EMBO/sd-character-level-ner huggingface dataset:
Figure 1Media from soluble TREM2-Fc transfected HEK293T cells incubated with Aβ42 fibrils. Top left panel shows original media samples. Top right panel shows the presence of soluble TREM2 associated with Aβ42 pellet. The bottom two panels represent the same samples as the top, but probed with 6E10 RIPA lysate from TREM2 transfected HEK293T cells incubated with Aβ42 fibrils. Top left panel shows original RIPA lysate samples and top right panel shows the presence of TREM2 associated with Aβ42 pellet. The bottom two panels represent the same samples as the top, but probed with 6E10 Western blo had been cropped to facilitate the reading and originals with the supernatant fractions are available in Fig EV1. All experiments were replicated 3 independent times Media from soluble TREM2-Fc and APOE3 co transfected HEK293T cells incubated with agarose beads anti-Human IgG Fc. Left panels show original media samples. Right panels show the purified soluble TREM2-Fc (bottom) and the presence of APOE3 with the purified soluble TREM2-Fc (top) Western blo had been cropped to facilitate the reading and originals with the supernatant fractions are available in Fig EV1. All experiments were replicated 3 independent times Media from soluble TREM-Fc family member transfected HEK293T cells incubated with Aβ42 fibrils. Top panel shows original media sample and the presence of TREM family members associated with Aβ42 pellet. Bottom panel shows original media and Aβ42 pellet probed with 6E10 Media from soluble TREM-Fc family members and APOE3 co transfected HEK293T cells incubated with agarose beads anti-Human IgG Fc. Top panel shows original media sample and the presence of soluble TREM family members associated with APOE3. Bottom panel shows original media and purified soluble TREM-Fc family members probed with anti-V5
Figure 2(A) Experimental layout to test the DNA topology simplification activity of cellular topo II upon the addition of ICRF-193. (B) Lanes 1 and 2: negatively supercoiled plasmid (YEp24) and its equilibrium distribution of Lk topoisomers upon its relaxation with Topo I. Lanes 3 and 4: distribution of Lk topoisomers of YEp24 upon its relaxation in lysates of ∆top1 TOP2 yeast cells in absence and after the addition of ICRF-193. Plots compare the relative intensity of individual topoisomers of the Lk distributions in lanes 2, 3 and 4. (C) Top: 2D gel electrophoresis of the Lk distributions of the YEp13 minichromosome present in the lysates of ∆top1 TOP2 yeast cells before and after the addition of ICRF-193 (see details in Fig EV3). Plots compare the relative intensity of the Lk distributions (divided into ten sections). Bottom: 2D gel electrophoresis of the same samples upon nicking the DNA in order to reveal the occurrence of knots (see details in Fig EV4). The graph shows Pkn of YEp13 (mean ± SD from three independent experiments). P-values (Student's t test): ns, p > 0.05. (D) Experimental layout to compare the activities of TOP2 and Top2-∆83 on DNA and chromatin. Arrowheads indicate the extrachromosomal expression of TOP2 and Top2-∆83 under the inducible pGAL1 promoter. (E) Lk distributions of the control plasmid (YEp24) relaxed by lysates of ∆top1 top2-4 yeast cells that expressed TOP2 or Top2-∆83. Plots compare the relative intensity of individual Lk topoisomers. (F) Top: 2D gel electrophoresis of the Lk distributions of the YEp13 minichromosome produced in the presence of TOP2 or Top2-∆83. Plots compare the relative intensity of the Lk distributions (divided into ten sections). Bottom: 2D gel electrophoresis of the same samples upon nicking the DNA in order to reveal the occurrence of knots. Graph: Pkn of YEp13 (mean ± SD from three independent experiments).
NKG2D binding to the MICA-129Met and MICA-129Val isoform and triggering of phosphorylation of SRC family kinases.Figure 2The linear regression of MICA expression intensity and binding of a recombinant NKG2D-Fc fusion protein both determined as MFI by flow cytometry is displayed for L-MICA-129Met (n = 79, left panel) and L-MICA-129Val clones (n = 81, right panel). The coefficients of determination (R2), the regression coefficients (reg. coeff.), and the P-values for Pearson correlation are indicated. Purified IL-2-stimulated (100 U/ml for 4 days) NK cells (106) were stimulated with immobilized MICA-129Met-mIgG2a-Fc or MICA-129Val-mIgG2a-Fc or OVA-mIgG2a-Fc fusion proteins (10 μg/ml) for 3, 10, or 30 min. The protein lysates of these cells were separated by SDS-PAGE, and the blot was probed subsequently with an anti-phospho-Tyr mAb, an anti-phospho-SRC family (Tyr419) kinases Ab, and an anti-β-actin mAb as a loading control. The arrow points toward phosphorylated SRC family kinases.Blots obtained from three independent experiments were analyzed by densitometry, and the means plus SD of the ratio between phospho-SRC family kinase and β-actin signals is displayed. The difference between NK cells stimulated for 10 min by MICA-129Met-Fc or MICA-129Val-Fc proteins was assessed by t-test. Purified IL-2-stimulated NK cells (100 U/ml for 4 days, 106) were incubated with the SRC kinase inhibitor PP2 (25 μM), the vehicle DMSO, or medium only (Ø) for 30 min before being added to immobilized MICA-129Met-Fc, MICA-129Val-Fc, or OVA-Fc fusion proteins (10 μg/ml) for 10 min. The protein lysates of these cells were separated by SDS-PAGE, and the blot was probed subsequently with an anti-phospho-Tyr mAb and an anti-β-actin mAb as a loading control. The blot is representative for two independent experiments. In parallel, degranulation of the NK cells was measured by anti-CD107a staining in flow cytometry. The difference between DMSO- and PP2-treated cells with respect to CD107a+ cells and the MFI of CD107a is indicated in the histograms. The results are representative for two independent experiments.
Figure 4, Gains of OTUB1 and KRAS mutation are mutually exclusive in lung adenocarcinomas. OncoPrint showing the distribution of KRAS somatic mutations and OTUB1 copy number alterations in TCGA lung adenocarcinomas and squamous cell carcinomas obtained from cBioPortal (Cerami et al, 2012; Gao et al, 2013). Co-occurrence analysis showing significant mutual exclusivity between KRAS mutation and OTUB1 gain. B,C, OTUB1 overexpression in TCGA lung carcinomas is associated with 11q13.1 copy number alteration. Pearson correlation of OTUB1 copy number (log2 ratio) with OTUB1 mRNA levels (RNAseq normalized read counts, log2 transformed) was analyzed. D,E,F, OTUB1 expression in TCGA lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (SCC) patients. Patients were stratified according to their OTUB1 mRNA levels and/or their KRAS status as described in Materials and Methods. Box whisker plots represent OTUB1 mRNA expression levels in TCGA lung carcinoma patients determined by RNAseq analysis. p-values were determined by two-sided t-Test. Total number of patients, n. G, Gain of 11q13.1 locus is an early event in lung adenocarcinoma development. TCGA lung adenocarcinoma patients with diploid or gain of the OTUB1 locus were stratified according tumor stages (T1-T4). Total numbers of patients, n. Statistical comparison of the sample distributions were compared using Chi-square test. H, OTUB1 mRNA overexpression is an early event in lung adenocarcinoma development. TCGA lung adenocarcinoma patients were stratified by tumor stages (T1-T4) and OTUB1 expression levels as described in Materials and Methods. Total numbers of patients, n. Statistical comparison of the sample distributions were compared using Chi-square test. I, KRAS mutation is a late event in lung adenocarcinoma progression. TCGA lung adenocarcinoma patients with different KRAS mutation status were stratified by tumor stages (T1-T4). Total numbers of patients, n. Statistical comparison of the sample distributions were compared using Chi-square test.
Domain architecture and structure of Miro.Figure 1Domain architecture and structure of Miro. (A) Bar diagram of Miro domain structure. Numbering corresponds to dMiro isoform D, domain names are described in the text (B) Ribbon diagram of the MiroS structure in the 'side view'. Note three distinct domains: ELM1 (blue), ELM2 (green), cGTPase (grey/yellow), joined by two linkers: Lnk1 (orange), and Lnk2 (red). The structure shown is MgGDP‐MiroS, with the Ca2+ ion from the Ca‐MiroS structure overlaid for orientation ( Ca2+/Mg2+/GDP as yellow/purple/multicoloured spheres). cGTPase, C‐terminal GTPase; dMiro, Drosophila Miro; ELM, EF hand pair with ligand mimic.
PCDH7 is required on the cell cortex for building rounding pressurePCDH7 antibody detects a single band of ˜110 kDa in luciferase RNAi (luc), PCDH1 RNAi HeLa cell lysates, but this band is not detectable in PCDH7 and PCDH1 and PCDH7 RNAi cells.Figure 5A PCDH7 antibody detects a single band of ˜110 kDa in luciferase RNAi (luc), PCDH1 RNAi HeLa cell lysates, but this band is not detectable in PCDH7 and PCDH1 and PCDH7 RNAi cells. α‐tubulin was used as a loading control (left). HeLa Kyoto cells stably expressing PCDH7‐LAP‐tagged BAC transgenes treated with luciferase RNAi (control) and 150 nM (1×) and 300 nM (2×) PCDH1 esiRNA and blotted against anti‐GFP, anti‐PCDH1, and anti‐actin antibodies. PCDH1 bands were reduced in PCDH1 RNAi cells, whereas mouse GFP::PCDH1 was not affected by the esiRNA treatment (right). B Rounding pressure plots of MYH9 (positive control, n = 7), luc (negative control, n = 12), PCDH7 (n = 11), PCDH1 (n = 12), and PCDH1 and PCDH7 (n = 11) RNAi cells. Bars show mean ± SEM (left). Rounding pressure plots HeLa Kyoto cells stably expressing PCDH7‐LAP‐tagged BAC transgenes treated with MYH9 (positive control, n = 7), luc (negative control, n = 14), PCDH1 (n = 13) esiRNA. *P 0.05; **P 0.01; ***P 0.001; ns, P > 0.05 (non‐significant).
a) Chemical structure of the NMS-859 covalent inhibitor.figf2(a) Chemical structure of the NMS-859 covalent inhibitor. (b) LC/MS analysis of VCP after 1 h incubation at 20 °C with 1% DMSO (top) or 100 μM NMS-859 (bottom), showing a 313-Da increase corresponding to the addition of NMS-859 lacking the Cl atom. (c) Relative positions of ADP and Walker A residues Cys522 and Lys524 (the latter two are within hydrogen-bonding range), as found in the C chain D2 ADP-binding pocket of VCP (PDB code 3CF1).
Figure 5  (A) The number of motile and non-motile sporozoites moving in the dermis of mice was manually counted in time courses spanning 120 minutes and is displayed as a percentage of total sporozoites observed 5 min after inoculation, for P. berghei (left panel), P. yoelii (center panel), and P. falciparum (right panel). (B) Fold-change in the number of motile sporozoites at 60 and 120 min post-inoculation compared to the number of motile sporozoites at 5 min. The data shown only includes complete imaging sessions over 120 min after intradermal injection of sporozoites. (P. berghei: n=2, P. yoelii n=4, P. falciparum n=2).
Figure 3A. Experimental scheme and representative images of DNA fibers. Scale bar: 5 μm.B. Analysis of fibers showing proportions of replication forks that showed restart after HU treatment or remained stalled. N=2, >500 fibers scored per experiment.C. Proportion of total forks showing de novo initiation after HU treatment. N=2, >500 fibers scored per experiment.D. Length of initial replication fork tracts (CldU tracts). N=2, >200 fibers measured per experiment.
Figure 1Schematic representation of the domain and degron architecture of Nek2A. The MR tail is the main contributor for Nek2A ubiquitination. Ubiquitination reactions performed with APC/CCdc20 for Nek2A wild type (Nek2AWT) and degron mutants. Nek2AK is the KEN box mutant (391KEN393/KAA), Nek2AK/D is the (KEN and D box mutant (391KEN393/KAA, 361RKFL364/AKFA), Nek2AMR is the 443∆MR mutant. The APC/C requires the Cdc20 coactivator subunit to ubiquitinate Nek2A. Nek2A ubiquitination reactions performed with the APC/C in absence and in presence of increasing concentrations of Cdc20 using the E2 UbcH10. The MR tail is essential for Nek2A binding to the APC/C whereas in contrast Cdc20 is dispensable. Size exclusion chromatography peak fractions of APC/C complexes with Nek2A. The input material (13% of total) is indicated (i). Nek2A binding does not stabilize binding of the coactivator subunit Cdc20 to the APC/C and does not compete with cyclin A for binding to the APC/C. Size exclusion chromatography peak fractions of APC/C complexes with Nek2A. Top: cylinder representation of the APC/C-Nek2A complex structure. The TPR lobe subunits are highlighted: Apc8 (cyan), Apc6 (pink), Apc3 (yellow) and Apc7 (green). The platform subunits and the small subunits from the TPR lobe are shown in transparency. Bottom: cryo-EM density of the Nek2AMR tail dipeptide and details of the Nek2AMR tail (orange) binding site on Apc8A.   Details of the IR tail of Cdc20MCC (orange) bound to Apc8A in APC/CMCC-closed. This is the same site as the Nek2AMR tail-binding site shown above. Shown is the cryo-EM density for the Cdc20MCC IR tail and contacting residues of Apc8A.
Figure 2A. Re-expression of mMCU rescues cell motility of MCU-silenced cells. Cells were transfected with siMCU or siControl. Ad-mMCU was used to re-express MCU (Ad-GFP was used as control). MCU protein expression was verified by western blot. The day after transduction a linear scratch was made (time-point 0h). Cell migration into the wounded area was monitored at 48 hour time-point and the covered area was measured (n=12). P-value: ***p<0.0001. B. MCU silencing blunts cell invasiveness. Stable shMCU and shControl-expressing spheroids were plated and let grow into collagen-I (0 hour time-point). Spheroid area was measured at 0-hour and 48-hour (n=8). Scale bar 300 μm. P-value: ***p=0.0003. C. MCU silencing reduces the clonogenic potential of MDA-MB-231 cells. Stable shMCU and shControl-expressing cells were plated at low confluence (2x103/well of a 6-well plate). After 7 days the number of colonies was counted (minimum 30 cells/colony, n=8). P-value: **p=0.0027. D. MCU depletion does not induce cell death. Cells were transfected with siMCU or siControl. 72 hours later, cell apoptosis and necrosis were measured by FITC-Annexin V and Propidium Iodide (PI) detection (Q1: PI positive, Q2: PI and FITC-Annexin V positive, Q3: PI and FITC-Annexin V negative, Q4: FITC-Annexin V positive. n=6). E. MCU depletion does not alter cell cycle. Cells were transfected with siMCU or siControl. 72 hours later, cell cycle distribution was monitored by Propidium Iodide (PI) detection (n=6).
a) Total GPx activity was measured in mGpx1-overexpressing (mGpx1) or control (WT) cell lines with (induced) or without (uninduced) Htt103Q expression.figf2a) Total GPx activity was measured in mGpx1-overexpressing (mGpx1) or control (WT) cell lines with (induced) or without (uninduced) Htt103Q expression. Overexpression of mGpx1 increases total GPx activity in PC12 cells (****P 0.0001 compared to uninduced controls; n = 7 per condition). (b) Quantification of caspase-3/7 activation in response to Htt103Q expression in cells overexpressing mGpx1. Overexpression of mGpx1 reduces Htt103Q-mediated caspase-3/7 activation (****P 0.0001). Values represent the ratio of mGpx1 activity in cells expressing Htt103Q for 72 h compared to cells not expressing Htt103Q (n = 12 (WT); n = 9 (monomeric red fluorescent protein (mRFP)); n = 8 (mGpx1) per condition). (c) Effect of the GPx mimetic ebselen on Htt103Q-mediated caspase activation. A single 10 μM dose of ebselen significantly reduced caspase-3/7 activation in Htt103Q-expressing cells (****P 0.0001). Values represent the ratio of caspase-3/7 activity in cells expressing Htt103Q for 72 h compared to cells not expressing Htt103Q. Statistical analysis was performed using an unpaired, two-tailed Mann-Whitney test (n = 12 per condition). (d) mGpx1 overexpression (****P 0.0001) and ebselen treatment (****P 0.0001) significantly reduce Htt103Q-mediated ROS production (n = 9 per condition). Unless stated otherwise, all statistical analyses were performed using one-way analysis of variance (ANOVA) with post-hoc tests. NS, not significant. **P 0.01. All data are shown as the mean ± s.e.m.
Figure 5CD4+ and CD8+ T cells were isolated by negative selection from WT or EGR4-/-mice. (A) CD4+ and CD8+ T cells were loaded with Fura-2AM and plated on poly-D-lysine (2 hrs) before treating with biotinylated anti-CD3ε, followed by streptavidin where indicated. The average response of the majority of CD4+ (WT 74.2±3.2%; EGR4-/- 77.8±2.9%) and CD8+ (WT 79.4±3.5%; EGR4-/- 78.2±2.5%) cells that responded to streptavidin crosslinking of CD3 (mean±SEM; 40-60 cells/coverslip; 7 to 8 separate experiments). CD4+ and CD8+ T cells were isolated by negative selection from WT or EGR4-/-mice. (B) Cells were plated on anti-CD3/CD28 antibodies for 2 hrs; BTP2 (10 μM) was added where marked by the arrow. CD4+ T cells were isolated by negative selection from WT or EGR4-/-mice. (C) CD4+ T cells were incubated with anti-CD3/CD28 antibodies for 6 hrs before measuring expression of IFNγ, IL-2, IL-17 and IL-4 by FACS analysis. Data are mean±SEM; a minimum of 3 biological replicates were examined; each biological replicate includes 2 technical replicates.
JQ1 pre-treatment inhibits N9 microglial cell inflammation, proliferation, and migration.Fig. 5 JQ1 pre-treatment inhibits N9 microglial cell inflammation, proliferation, and migration. N9 microglial cells were pre-incubated with vehicle (DMSO), JQ1 (0.5 μM), RVX208 (30 μM), or Olinone (30 μM) for 12 h followed by stimulation with LPS (1 μg/ml) for another 2 h, and then subjected to qRT-PCR (a) for determination of inflammatory cytokine expression. Proliferation assay (b, BrdU ELISA) and migration assay (c, Transwell) were performed after incubation for additional 24 h. Quantification: mean ± SEM; n = 3 experiments; *P < 0.05, **P < 0.01 compared to vehicle + LPS
Figure 1MCF10A cells were treated with 5uM RSL3 for times that ranged from 0 to 120 minutes. mRNA was isolated from each time point and PROM2 expression was quantified by qPCR. Shown is an average of three independent experiments with standard deviation (n=3 experiments per time point). P‑values were obtained by unpaired Student's t‑test with *P<0.05, **p<0.01, ***p<0.005. Exact p‑values are reported in the Appendix Table S1. MCF10A cells were pretreated for 15 minutes with either DMSO or 2uM fer-1, followed by 60 minutes with either DMSO, 5uM RSL3 or RSL3 and fer-1. mRNA was isolated and PROM2 expression was quantified by qPCR. Shown are three independent experiments with standard deviation (n=3 experiments per group). P‑values were obtained by unpaired Student's t‑test with *P<0.05, **p<0.01, ***p<0.005. Exact p‑values are reported in the Appendix Table S1. MCF10A cells were treated with 25uM 4HNE for times that ranged from 0 to 120 minutes. mRNA was isolated from each time point and PROM2 expression was quantified by qPCR. Shown is an average of three independent experiments with standard deviation (n=3 experiments per time point). P‑values were obtained by unpaired Student's t‑test with *P<0.05, **p<0.01, ***p<0.005. Exact p‑values are reported in the Appendix Table S1. MCF10A cells were pretreated for 15 minutes with either DMSO or 2uM fer-1, followed by 60 minutes with either EtOH, 25uM 4HNE or RSL3 and fer-1. mRNA was isolated and PROM2 expression was quantified by qPCR. Shown are three independent experiments with standard deviation (n=3 experiments per group). P‑values were obtained by unpaired Student's t‑test with *P<0.05, **p<0.01, ***p<0.005. n=3 experiments per group. Exact p‑values are reported in the Appendix Table S1. MCF10A cells were treated with either EtOH, 25uM 4HNE or 5uM RSL3 for 60 minutes. Isolated protein was assessed by immunoblotting for 4HNE, prominin2, and β-actin expression. Shown is one replicate of three independent experiments (n=3). MCF10A cells were treated with DMSO or 100uM MDA for 60 and 120 minutes. Isolated protein was assessed by immunoblotting for prominin2, and β-actin expression. Shown is one replicate of three independent experiments (n=3).
Fig.Figure 8Representative immunoblots from protein extract of GC muscles of 6-month-old control, HSA-Cre untreated and HSA-Cre TUDCA-treated mice, with the indicated antibodies. Quantitative RT-PCR measurement of Fgf21 and GDF15 expression in GC muscle of 6-month-old control, HSA-Cre untreated and HSA-Cre TUDCA-treated mice. Histological analysis of TA muscles of 6-month-old control, HSA-Cre untreated and HSA-Cre TUDCA-treated mice by Oil Red O staining to detect the neutral lipids. Scale bars: 40 μm. TAG level measurement in GC muscles of 6-month-old control, HSA-Cre untreated and HSA-Cre TUDCA-treated mice. In vivo force measurements performed on GC muscles of 6-month-old control, HSA-Cre untreated and HSA-Cre TUDCA-treated mice. Representative staining of IgG positive fibers on frozen sections of TA of 6-month-old control, HSA-Cre untreated and HSA-Cre TUDCA-treated mice. The sections were stained with a secondary fluorescent anti-mouse IgG antibody. Quantification of the number of necrotic fibers over the total number of fibers.
Figure 4A HDX-MS reveals a constrained BH4 domain in inactive BAK that is rapidly deuterated after activation. BAK BH4 residues in the α1 (V34, F35, S37 and Y38) corresponding to the BH4 motif Φ1Φ2XXΦ3Φ B BAK wild-type, or BAK engineered with the indicated mutations (on a BAK∆Cys background) were stably expressed in Bax-/-Bak-/- MEFs and expression was assessed by immunoblotting following 3 h doxycycline treatment. C Bax-/-Bak-/- MEFs expressing the indicated BAK variants were treated with doxycycline (Dox, 3 h), incubated for a further 24 h in the presence or absence of BH3-mimetic compounds, and cell death was assessed by PI uptake. D Mutation of BH4 promotes BAK activating conformation change. Cells were treated with doxycycline (3 h) to induce BAK expression followed by incubation with BH3-mimetics where indicated (for 2 h) and conformation change of BAK was assessed by intracellular flow cytometry with an antibody that recognises activated BAK (G3172).
Figure 3(A-D) Pooled patient samples at high (5.7x104 copies), medium (3.6x102 copies) or low (2x101 copies) concentrations (A, B), or stocks of SARS-CoV-2 WA1 (C, D) were subjected to -80ºC storage interrupted by (A, C) successive freeze-thaw cycles or (B, D) storage at 4ºC, room temperature (RT) or 37ºC for the indicated time periods. N=1 for A, B. For C and D datapoints are from 3 different viral stocks titered in duplicate and plotted as mean with error bars plotted as +/- SEM. For C raw values are plotted while values are normalized to starting titer for D. Limit of detection for infectious titer in C is indicated by a dashed line.
Figure 4A Enrichment of histone H3, AcH3, total RNPII, ser5-p RNPII, ser2-p RNPII, and NELF-E in the basal state (0 hours) and after treatment (2 and 4 hours) with 20 ng/ml TNF quantified using ChIP and shown as % input (non-IP control). Data are presented as mean ± standard error of the mean (s.e.m.) of three biological replicates. Significance calculated by Dunnett's multiple comparison test (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). B Hierarchical clustering of ChIP data before and after TNF treatment separates promoters with TNF-mediated increases in burst frequency or burst size. For each protein target, color bar indicates the % maximum ChIP value measured across all genes and time points.