EMBO/sd-character-level-ner huggingface dataset:
Figure 3A. Uptake of FITC-insulin and CAV1 expression in primary human umbilical venous ECs (HUVECs) upon Notch induction. Scale bar 20 µm. B. Uptake of FITC-insulin and CAV1 expression in (HUVECs) upon Notch blockage. Scale bar 20 µm. C. Representative western blot of AKT phosphorylation in gastrocnemius muscle 7 minutes after intravenous injection of PBS or 1.5 U/kg insulin in control (n=3) and Rbpji∆EC (n=3) mice five weeks after recombination. D. Densitometric analysis of western blot in C. n=3, data represent mean ± SEM, unpaired t-test. E. Representative western blot of AKT phosphorylation in liver 7 minutes after intravenous injection of PBS or 1.5 U/kg insulin in control (n=3) and Rbpji∆EC (n=3) mice five weeks after recombination. F. Densitometric analysis of western blot in E. n=3, data represent mean ± SEM, unpaired t-test.
Figure 5To assess the effects of IFT restoration on TZ assembly, ts-che-3 mutant animals were shifted from the restrictive to the permissive temperature at different time points during their development (days 1, 4 and 8 of adulthood). The TZ markers MKS-6 (a) and NPHP-4 (b) show strong ectopic ciliary accumulations when raised at the restrictive temperature until day 1 of adulthood. After a shift to the permissive temperature for 24 hours, day 2 adults show "repaired" TZ protein localisation resembling that of wild-type animals. For both markers, Day 4 adults (maintained at the restrictive temperature) show less ectopic TZ protein localisation than Day 1 adults. The correction of ectopic localisation following shift to the permissive temperature for 24 hours was reduced in day 4 (to day 5) of adulthood compared to that observed in day 1 (to day 2) adults. For MKS-6, but not NPHP-4, day 8 adults (maintained at the restrictive temperature) also display less ectopic accumulation than day 1 adults. The correction of ectopic TZ protein localisation in animals shifted to the permissive temperature after 8 days of adulthood is also much smaller in magnitude for both markers, indicating that this repair is most effective in day 1 adults. In summary, as the adults age, ectopic TZ protein localisation is reduced, while the ability to correct this phenotype by restoring IFT function al
Figure 6(A, B) The schematic illustrates the screening process of 702 drugs related to the mitochondrial respiratory capacity assessed by Seahorse XFe96 in C2C12 myocytes. Palmitate is used as a substrate. A total of 41 hits were identified. Maximum respiration (Max R, % of untreated cells) of compounds categorized by their therapeutic use (B). (C, D) The schematic illustrates the screening process of the 41 hits on FFA β-oxidation using 9,103H(N) palmitic acid as a substrate in C2C12 myocytes. Ten enhancers were identified. D. Correlation between the maximum respiration (x) and palmitate β-oxidation (y). Colors identify classes of pharmaceuticals as in B. Edaravone was selected as a hit. E. Representative respiratory profile of C2C12 myocytes treated (green trace) or not treated (black trace) with 2 µM edaravone. The CPT1 inhibitor etomoxir was used as a negative control. OCR, oxygen consumption rate; OL, oligomycin; DNP, 2,4-dinitrophenol; Rot, rotenone; Ant A, antimycin A. Quantification in right histogram. Bars are the mean ± s.e.m. of 12 replicas/condition. Data information ;0.05 when compared to wt by ANOVA and Student's t-test. (F, G) FFA β-oxidation in C2C12 myocytes transfected with CRL or ATPIF1H49K plasmids (F) and in primary myotubes from wt and LowOXPHOS mice (G), treated (green bars) or not with 2 µM edaravone. Bars are the mean ± s.e.m. of n= 3 experiments, 9 replicas/condition. Data information: *p <0.05 when compared to wt by ANOVA and Student's t-test.
Figure 5ID2 interaction with DNA is proposed to promote a dynamic equilibrium where ID2 can exist in both an open and a closed conformation. FANCD2 ubiquitination by Ube2T and the FA-core complex, within a closed ID2 conformation status, can shift this equilibrium in favour of the closed conformation, with the action of USP1-UAF1 counteracting such shift. When FANCI is also ubiquitinated by Ube2T and the FA-core complex, both ubiquitins are largely resistant to USP1-UAF1 deubiquitination, and thus the doubly ubiquitinated ID2 can remain in the closed conformation. The latter conformation has a tighter DNA affinity and hence locks ID2 onto DNA. The R1285Q mutation on FANCI likely restricts formation of a closed ID2 conformation, which in turn negatively impacts on ID2 ubiquitination and locking onto DNA.
Figure 1Plasmid reporter constructs developed to monitor non-AUG-dependent translation using the photoconvertible fluorescent protein, Dendra2. The endogenous C9orf72 5ʹ gene region is immediately upstream and Dendra2 is immediately downstream of the NRE insertion site. Each construct is modified +1 or +2 base pairs to fluorescently monitor all individual ORFs that are in-frame with the Dendra2 with an n-terminal HA affinity tag. All ORFs for the sense non-AUG-dependent translation of the (G4C2)n~188, DPR coding for (GA), (GP), (GR) can be detected and quantified using a filter-trap binding assay probing for the c-terminal HA tag of the C9 DPR reporter construct. The (GA) ORF is the most abundantly detected DPR and GR the lowest in HEK293T cell 24 h post transient transfection of the C9 DPR reporter construct. n = 5. The C9orf72 NRE non-AUG-dependent reporter constructs recapitulate localization patterns and pathological features identified in C9orf72 NRE models. The Dendra2 (green) c-terminal fusion was used to monitor cellular localization and fluorescence intensity for each DPR translated from the G4C2 strand in HEK293T cells transiently cotransfected with an NES-mIFP-mIFP-NES (NES-mIFP) fusion protein cytoplasmic marker. C9 DPR reporter constructs show cell-type variability in the number of cells that are DPR positive and the overall DPR levels. Transient cotransfections of HEK293T cells robustly undergo G4C2 -Dendra2 non-AUG-dependent translation compared to NSC34 or rat primary cortical neurons. DPR positive cells and DPR fluorescent intensity were analyzed and calculated 24 h post-transfection only in cells expressing the co-transfected NES-mIFP construct. DPR fluorescent intensity was normalized to NES-mIFP fluorescent intensity to adjust for cell specific AUG-dependent translation levels; represented as fold-change relative to HEK293T. HEK293T: n = 5 with m > 500 cells analyzed per n. NSC34: n = 5 with m > 500 cells per n. Rat primary cortical neuron: n = 5 with m > 40 cells per n. (***P < 0.001, **P < 0.01, *P <0.05) See also Figure EV1-2.
Figure 1Immunostaining from Daam2LacZ/+ animals with Aldh1l1-EGFP reporter indicates that Daam2 (LacZ+, Red) is expressed in astrocytes (Green). (A) Daam2 expression is observed in multiple brain regions at postnatal day 21. Immunostaining from Daam2LacZ/+ animals with Aldh1l1-EGFP reporter indicates that Daam2 (LacZ+, Red) is expressed in astrocytes (Green). (B) Expression of Daam2 in cortex persists throughout development (P7 to adult). C. Quantification of Daam2-expressing astrocytes (LacZ+) with Aldh1l1-EGFP labeled astrocytes in cortex during development. Data is presented as mean ± SEM. Two brain sections, N=4 for each age. D. Schematic of the generation of astrocyte specific deletion of Daam2 in Aldh1l1-Cre and Daam2-floxed mice. Quantitative RT-PCR in FAC-sorted astrocytes confirms significant reduction of Daam2 transcript from Daam2 cKO mice. Experiments is performed as duplicates from N=3-4. Data is presented as mean ± SEM. Student's t-test is used for statistical analysis. ***p<0.001. E-F. Quantification of astrocyte marker, Sox9+ cells in cortex (E) and OB (F) of Daam2 cHet and Daam2 cKO mice. Data is presented as mean ± SEM. 4-6 brain sections, N=4. Student's t-test is used for statistical analysis. G. Representative images of astrocytes labeled with Aldh1l1-EGFP reporter in cortex and OB of Daam2 cHet and cKO mice at postnatal day 28 (P28). Scale bar: 50 μm. H-K. Overall complexity of astrocytes with Aldh1l1-EGFP reporter was measured by Sholl analysis (Two-way ANOVA) and total process length (Student's t-test). Data is presented as mean ± SEM. n= 6-10 cells from N=3-5 mice per genotype, *p<0.05, p****<0.0001
Figure 3A. Pure tone audiogram recorded from one of the probands, showing severe to profound bilateral asymmetric hearing loss. B. Capillary sequence traces from fathers, mothers and probands, showing the heterozygosity of the parents for the two separate variants and the compound heterozygosity for each proband. C. ConSeq (http://conseq.tau.ac.il/) analysis of the residues; the locations of the three human variants (p.Ala160Thr, p.Met163Leu and p.Ala224Val; boxed in black) are average to highly conserved. "b" and "e" indicate buried and exposed residues (according to the neural network algorithm) and "f" and "s" indicate predicted functional and predicted structural residues. The GRAM and WW binding domains (WW1 and WW2) are marked by blue and green boxes. The GRAM domain is thought to be an intracellular protein- or lipid-binding signalling domain, and may play an important role in membrane-associated processes. D. Alignment of the protein sequence from a range of vertebrates. The human variants are boxed in black.
Figure 5A. Aged mice (>12 months) were injected i.v. with siScr or siMST. Twenty-four h after siRNA injection a 70% PH was performed and remnant/regenerating liver tissue was harvested 40 h later. p.i. of the livers was calculated by IHC staining of Ki67 positive hepatocytes and based on the percentage of positive cells liver were classified (-) non regenerating or (+) regenerating. Non-surviving animals were sacrificed before the 40 h end time point. "n" indicates the number of animals per group.B. Ki67 immunostaining of the regenerating aged mice livers, 40 h post PH. Sections were scanned using Histotech Pannoramic MIDI Scanner and quantitated with Quant Center 2.0 software. p.i. values are provided on the image. C. Representative photomicrograph of an aged liver treated with siMST and stained with H&amp;amp;E and by IHC for CK19 and Ki67. Arrows are indicating ductal regions with no signs of reaction or oval cell expansion. D. Quantitative RT-PCR analysis of RNA isolated from mouse liver for FoxM1B, Birc5 and cyclin B1 at 0 and 40 h post PH. Log2 fold change was calculated using non-transfected/non-resected mouse liver as a control. Representative results of a single experiment with n = 6 animals per group are shown. Paired, two-tailed Student t-test was used to calculate the significant change in signals between liver tissues at the time of resection (0h) compared to 40 h post PH. E. Percentage of Ki67 positive hepatocytes in aged and aged + siMST 40 hours post resection. Representative results of a single experiment with n = 6 independent aged animals for group are shown. Two-tailed Student t-test was used to calculate the significance of percentage of positive hepatocytes of siMST treated animals compared to non-treated controls. F. Endpoint liver weight was taken of the remnant lobe and expressed as a percentage to total body weight. Representative results of 1experiment with n = 6 independent animals for group expect for control group were n = 6 are shown. Estimated liver-to-body weight ratio before PH in mice = 3.85 ± 0.05 (SD). Unpaired, two-tailed Student t-test was used to calculate the significance of each aged group in comparison to the control young group. G. Hepatocyte geometric diameter was calculated (see material and methods). Representative results of 1 experiment with n = 3 independent animals per group. Unpaired, two-tailed Student t-test was used to calculate the significant change in hepatocyte size in liver tissue with and without siMST 40 h post PH.
Figure 1(A) Western blot of autophagy proteins LC3 and p62 at various time points after infection of human NSCs with strains MR766 or Paraiba. (B-C) Quantitative representation of LC3-II and p62 protein levels during ZIKV infection at indicated time points. Mean ± SEM, n=3 biological replicates. (D) GFP-LC3 puncta of mock- and ZIKV-infected hNSCs. Scale bar = 100 µm. (E) Tandem EGFP-LC3 and mCherry punctae co-localization in control and ZIKV infected hNSC. Scale bar = 100 µm, Boxed magnified images with scale bar = 50 µm (F) Number of GFP-LC3 puncta in MR766 and Paraiba infected hNSCs, n =>50 cells. Box plots show the mean and the smallest; largest values in whiskers represent the 10th and 90th percentiles in 50 randomly selected cells per group; and solid horizontal line indicates median. ***p < 0.001 by Student's t test. (G) RT-qPCR of ZIKV RNA in hNSCs incubated with vehicle or 100 nM rapamycin, 20 μM chloroquine, 10 nM bafilomycin A1, or 5 mM 3-methyladenine for 24 h. Mean ± SEM of 3 biological replicates. *p < 0.05, ***p < 0.001 by Student's t test. (H) GFP-LC3 puncta in ZIKV with or without rapamycin and 3-MA treated hNSC cells. Cells were treated with or without rapamycin (100 nM) or 3-methyladenine (5 μM). Box plots show the mean and the smallest; largest values in whiskers represent the 10th and 90th percentiles in 50 randomly selected cells per group; and solid horizontal line indicates median. *p < 0.05, **p < 0.01, ***p < 0.001 by Student's t test. (I) RT-qPCR of ZIKV RNA in infected hNSCs transfected with siRNAs targeting LC3, BECN or ATG9A 48 h post infection. Mean ± SEM of 3 biological replicates. *p < 0.05 by Student's t test. (J) Venn diagram of genes significantly downregulated in HeLa cells treated for 4 h with perifosine (50 μM), rapamycin (50 nM), and resveratrol (100 μM), compared with ZIKV-infected hNSCs (ZIKV data from (Tang et al., 2016)). (K) Sankey diagram of pathways associated with genes commonly enriched in ZIKV-infected hNSCs and perifosine-, resveratrol-, and rapamycin-treated HeLa cells. The width of each band is proportional to the number of genes in the group/pathway. (L) Transmission electron microscopy of hNSCs infected by ZIKV MR766 and Paraiba strains. Mitochondria (MT), endoplasmic reticulum (ER), Golgi bodies (GB), ribosomes (RS), phagophores (PH), lipid droplet (LD). Scale bars, first row, 1 μm (left), 500 nm (control right); 200 nm (Right).
Fig.Figure 4 A. Average enrichment of Upf1-23 and Upf1-decapping components in purified Upf1-TAP in the presence (black bars) and absence (blue bars) of NMD4. Pink and dark blue vertical lines highlight proteins of the Upf1-23 and Upf1-decapping complexes respectively. White dots correspond to individual enrichment values obtained in replicate experiments, 6 for Upf1-TAP and 3 for the nmd4Δ condition. Error bars correspond to SD  B. Average enrichment of Upf1-decapping components in Nmd4-TAP in the presence (black bars) and absence (blue bars) of UPF1. White dots correspond to individual enrichment values obtained in replicate experiments, 4 for Nmd4-TAP and 2 for the upf1Δ condition. Error bars correspond to SD  C. Schematics of Upf1 fragments used for the in vitro interaction assay; yUpf1-HD is the helicase domain (220-851) of the yeast Upf1 protein, hUpf1-HD is the helicase domain (295-914) of human Upf1 (not to scale)  D. CBP-Nmd4 was mixed with hUPF1-HD or yUPF1-HD (all the proteins overexpressed in E. coli and purified). Protein mixtures before (input, 20% of total) or after purification on calmodulin affinity beads were separated on 10% SDS-PAGE (w/v) acrylamide gels  E. Upf1 interaction with a 30-mer 3' biotinylated RNA fragment was tested by mixing purified Upf1 helicase domain with Nmd4 and testing the fraction of recovered protein on streptavidin beads after washes with 150 mM and 300 mM NaCl containing buffer solution
Figure 1A. MS/MS analysis of peptides corresponding to amino acids 380-399 of STAT2. 293T cells expressing flag-tagged STAT2 were used. B. Abundance of T387 phosphorylated STAT2 in 293T cells expressing flag-tagged STAT2. C. Location of T387 in STAT2. D. Conservation of T387.
Trafficking of MDVs to the lysosome is dependent on Tollip.Figure 7(A) A subset of TOM20+ve/PDH-ve MDVs are trafficked to the lysosome. siRNA knockdown of Tollip was conducted in SH-SY5Y cells over a 72 hours period prior to treatment with 5 μM antimycin A/10 μM oligomycin B (AO) or 25 μM antimycin A (AA) for 2 hours. Cells were then fixed and stained with antibodies specific to LAMP1 (green), PDH E2/E3bp (red) and TOM20 (blue). Arrows denote TOM20+ve/PDH-ve MDVs colocalising with LAMP1, whereas arrowheads denote MDVs that do not colocalise with LAMP1. (B) From immunofluorescence images, the total number of TOM20+ve/PDH-ve MDVs were counted as well as the number of TOM20 MDVs that colocalised with LAMP1 to calculate the % of TOM20 positive MDVs colocalising with LAMP1 per cell (n = 25-32 cells per condition from 3 independent experiments). (C) siRNA knockdown was performed in SH-SY5Y cells, then 18 hours prior to fixation cells were transfected with GFP-Tollip wild-type (WT), ∆Nterm, CUEmut, or R78A. Cells were fixed and immunostained for PDH E2/E3bp (red) and TOM20 (blue). Arrowheads denote TOM20+ve/PDH-ve MDVs. (D) Quantification of TOM20 MDVs per cell was performed by eye (n = 11-14 cells per mutant from 2 independent experiments). Cells expressing a GFP-Tollip construct were identified using the GFP channel. Surrounding cells not expressing GFP were counted as the Tollip siRNA only population.
Figure 6A-C Immunolabelling of γ-Tubulin and SYCP3 during prophase I in control, Plk1(∆/∆) and BI2536-treated spermatocytes. Images are show for pachytene, diplotene and diakinesis in Control (A), Plk1(∆/∆) (B) and BI2536-treated spermatocytes (C). First columns show the double immunolabelling of PCM -PCNT- (green) and SYCP3 (red), and second columns shows γ-Tubulin (γ-Tub, magenta) and SYCP3 (red). Chromatin has been stained with DAPI (blue). Amplifications at 300% magnification for all labellings are displayed for each image. Data for BI2536 represent results for 8h at 100µM treatment on organotypic cultures of seminiferous tubules (D) Immunolabelling of CETN3 and γ-Tubulin in control and Plk1(∆/∆). Double immunolabelling of CETN3 (red) and γ-Tubulin (green) in pachytene (a-b) and diakinesis (c-d). Chromatin has been stained with DAPI (blue). (E) Quantification of the number of cells without γ-Tubulin at centrosomes (γ-Tub -) in pachytene and diplotene spermatocytes in control, Plk1(∆/∆) and BI2536-treated spermatocytes. Data represent average of two biological replicates per condition.
Figure 2A. Volcano plot of gene expression changes in bEnd.3 cells following 24h of treatment with 30nM F4L5.13. Genes with significant changes in expression level are highlighted in blue and genes co-regulated by treatment with 30nM NDP for 24h are highlighted in red. Statistical analysis was performed using the DESeq2 R package and adjusted p-value (Wald test) threshold less than 0.1 was used. Labelled genes are associated with enriched GO biological processes indicated below. B. Enrichment Map of overrepresented GO biological processes and pathways in genes regulated by F4L5.13. Node sizes represent the number of genes belonging to individual terms (g:SCS FDR adjusted p-value < 0.05, gProfiler). C. Venn diagrams showing overlap in differentially expressed genes following treatment with F4L5.13 or NDP. D D. Heatmaps of differentially expressed genes mapping to the indicated biological processes and pathways across treatment groups in RNA-seq experiment. Mean of n=3 samples per group.
Fig.Figure 6The SARS-CoV-2 spike protein S consists of the two subunits S1 and S2 and binds through S1 to ACE2 on host cells, which is required for viral fusion with host cells (A) (A) TMPRSS2 may cleave S in trans and allow fusion of SARS-CoV-2 with the host cell plasma membrane. In contrast, ADAM17 may either cleave S in trans at the plasma membrane or may cleave it once the fusion peptide of S has inserted into the host plasma membrane after endocytosis, which is conceptually similar to a cleavage in cis. After ADAM17 cleavage fusion of SARS-CoV-2 with the host cell is likely to happen after endocytosis. Inhibition of ADAM17 reduces viral uptake and fusion. , B. The SARS-CoV-2 spike protein S consists of the two subunits S1 and S2 and binds through S1 to ACE2 on host cells, which is required for syncytia formation (B). (B) For syncytia formation, the S protein expressed in an infected cell binds at the plasma membrane to ACE2 on another cell. This requires S priming by TMPRSS2 or ADAM10. The priming step may happen in trans while SARS-CoV-2 is still bound to ACE2 or after insertion of the S2 subunit - which contains the fusion peptide - into the membrane of the ACE2-expressing cell, which may be seen as a cleavage in cis. Inhibitors of TMPRSS2 (e.g. camostat) or ADAM10 reduce cell fusion and syncytia formation. The figure was created with BioRender.
Figure 5The relative enrichment of H3K4me3 (A) were evaluated in SH-SY5Y cells expressing dCas9-5xGCN4 with (sgA-dCas9) or without scFV-sfGFP-JARID1A (sgA-dCas9-JA). A ChIP data demonstrated a significant decrease of H3K4me3 at the SNCA promoter. The relative enrichment by H3K4me3 was normalized to the respective inputs. Four independent repeats were performed. *P < 0.05. Data were analyzed using non-parametric t-test followed by Mann-Whitney post-hoc corrections. Two-tailed p-values were calculated. Data information: All data are presented as mean ± SEM. The corresponding α-synuclein levels (B were evaluated in SH-SY5Y cells expressing dCas9-5xGCN4 with (sgA-dCas9) or without scFV-sfGFP-JARID1A (sgA-dCas9-JA). B The levels of α-synuclein (α-SYN) in SH-SY5Y cells were evaluated using RT-PCR. The level of α-SYN was normalized to β-actin expression. A significant reduction of α-SYN in cells expressing scFV-sfGFP-JARID1A was observed. Four independent repeats were performed. *P < 0.05. Data were analyzed using non-parametric t-test followed by Mann-Whitney post-hoc corrections. Two-tailed p-values were calculated. Data information: All data are presented as mean ± SEM. The corresponding α-synuclein levels C) were evaluated in SH-SY5Y cells expressing dCas9-5xGCN4 with (sgA-dCas9) or without scFV-sfGFP-JARID1A (sgA-dCas9-JA). C α-SYN levels in SH-SY5Y cells were also evaluated using western blot. The levels of α-SYN was normalized to β-actin. Significant reduction of α-SYN was observed in cells expressing JARID1A. Three independent repeats were performed. Percentage of reduction is shown in the graph. **P <0.01. Data were analyzed using non-parametric t-test followed by Mann-Whitney post-hoc corrections. Two-tailed p-values were calculated. Data information: All data are presented as mean ± SEM.
Figure 1A SARS-CoV-2 genome annotation. Two-thirds of the viral RNA, mainly located in the first open reading frame (ORF 1a/b), encodes 16 nonstructural proteins (NSPs). The rest of the viral genome encodes four main structural proteins, namely, the spike (S), envelope (E), nucleocapsid (N), membrane (M) proteins, and several accessory factors. SARS‐CoV‐2 enters the cell primarily via binding to ACE2, followed by its priming by TMPRSS2. In addition to ACE2 and TMPRSS2, other potential SARS-CoV-2 receptors, proteases, and cofactors for infection are indicated. B Experimental workflow for the establishment of a SARS-CoV-2-host interactome to identify potential drug targets and treatment. C Virus-host protein interaction networks were constructed by AP-MS (top) and BioID-MS (bottom). Only protein-protein interactions (PPIs) with a Bayesian false discovery rate (BFDR) ≤0.01 (assessed by SAINTexpress) are shown.