EMBO/sd-character-level-ner huggingface dataset:
Figure 6A, B ATAC-seq analyses revealed progressive increased telomeric DNA accessibilities in sgA#10 transduced IMR90 cells. (A) The graph plots show the cumulative percentage of total reads that contained at least indicated number of TTAGGG (or CCCTAA) tandem repeats. (B) The percentage of total reads that contains > 3 tandem telomeric repeats are expressed as means ± s.d., N = 2, unpaired t test. C, D ChIP assays (C) show reduced nucleosome densities in sgA#10 transduced IMR90 cells (D). Input and ChIP DNAs processed against indicated antibodies were assayed by dot-blotting and hybridized with 32P-labeled probes specific for TelG. (D) The relative enrichment was calculated after normalization of ChIP DNA signals to the respective input DNA signals. Data are expressed as means ± s.d., N = 3, paired t test.
Figure 2ELISA of IL-1β secretion by WT or Spata2-KO BMDMs that were either untreated (-) or LPS-primed and then treated with the indicated NLRP3 inducers ELISA of IL-1β secretion by WT or Spata2-KO BMDMs that were either untreated (-) or stimulated with the NLRC4 inducer (Salmonella infection) or AIM2 inducer ( Poly(dA:dT) transfection) (B). Immunoblot analysis of the mature IL-1β p17 and the active Caspase-1 p20 in cell supernatants (Sup) and the indicated proteins in the cell extracts (Cell ext) of WT or Spata2-KO BMDMs stimulated as in (A). WT and Spata2 CRISPR knock out (KO) iBMDMs stably expressing GFP-ASC were primed with LPS and stimulated with Nigericin for 1 h. GFP-ASC specks were imaged by confocal microscope, and percentage of ASC speck-positive cells was quantified. Scale bar, 10 µm. BMDMs from Spata2+/+ (WT) and Spata2-/- (KO) mice were pretreated with Caspase-1 inhibitor, Z-YVAD-FMK, for 30 min, primed with LPS, and then stimulated with Nigericin for 1 h. ASC specks were assessed by Immunofluorescence with antibody against ASC and imaged by confocal microscope, and percentage of ASC speck-positive cells was quantified. Scale bar, 10 µm. Immunoblot analysis of ASC in soluble cell lysate and DSS cross-linked insoluble fraction of WT or Spata2-KO BMDMs primed with LPS and stimulated without or with nigericin (Nig) for 1 h. Relative levels of ASC in cross-linked samples from LPS+ Nig treated cells were quantitated and shown below. Spata2+/+ (WT) and Spata2-/- (KO) mice were injected intraperitoneally (i.p.) with alum (20 mg/kg bodyweight) and sacrificed 6 h latter to collect peritoneal lavages. Absolute numbers of neutrophils recruited to the peritoneum were counted by FACS (G) Spata2+/+ (WT) and Spata2-/- (KO) mice were injected intraperitoneally (i.p.) with alum (20 mg/kg bodyweight) and sacrificed 6 h latter to collect peritoneal lavages. levels of IL-1β (H) and IL-6 (I) in the peritoneal fluids were measured by ELISA.
BRAVO interacts with WOX5.Figure 4Interaction of BRAVO with WOX5 (B), BES1 (C), BES1D (D) and TPL (E); and interaction of WOX5 with BRAVO (G), BES1 (H), BES1D (I) and TPL (J) measured by FRET-FLIM. GFP fluorescence lifetime τ [ns] was measured in transiently expressing Nicotiana benthamiana leaf epidermal cells. GFP fluorescence lifetime fitted pixel-wise with a mono-exponential model of BRAVO and WOX5 interactions. mV, mVenus; mCh, mCherry. Scale bar: 5 µm. Fluorescence-weighted average lifetimes of BRAVO and WOX5 interactions fitted with a double-exponential model of the indicated samples are summarized in box plots. Statistical significance was tested by one-way ANOVA with a Sidakholm post-hoc test. Different letters indicate statistically significant differences (p-value < 0.01). For each combination two to three independent experiments were carried out and in total more than 20 biological replicates were measured. In the boxplot, box width represents the interquartile range (IQR=Q3-Q1), with the horizontal line denoting the median, while whiskers extend from Q1-1.5IQR to Q3+1.5IQR. White dots are the outliers. Yeast two-hybrid assay showing BRAVO interacting with WOX5, BES1-D and TPL; and BES1-D interacting with TPL. In the left column yeast cells were grown on control media, and in the right column yeast cells were grown on control media lacking Leu, Trp and His, indicating an interaction between the proteins.
Figure 1(A) A schematic diagram of the single-cell sequencing process. (B) Visualization of major classes of spinal cord cells by integrated UMAP plots of cell clusters. Cell types are labelled and circled with dotted lines (top). Violin plots show the most distinct and commonly used marker genes for each cell cluster (bottom). Cell-type annotations are listed on the right and shown in varying colours. (C) Three-dimensional visualization of neural subtype developmental trajectories for individual samples from GW7, GW10, GW14, GW18, and GW23. Stage-dependent differentiation trends changed with development. The red arrow indicates the proliferative neural progenitor cell cluster (MKI67, PAX6, NES, and SOX2). Neural cell types are named and circled with dotted lines. Gray represents non-neural cell types (e.g., vascular and meningeal). (D) Immunofluorescence staining of the spinal cord at GW7, GW8, GW10, GW11, and GW23 for RBFOX3 (red), OLIG2 (green), and GFAP (white). The appearance of astrocytes and oligodendrocytes was observed from GW8 onward. Scale bars: 100 μm.
Accumulation of PlcA/B‐induced granules as a result of reduced autophagic flux and PI3P depletion.Figure 6(A) HeLa cells were transfected with GFP‐RFP‐LC3 and either were left uninfected (t=0) or were infected with Listeria WT or PlcA/B− for 1-4 h. GFP+ RFP+ and GFP− RFP+ puncta appear yellow and red, respectively. Percentage of cells infected with Listeria WT or PlcA/B− for 1-4 h displaying GFP− RFP+ red puncta were quantified. Values are means±s.e.m. n=3. **P0.01; ***P0.001. (B) HeLa cells left unstimulated (CTR) or infected with Listeria WT or PlcA/B− for 4 h were lysed and PI3P levels measured by competitive ELISA. (C) HeLa cells transfected with GFP‐PKCδ and infected with Listeria WT for 4 h were analysed by IF using an antibody against NDP52. The arrow indicates an NDP52+ granule. (D) HeLa cells infected with Listeria PlcA/B− for 3 h, in the absence or presence of Wortmannin added during the last 10 min of infection, analysed by IF using antibodies against NDP52 and WIPI‐2. The arrow indicates an NDP52+ granule. Scale bars: 5 μm.
68Ga]Pentixafor PET/MR images.Figure 5[68Ga]PentixaforPET/MRimagesA-C Coronal views of [68Ga]Pentixafor (A), T2 STIR weighted MRI (B) and CTbone window (C) of a maleuptake with histologically proven multiple myeloma. The increased [68Ga]Pentixaforbone correlates with the hyperintense T2 STIR signal; however, the myeloma manifestations are underestimated in the corresponding bone window CT.
Pro-longevity mitochondrial stress reduces germ cells proliferation and apoptosis in basal conditions.Figure 1A, B Quantification of mitotic (A) and meiotic (B) cells in the distal germline in wild-type animals fed bacteria transformed with either empty vector (con) or vector-expressing dsRNA against frh-1 (frh-1) or isp-1 (isp-1) Data information: Data are presented as mean ± SE (A) N=3 at least 5 worms per replicate and condition, (B) N=3 at least 10 worms for conditio * P < 0.05 and **** P < 0.0001 vs con (One-way ANOVA Tukey"s multiple comparisons Quantification of dissected distal germline from wild-type animal stained either anti-phosphohistone H3 (anti-PH3) (C relative to the total number of mitotic cells of wild-type animal Data informatio Data are presented a as mean ± S N=2 at least 5 worms per replicate and conditio * P < 0.05 and **** P < 0.0001 vs con (One-way ANOVA Tukey"s multiple comparisons Quantification of dissected distal germline from wild-type animal stained eithe anti-CDK-1 (anti-CDK-1 (D relative to the total number of mitotic cells of wild-type animal Data information: Data are presented a mean ± S N=2 at least 5 worms per replicate and conditio * P < 0.05 and **** P < 0.0001 vs con (One-way ANOVA Tukey"s multiple comparisons Quantification of dissected distal germline from wild-type animal stained eithe 5-ethynyl-2′-deoxyuridine (EdU -green) (E) relative to the total number of mitotic cells of wild-type animal Data information: Data are presented a mean ± S N=2 at least 5 worms per replicate and conditio * P < 0.05 and **** P < 0.0001 vs con (One-way ANOVA Tukey"s multiple comparisons F Average number of apoptotic corpses in the meiotic compartment relative to the total number of meiotic cells in wild-type worms fed as in (A) Data information: Data are presented as mean ± SE (F) N=5 at least 10 worms per replicate and condition. * P < 0.05 and **** P < 0.0001 vs con (One-way ANOVA Tukey"s multiple comparisons
Figure 6(A, B) Alignment of the ICP27 homologs of the human herpesviruses (A), and a series of alpha-herpesviruses (B). The diagram to the right illustrates the degree of similarity with HSV-1 ICP27 and the distribution within the molecules. (C) THP1 cells were infected with HSV-1 (KOS), HSV-2 (HG52), HSV-1 ΔICP27, or HSV-1 ΔICP27 rescued with HSV-2 ICP27 (MOI 3). Supernatants were harvested 18 hpi for measurement of type I IFN bioactivity. (D) RAW264.7 cells were infected with HSV-1 (KOS), PRV (vBecker3) and the corresponding mutants lacking ICP27 (UL54 in the case of PRV). Supernatants were harvested 18 hpi for measurement of type I IFN bioactivity. (E) Alignment of the RGG box of in ICP27 proteins of the simplexvirus genera of alpha-herpesviruses. (F) THP1 cells were infected with HSV-1 (KOS), ΔRGG, or ΔICP27 (MOI 3). Supernatants were harvested 18 hpi for measurement of type I IFN bioactivity. ICP27 expression in the infected cells was determined by Western blotting. (G) THP1 cells were infected with HSV-1 KOS or HSV-1 ΔRGG (MOI 10) for 8 h and total lysates were generated. STING was immunoprecipitated, and subjected to Western blotting using antibodies against ICP27 and STING.
UNC‐51‐like kinase (Ulk1) and zipper‐interacting protein kinase (ZIPK) are essential for starvation‐induced myosin II activation.Figure 5(A) Ulk1 interacted with ZIPK. 293T cells transfected with HA‐tagged ZIPK together with Flag‐tagged Ulk1 or Ulk1‐KI were subjected to immunoprecipitations with anti‐Flag antibody. The immunoprecipitated proteins and the total cell lysates were analysed by immunoblotting with antibodies as indicated. (B) HA‐tagged kinase‐inactive ZIPK‐KA (K42A) was expressed in 293T cells with Flag‐tagged Ulk1 or Ulk1‐KI. Cell lysates were incubated with or without alkaline phosphatase (CIP) and analysed by immunoblotting with antibodies as indicated. (C) Myosin II was activated upon nutrient deprivation in MCF7 cells. MCF7 cells were cultured in serum containing DMEM medium (nutrient‐rich condition) or in Earle's balanced salt solution (EBSS; starved condition) for 2 h. The myosin II activity was significantly downregulated after EBSS‐starved cells were replenished with fresh DMEM medium containing 10% FBS for 2 h. The myosin II activity was quantified and expressed as a fold changes compared with the DMEM controls. Each value represents mean±s.e. of triplicates. (D) MCF7 cells stably infected with lentivirus expressing control (shLuc), Ulk1 or ZIPK shRNA were cultured in nutrient‐rich DMEM medium (F) or EBSS (S) for 2 h. Effects of Ulk1 and ZIPK knockdown on starvation‐induced myosin II activation were analysed by immunoblotting with antibodies as indicated. Data are mean±s.e. of triplicates.
Therapeutic use of AAV-BR1: Normalizing endothelial cell survival and blood-brain barrier permeability in neonatal incontinentia pigmenti mice (Nemo-/+) with AAV-BR1-NEMO.Figure 7BR1-mediated expression of NEMO or eGFP was driven by the CAG promoter.A Immunostaining of cerebral microvessels. Treatment with AAV-BR1-NEMO normalized string vessels (white arrows, highlighted in white square inset) in Nemo-/+ mice as compared to Nemo-/+ mice treated with the AAV-BR1-eGFP control vector at postnatal day 8 (P8). The upper left panel shows the staining in untreated wild type control mice. String vessels were identified as capillaries that have lost CD31-positive (green) endothelial cells but stain for the basement membrane protein collagen IV (red). Scale bars represent 200 µm. The lower right panel summarizes quantitative analysis of string vessels in Nemo-/+ and control mice (NemoFl or wild type) at P0 (n=3 animals/ group) and at P8 (n=6 animals/ group, *p = 0.0125). String vessels in the cerebral cortex were quantified as percentage of total vessel lengths. BR1-mediated expression of NEMO or eGFP was driven by the CAG promoter.B Quantification of active caspase 3-positive endothelial cells at P8 (n= 5 animals/ group, *p = 0.0201). BR1-mediated expression of NEMO or eGFP was driven by the CAG promoter.C Albumin in brain tissue as indicator for BBB leakage. In Nemo-/+ mice treated with AAV-BR1-NEMO, less albumin was found in brain tissue, indicating less BBB leakage. Representative Western blot with albumin from P8 Nemo-/+ mice treated with AAV-BR1-NEMO or AAV-BR1-eGFP control vector, respectively, as well as untreated wild type (WT) control mice. Right panel: quantitative analysis of the Western blots (n = 4 animals/ group, *p = 0.0283) BR1-mediated expression of NEMO or eGFP was driven by the CAG promoter.D Body weight of vector-treated mice. WT control or Nemo-/+ mice at P8 treated with AAV-BR1-NEMO or AAV-BR1-eGFP (n= 18 animals/ group, *p = 0.0038).
Figure 3USP36 interacts with Ubc9 in cells. H1299 cell transfected with the indicated plasmids were subjected to co-IP with anti-Flag antibody followed by IB. USP36 interacts with Ubc9 in vitro. Purified His-Ubc9 was incubated with GST, GST-USP361-800 or GST-USP361-420. Bound protein was detected by IB. GST and GST-fusion proteins were shown in the bottom panel by coomassie staining. USP36 interacts with SUMO in cells. H1299 cells transfected with either control or Flag-USP361-800 were assayed by co-IP using anti-Flag, followed by IB with anti-SUMO2/3 antibodies. USP36 interacts with SUMO2 chains in vitro. Recombinant SUMO2 chains were incubated with GST or indicated GST-USP36 fragments. Bound proteins were assayed by IB using anti-SUMO2/3 antibodies. Coomassie staining of the recombinant proteins was shown in bottom panel. USP36 SUMOylates PARP1 in vitro. Recombinant T7-PARP1 protein (0.1 μM) was incubated with SUMO E1 (30 nM), Ubc9 (50 nM), SUMO2 (4 μM) in the presence of USP361-800 (50 nM) and/or ATP (2.5 mM) at 30ºC for 5 hours and then assayed by IB.
Figure 8(A) Western blot analysis of MBP-Zwilch pull downs of full length (FL) his6x-SpindlyWT and phosphomimetic mutant his6x-SpindlyS499D. Immobilized MBP was used as negative control. B:Beads, FT: Flow-through. (B) Western blot analysis of MBP-Zwilch pull downs of his6x-SpindlyWT or his6x-SpindlyS499D fragments (aa1-510). Immobilized MBP was used as negative control. B:Beads, FT: Flow-through. (C) Graph represents the levels of his6x-Spindly relative to MBP-Zwilch from at least three independent pull down experiments. The values obtained for his6x-SpindlyWT were set to 1. Data information: XXXX Data are shown as mean ± SD. Scale bar: 5 μm. (D) Selected frames from live imaging analysis of SpindlyS499A-EGFP or SpindlyS499D-EGFP behavior during KT alignment in Drosophila S2 cells expressing PoloWT-mRFP. SpindlyWT-EGFP expressing cells were used as control. All Spindly-EGFP constructs were resistant to the RNAi targeted against endogenous Spindly. Insets show magnifications of the outlined regions that highlight KTs that delay in congressing to the spindle equator (yellow dashed line). Time 0 is the first shift in KT direction from poleward to anti-poleward movement. (E) Graph represents the variation in SpindlyWT-EGFP, SpindlyS499A-EGFP and SpindlyS499D-EGFP levels at KTs highlighted in (D) and its correlation with efficiency of alignment. All values were determined as described in Fig.6D. Time 0 is the first shift in KT direction from poleward to anti-poleward movement (n≥5 KTs from at least 4 cells for each condition, n≥2 independent experiments). Data information: XXXX Data are shown as mean ± SD. Scale bar: 5 μm.
A) Proteasomal chymotrypsin and cathepsin activity in lysates from young (Y) and old (O) I90 cells was determined using specific fluorescence probes, as described in the Material and methods section.Figure 5(A) Proteasomal chymotrypsin and cathepsin activity in lysates from young (Y) and old (O) I90 cells was determined using specific fluorescence probes, as described in the Material and methods section. Values are expressed are mean±s.e.m. *P0.05 and **P0.01 versus young, n=3. (B) Real‐time PCR analysis of LC3, WIPI1 and SQSTM1 mRNA levels in young and old I90 cells. Depicted is the mean expression ratio (log2) ±s.e.m. of target genes in old cells relative to young cells. **P0.01 and ***P0.001 versus young, n=3. (C) Immunoblot analysis of LC3 and SQSTM1 in young and old I90 cells. (D) Young and old I90 cells were treated for 2 h with bafilomycin A1 (BafA1, 2 μM) or DMSO as control followed by immunoblot analysis of indicated proteins. (E) Immunoblot analysis of WIPI1 expression in young and old I90 cells. (F) Indirect immunofluorescence staining of endogenous LC3 (green), SQSTM1 (red) and WIPI1 (white) in I90 cells of young and old age. DAPI (blue) was used to stain DNA. Representative pictures are shown. Bar: 20 μm. Diagrams show percentage of cells with indicated characteristics counted as in Figure 3E. (G) Old and young I90 cells were treated for 1 h with DMSO as control (C), lactacystin (L, 2 μM) or NH4Cl (20 mM) plus leupeptin (Leu, 5 μM) (N, NH4Cl/Leu). Western‐blot analyses were performed for detection of indicated proteins. In the diagram (right panel), levels of polyUb‐proteins and SQSTM1 are depicted after normalisation to corresponding Tubulin levels. Values are expressed as mean±s.e.m. *P0.05 versus old control, #P0.05 versus young control, n=3. (H) I90 cells of old (upper panel) and young (lower panel) age were transfected with atg7 or nonsense (nons) siRNA. After transfection for 4 days , the cells were treated with NH4Cl/Leu or DMSO for 1 h followed by fractioning of cell lysates in TritonX‐100 (TX‐100) soluble and insoluble material. Equal protein amounts of both fractions were directed to immunoblot analysis for analysis of indicated proteins. Gapdh and Histone H3 were used as loading controls of soluble and insoluble fractions, respectively. (I) Transmission electron microscopic analysis of young (a) and old (b) I90 cells. Magnifications of marked areas in (a) and (b) are shown in (c) and (d), respectively. An arrow indicates an autolysosome, and arrow heads indicate autophagosomes. M, mitochondrion. N, nucleus.
a) A HeLa cell expressing EGFP-Ub and TagRFP-p62, and labelled with AlPcS2a, was 635-nm illuminated within the white square region, resulting in ubiquitination of and p62 recruitment to the photodamaged lysosomes, as shown in b.f4(a) A HeLa cell expressing EGFP-Ub and TagRFP-p62, and labelled with AlPcS2a, was 635-nm illuminated within the white square region, resulting in ubiquitination of and p62 recruitment to the photodamaged lysosomes, as shown in b. (b) Magnified view of the 635-nm illuminated cell region in a. Ubiquitin and p62 initially distributed uniformly within the region (t=0 min, top panels), but formed distinct foci after light activation (bottom panels). (c) A HeLa cell expressing EGFP-LC3B and lamp1-RFP, and labelled with AlPcS2a, was 635-nm illuminated within the white square region, resulting in LC3 recruitment onto photodamaged lysosomes, as shown in d. (d) Magnified view of the 635-nm illuminated cell region in c. LC3 is initially distributed away from lysosomes (lamp1, t=0 min, top panels), but appears on lamp1-positive structures 40 min after light activation (bottom panels). Scale bars, 10 μm (a,c); 3 μm (b,d).
RPA is SUMO modified on two major sites.Figure 1Diagrams showing the domain organization of SUMO (Smt3), Siz2, Rfa1, Rfa2 and Rfa3. Orange circle indicates known (Rfa1 and Smt3) or predicted (Siz2) SUMO modification sites. Dash lines represent truncation mutants used in this study in addition to the full-length proteins. SYPRO-stained SDS-PAGE showing purified recombinant SUMO, E1∆CT, E2, Siz2 and RPA proteins. Mass in kilodaltons is annotated for all bands for the molecular weight marker. SUMO conjugation assays performed under multiple turnover conditions using wild-type or mutant forms of RPA, SUMO and Siz2. Only the 80-minute time points are presented to highlight the formation of Rfa1-SUMO and Siz2-SUMO species. The complete time series are presented in Appendix Fig S1. Bands for Mw marker are fully annotated in Figure 1B.
Figure 7Numerous cellular stresses and/or increased neuronal activity mechanistically converge to increase phosphorylation of eif2α, and thereby drives the non-AUG dependent translation of DPRs from C9orf72 NRE containing transcripts. Phosphorylation of eif2α is a key component of the ISR and is regulated by various ISR-related kinases. One of these kinases, PERK, also shows increased levels in response to cellular stressors, neuronal excitotoxic stress, or aberrant neuronal excitation. Perturbing or blocking specific components of the ISR, using compounds such as the FDA approved trazodone or dibenzoylmethane (1,3DBM), reduces stress-induced C9orf72 NRE-linked DPR levels and provides promising therapeutic strategies to combat NRE-linked neurodegeneration.
Binding of the SPIN90 ARM domain to the ARPC4 long helix may stimulate movement of Arp2 into or toward the short pitch conformation.Figure 5 A. Plot of maximum polymerization rate versus concentration of the small molecule Arp2/3 complex inhibitor CK-666. Reactions contained 3 μM 15% pyrene labeled actin, 50 nM Bos taurus Arp2/3 complex and the indicated concentrations of CK-666  B. Cartoon of "rotation model" for movement of Arp2 into the short pitch conformation. White arrow shows the approximate position of a hinge point for the rotation  C. Structural superposition showing the proposed hinge movement in ARPC4 and the position of bound SPIN90 relative to the hinge. Residues 156-168 from ARPC4 and residues 247-260 from ARPC2 in an inactive crystal structure (4JD2) were overlaid with the same residues from the molecular dynamics (MD) short pitch model of Arp2/3 comple ARPC4 from 4JD2 (blue) and from the MD simulation (cyan) are shown. The helical axis of the long alpha helix (αD) in ARPC4 is shown as a black line in the 4JD2 structure and as a green line in the MD simulation structure. The red line represents the helical axis of helix αD in ARPC4 in the SPIN90-bound co-complex overlaid as described above. For clarity, the cartoon representation of the ARPC4 subunit in the co-complex structure is not shown her The orientation of this panel is similar that depicted in panels (B and D)  D. Conceptual model of SPIN90-bound Arp2/3 complex in the short pitch conformation. The model was made by superposing subdomains 1 and 2 of Arp3 from the co-complex structure onto an actin subunit from a high-resolution structure of an actin filament (3J8I Arp2 (red ribbon) was then moved into the short pitch position based on the filament structure. Arp2 in its original splayed position (as observed in the crystal structure) is shown in grey ribbon representation. Shortest Cα-Cα distances from SPIN90 to either subdomain 2 or subdomain 4 in Arp2 in the hypothetical short pitch conformation are indicated  E. Comparison of the position of Arp2 relative to Arp3 in the short pitch arrangement (grey Arp2), the inactive splayed arrangement (red Arp2), and the SPIN90 bound x-ray crystal structure (cyan). The short pitch arrangement of Arp2-Arp3 was modeled by overlaying subdomains 1 and 2 of Arp3 on the same subdomains in an actin subunit in the actin filament structure 3G37(Murakam ), and then superposing Arp2 from the 4JD2 structure onto the actin subunit in the short pitch position. The SPIN90-bound complex was then overlaid onto Arp3 subdomains 1 and 2 to determine the relative position of Arp2 in the SPIN90-bound complex. Blue line shows a potential trajectory of the center of mass of Arp2 when it moves from the splayed to the short pitch position