+-binding. The important α-helixes are highlighted as cylinders. The red sphere represents Ca2+. B Structural alignment of Ca2+-free, partial Ca2+-bound and fully Ca2+-bound MICU1-MICU2 model heterodimers as well as important substructures with conformational changes are indicated by numbered dashed circles. Three different MICU1 and MICU2 molecules are individually shown in similar colours, and the colours changing from light to dark correspond to an increase in Ca2+ numbers. Circles 1, 2, and 3: close-up views of MICU2 EF-hand 1, MICU1 EF-hand pair 2 and MICU1 EF-hand 1 conformational changes upon Ca2+-binding, respectively. Important α-helixes are highlighted as cylinders. The red sphere represents Ca2+. C Conformational changes of the alkaline groove between T4L fusion Ca2+-free MICU1-MICU2 and partial Ca2+-bound MICU1-MICU2 heterodimers. Inset, KRKK residues in the alkaline groove rotate upon Ca2+ binding. D Electrostatic surface potential of the alkaline groove in partial Ca2+-bound structure. The gradient shown is from -3 (acidic, red) to +3 (basic, blue) kT/e.
Figure 3A WB analysis showing KO efficiency for CRISPR134B MEF. WB analysis showing KO efficiency for CRISPR62 MEF. C-E CRISPR WT MEF, CRISPR134B MEF, CRISPR62 MEF, respectively. Scale bars: 10 μm. F Quantification of C-E (n=10, 8, 7 cells, respectively). One-way ANOVA and Dunnett's multiple comparisons test, ns P>0.05, **** P<0.0001.