EMBO/sd-character-level-ner huggingface dataset:
Fig.Figure 1(A) Blood ammonia in 8-9-week-old C57BL/6J wild-type (WT) mice (n=8) and Becn1F121A mice with constitutive activation of autophagy (n=8) at baseline and 30 min after i.p. injection of NH4Cl (10 mmol/kg). **p<0.01 (Unpaired t-test). ns: not statistically significant difference. (B) Urinary orotic acid of 12-week-old spf-ash mice treated with TB-1 (15 mg/kg, i.p) or vehicle at various times as indicated by the arrows (n=5 mice/group). *p<0.05 (Two-way ANOVA). (C; D) Western blotting and densitometric quantifications of autophagy markers (LC3II: autophagosomes; p62 and NBR1: cargo receptors) in livers of spf-ash mice harvested after ten days of treatment with TB-1 or vehicle. β-actin was used as loading control. n=5 mice/group. **p<0.01, *p<0.05 (Unpaired t-test). (E) Survival curves of spf-ash mice fed with a high protein diet (HPD) for ten days and treated with TB-1 alone or combined with scavenger drug and L-Arginine, or treated with scavenger drug and L-Arginine, or left untreated. WT control were included (n=5/group). ***p<0.001 (Log-rank Mantel-Cox test). Data information: Treatments Scavenger (Na-benzoate 250 mg/kg/day, i.p.) and L-arginine (L-Arg, 250 mg/kg/day, i.p.); TB-1 (15 mg/kg every 2 days, i.p). WT mice were age-, gender- and strain (C3H)-matched. All values are shown as averages ± S.E.M. (F) Blood ammonia levels determined after 4 days under HPD (n=5 mice/group); ***p<0.001, **p<0.01, *p<0.05 (One-way ANOVA). Data information: Treatments Scavenger (Na-benzoate 250 mg/kg/day, i.p.) and L-arginine (L-Arg, 250 mg/kg/day, i.p.); TB-1 (15 mg/kg every 2 days, i.p). WT mice were age-, gender- and strain (C3H)-matched. All values are shown as averages ± S.E.M.
Figure 2A. Expression of CSR1 to 35 was evaluated by qRT-PCR in RNA isolated from control cells or cells infected with a moi 0.3 of HCV for 6 days. GAPDH levels were also measured and used as a reference. Fold increase of each CSR in infected versus control cells is indicated at the top of each bar. A line has been drawn to mark the position of 7 fold, our arbitrary cut-off value. Black bars, candidates expressed to good levels that increase more than 7 fold; white bars, candidates expressed poorly according to the parameters described in Appendix Table S.2; grey bars, candidates expressed to good levels induced less than 7 fold. CSR28 bar was shaded to highlight that this candidate was not evaluated further. B. HuH7 cells were infected with a moi 0.3 of HCV for 0, 12, 24, 48, 72h and analyzed as in A. The experiments were performed in triplicate at least twice and each value shows the average of the results (n=6). Error bars indicate standard deviations and asterisks mark significant differences obtained with a two-tailed non-parametric Mann-Whitney U-test (ns, non-significant, * denotes p<0.05, ** p<0.01).
A model of the mechanism underlying the effect of intronic piRNAs on NPF1 signaling.Figure 8When piRs-3-I2 and piRs-3-I3 mimics were injected, the decrease in pre-NPF1 levels and increase in mature NPF1 levels led to an increase in food consumption. When piRs-3-I2 and piRs-3-I3 inhibitors were injected, the increase in pre-NPF1 levels and decrease in mature NPF1 levels led to reduced food consumption. The purple region represents exons, and the black region represents introns.
Figure 3A. EVs isolated from MH-S AMs and A549 AECs were co-incubated in parallel with Luc-WSN/33 (MOI = 0.5) in MDCK-SIAT1 cells. Data represent mean luminescence AUC from individual wells normalized to the mean of the corresponding control condition from 3 independent experiments. B. EVs isolated from MH-S cells treated with increasing concentrations of CSE were co-incubated with Luc-WSN/33 in MLE-12 cells. Data represent mean luminescence at 12 h post infection from individual wells normalized to the mean of untreated AM-EVs from 3 independent experiments per condition.
| NTRAS controls vascular permeability by regulating TJP1 splicing.Figure 5Model depicting the molecular and physiological barrier function of NTRAS. By sequestering hnRNPL, NTRAS promotes TJP1 exon 20 inclusion and expression of TJP1α+, this way maintaining endothelial barrier function. In the absence of NTRAS, hnRNPL represses exon 20, thereby favoring TJP1α- expression and hyperpermeability. TJP1 alternative exon 20 and the alpha domain are highlighted in green, hnRNPL binding motifs are depicted as red boxes.
Figure 7Western blot analyses of expression levels of TTF-1 and Mucin 5B in H1299control and H1299RASSF1A cells with band intensity quantification ratio determine by ImageJ analyser. Representative images of H&E and immunohistochemical staining for lung differentiation markers TTF-1 and Mucin 5B for at least 2 independent regions of n=4 H1299control and n=2 H1299RASSF1A primary tumours at day 17 with quantification (right) of TTF-1 based on nuclear intensity. Scale bars 100µm. Statistical significance was determined by Student's t-test (2-tailed). Error bars represent the mean ± S.E.M Representative images of immunofluorescence staining of spheroids grown in collagen matrix (2mg/ml) for differentiation markers TTF-1 (Green) and Mucin 5B (Red). Scale bars 50µm, Zoom 10µm. Bright field images of H1299control and H1299RASSF1A spheroid differentiation on Matrigel matrix after 24 hours when 3D spheroids were seeded on three-dimensional substrate. Scale bars 200µm.
Figure 6 A, B ChIP-qPCR analysis of Scc1 association with early replication origin ARS305 or ARS607. HA-tagged Scc1 interaction with chromatin was monitored in wild type (Y39) and scc2-4 (Y4366) cells. Error bars represent mean value ± standard deviations of mean (n=3) C, D ChIP-qPCR analysis of Pol2 association with early replication origin ARS305 or ARS607. HA-tagged Pol2 interaction with chromatin was monitored in wild type (TB067) and scc2-4 (TB068) cells. Error bars represent mean value ± standard deviations of mean (n=3) E Cohesin loading complex is required for completion of chromosome replication after MMS treatment. Intact chromosomes were isolated from wild type (Y39) and scc2-4 (Y4366) cells cultivated at permissive temperature (25°C) and processed for PFGE. M, MMS  F Quantification of data presented in E. Error bars represent mean value ± standard deviations of mean for the intensity values of three distinct chromosome bands representing chromosome XII or III (n=3). Values for G1-synchronized cells were set to 100  G Sensitivity of scc2-4 to DNA damaging agents at permissive temperature (25°C) or semi-permissive temperature (30°C). Logarithmically growing cultures of wild type (Y39), and scc2-4 (Y4366) strains were 10-fold serially diluted and plated onto solid YPD containing indicated concentrations of DNA damaging agents
Figure 5Expanded sense G4C2 and antisense C4G2 repeats are translated into polyGA, polyGR and polyPG DPR proteins through initiation to near-cognate codons or a cognate ATG codon embedded in a poor Kozac sequence. Concomitantly, expanded G4C2 repeats promote epigenetic DNA changes that inhibit promoter 1b activity, ultimately resulting in decreased expression of the C9ORF72 protein. Reduced expression of C9ORF72 leads to suboptimal autophagy that promotes the toxic accumulation of polyGA, polyGR and polyPG DPR proteins, ultimately resulting into neuronal cell death.
Fig.Figure 1Fig. 1. A co-culture assay based on the split TEV technique for monitoring NRG1-ERBB4 signaling activity.(A) The ERBB4-PIK3R1 split TEV assay monitors NRG1-ERBB4 signaling in PC12 cells. The Nrg1 ligand (green) is stably expressed in the signal-sending cell population A. The signal-receiving cell population B (or split TEV assay cells) is transfected with plasmids encoding the assay components ERBB4 (red) fused to NTEV-tevS-GV (ERBB4-NTEV-tevS-GV), the adapter molecule PIK3R1 (purple, the regulatory subunit alpha of the PI3K) fused to CTEV (PIK3R1-CTEV), and a UAS-driven firefly luciferase reporter (Fluc). Upon Nrg1 binding to the extracellular domain of ERBB4 (1), ERBB4-NTEV-tevS-GV dimerizes and cross-phosphorylates itself (2). PIK3R1-CTEV binds to the phosphorylated ERBB4 receptor leading to the functional reconstitution of TEV protease activity and the concomitant release of the artificial co-transcriptional activator Gal4-VP16 (GV) through proteolytic cleavage at a TEV protease cleavage site (tevS) (3). In turn, released GV translocates to the nucleus and binds to UAS sequences (open box) to activate the transcription of a firefly reporter gene (4). Fig. 1. A co-culture assay based on the split TEV technique for monitoring NRG1-ERBB4 signaling activity.(B) Dose-response assay using increasing numbers of Nrg1 type I β1a-expressing PC12 cells. For each 96-well, 40,000 split TEV assay cells were co-plated with increasing numbers of Nrg1-expressing cells, and incubated for 24h. Half-maximal activation is reached at 5,000 Nrg1-expressing cells. The Z' factor is 0.5 indicating a large separation band for this assay. Fig. 1. A co-culture assay based on the split TEV technique for monitoring NRG1-ERBB4 signaling activity.(C) Adding 10ng/ml EGFld resulted in a two-fold activation. Per 96-well, 40,000 split TEV assay cells were co-plated with empty PC12 cells (no Nrg1 expression), 10,000 Nrg1-expressing cells, and 10,000 Nrg1-expressing cells plus 10ng/ml EGFld. Fig. 1. A co-culture assay based on the split TEV technique for monitoring NRG1-ERBB4 signaling activity.(D) Lapatinib antagonizes ERBB4-PIK3R1 signaling in a dose-dependent manner. Per 96-well, 40,000 split TEV assay cells were incubated with increasing amounts of lapatinib, followed by co-plating 10,000 Nrg1 type I β1a expressing cells. The inset depicts the IC 50 value in µM. After compound/stimulus addition, each assay was incubated for 24h. RLU, relative luciferase units; Fluc, firefly luciferase activity (black line); Rluc, Renilla luciferase activity (grey line); n=6; data are shown as mean, error bars represent SEM.
Figure 7(A) Ena1 and Ena2 loci with average amino acid sequence identity indicated between the population of EnaA-C ortho- and homologs. Ena1C shows considerably more variation and is in B. cytotoxicus different from both Ena1C and Ena2C (see Fig EV5), while other genomes have enaC present at different loci (applies to two isolates of B. mycoides). (B) Distribution of ena1/2A-C among Bacillus species. Whole genome clustering of the B. cereus s.l. group and B. subtilis created by Mashtree (Katz et al., 2019; Ondov et al., 2016) and visualized in Microreact (Argimon et al., 2016). Rooted on B. subtilis. Traits for species (colored nodes), Bazinet clades and presence of ena are indicated on surrounding four rings in the following order from inner to outer: clade according to Bazinet 2017 (when available) (Bazinet, 2017), and presence of enaA, enaB and enaC, resp. (for all three, ena1: teal, ena2: orange, different locus: cyan). When no homo- or ortholog was found, the ring is grey. Ena1A-C and Ena2A-C are defined as present when a homologous protein of the corresponding genome has high coverage (>90%) and >80% and 50-65% sequence identity, respectively, with Ena1A-C of the NMH 0095/75 strain. Interactive tree accessible at https://microreact.org/project/vn2oWw7zM3cwejEFNoRGWA/0024f86c
Figure 5A Schema of the expression vectors of ITCH deletion mutants and the carboxyl-terminal tail of CXCR4. TM7, the seventh transmembrane domain of CXCR4; HA, hemagglutinin tag; DsRed, DsRed-momoner (Clontech/TAKARA). B The immunoprecipitation analysis. Cytoplasmic ENTREP co-precipitated with either ITCH C830A, delWW12 or delWW34 but not with delWW14. Data shown are representative of at least two independent experiments. C The immunoprecipitation analysis. HA-CXCR4SS-DsRed hardly co-precipitated with either ITCHC830A or delWW mutants. HA-CXCR4DD-DsRed co-precipitated with ITCH C830A and delWW12 but not with delWW14 and delWW34. Data shown are representative of at least two independent experiments. D The immunoprecipitation analysis. HA-CXCR4DD-DsRed co-precipitated with ENTREP when co-transfected with either ITCH C830A or delWW12. Data shown are representative of at least two independent experiments. E The immunoprecipitation analysis. The increased transfection of ENTREP vector enhanced precipitation of HA-CXCR4DD-DsRed with ITCH C830A. Data shown are representative of at least two independent experiments. F The nickel-pull down assay using indicated vectors along with HISx6-tagged wild type ubiquitin vector. HA-tagged DsRed was used as a control for HA-CXCR4DD-DsRed. Arrowhead, FLAG-tagged ITCH (wild or C830A); arrow, FLAG-ENTREP wild cyt. Data shown are representative of at least two independent experiments. G Co-localization of ENTREP and CXCR4 in the endosome. Cos7 cells were transfected with ENTREP-FLAG, CXCR4-DsRed and Halo-ITCH wild vectors and treated with CXCL12. ENTREP-FLAG and CXCR4-DsRed co-localized in the RAB7-positive endosome. Inserts, the high-magnification images of the marked area indicating the colocalizaion of ENTREP-FLAG, CXCR4-DsRed and RAB7. Scale bar 10mm. The image is representative of at least two independent experiments.
Figure 7(A-C) Northern blots probing for the 5' NGD-IM in mutant cells expressing the R(CGN)12 reporter. (A) 5' NGD-IM detection in not4∆ski2∆, hel2∆ski2∆, slh1∆ski2∆, not4∆ slh1∆ski2∆. Quantification of full length and the 5'NGD-IM relative to the loading control (SCR1) is given below. Note the size difference of 5'-NGD IMs resulting from NGDRQC+ and NGDRQC- and that the shorter 5'-NGD intermediate (representing intermediates of the NGDRQC- pathway) was reduced in not4∆slh1∆ski2∆ mutant cells. Northern blots probing for the 5' NGD-IM in mutant cells expressing the R(CGN)12 reporter. (B) Northern blot as in (A) except uS10-WTski2∆ or uS10-K6/8Rski2∆ mutant cells with or without a deletion of Not4 (not4∆) were used. Note that the shorter 5'-NGD intermediates are also reduced in ski2∆not4∆uS10-K6/8R cells. Northern blots probing for the 5' NGD-IM in mutant cells expressing the R(CGN)12 reporter. (C) Cells expressing HA-tagged eS7A or the eS7A-4KR mutant in a ski2ΔeS7AΔeS7BΔ or ski2ΔeS7AΔeS7BΔslh1Δ backgrounds were probed for 5'-NGD intermediates by Northern blotting. Note that the four mutated lysine residues in eS7 were responsible for NGDRQC- in the R(CGN)12 reporter. (D) Western blot analysis showing that K83 and K84 of eS7A are main target sites for Hel2 and Not4-mediated ubiquitination: Similarly, as in Fig 6C, eS7AΔeS7BΔ mutant cells were used and wild type and single mutants of HA-tagged eS7A were expressed. Ubiquitinated eS7-HA was monitored using an anti-HA antibody. (E) Northern Blot analysis probing for the levels of 5' NGD-IM derived from the R(CGN)12 reporter expressed in eS7AΔeS7BΔski2Δ cells and HA-tagged eS7 or eS7 mutants. We observed, that ubiquitination at K83 or K84 of eS7A is mainly responsible for NGDRQC-. (F) The levels of polyubiquitinated eS7A were increased by the overproduction of wild-type Hel2 (FL) but neither Hel2ΔRING nor Hel2(1-315) mutants. eS7A-3HAhel2Δ mutant cells harbouring the indicated plasmids expressing wild-type Hel2 (FL), Hel2ΔRING or Hel2(1-315) mutant proteins were harvested. Protein samples were analysed by Western blotting with an anti-HA (Top panel) or anti-FLAG antibody (Bottom panel). (G) Hel2(1-315) polyubiquitinates eS7A but not uS10. Western blotting after an in vitro ubiquitination assay using the Hel2-(1-315) mutant and purified ribosomes containing HA-tagged uS10. (H) Model for quality control pathways induced by R(CGN)12-mediated translation arrest. If the RQC pathway is intact, the leading ribosome that is stalled by the arrest sequence undergoes RQC. The uS10 ubiquitination and ATPase activity of Slh1/Rqt2-dependent subunit dissociation induce the endonucleolytic cleavages in the sequence covered by the first and the second ribosome (disome). In the absence of uS10 ubiquitination and Rqt2, RQC in the first ribosome, as well as NGD in the disome, are eliminated. Instead, RQC-uncoupled NGDRQC- takes place upstream of the disome. The carboxyl-terminal region of Hel2 is crucial to induce RQC thereby NGD in the disome (NGDRQC+), but dispensable for RQC-uncoupled NGD (NGDRQC-).
Figure 4(A) Reconstituted Sb-CCM1 mutants can rescue the incompatibility of Sb-CCM1. Among 94 suppressor clones, 20 of them carry mutations in the Sb-CCM1 gene. These mutants were reconstructed in Sc+Sb-CCM1 cells to test their function. Superscripts next to Sb-CCM1 indicate the respective amino acid substitutions and numbers in parentheses indicate the number of independent clones in our suppressor screen. Sc, wild-type S. cerevisiae. (B) Sequence alignment shows that most of the Sb-CCM1 suppressor mutants contain de novo amino acid changes and only two mutants have switched their residues from the S. bayanus sequence to the S. cerevisiae sequence (F294L and D400N). Letters beside the phylogenetic tree (in which the line lengths are not proportional to the evolutionary distances) indicate the amino acid state at the 400th residue and parentheses contain the codons encoding for the amino acids. (C) The 400th residue plays a determinant role in the S. cerevisiae-S. bayanus incompatibility. Both Sb-CCM1E375Q and Sb-CCM1D400N mutants relieved the incompatibility in S. cerevisiae, but only Sb-CCM1D400N caused respiration defects in S. bayanus. The Sb-CCM1E375Q mutant was selected as a control in this experiment since it was a charged-to-polar substitution similar to Sb-CCM1D400N and also appeared three times in our screen. Sb, wild-type S. bayanus. (D) Protein structural analysis shows that all intramolecular suppressor mutations were located on the predicted PPR motifs. The predicted protein structure of Ccm1 is shown at left. Blue and green colors indicate the two α-helices in each predicted PPR motif and white color indicates additional α-helices in the N-terminus and the middle region. Red color indicates the positions of the suppressor mutations. Predicted PPR motifs occupy around 52% of the whole protein (453 of 871 residues). The box at right displays a magnified version of some PPR motifs where the substitutions are located.
Transcriptomic analysis of bronchioalveolar-like and small airway air-liquid interface cultures.Figure 3A) Heatmaps depicting top 30 up- and downregulated genes in 2D bronchioalveolar-like air-liquid cultures (Bronchioalveolar) compared to small airway air-liquid interface cultures (Small airway). Colored bars represent Z-scores of log2 trans- formed values. B) Heatmaps depicting pulmonary cell type marker genes in 2D bronchioalveolar-like air-liquid cultures (Bronchioalveolar) and small airway air-liquid interface cultures (Small airway). Color coded bars indicate which cell type is marked by the presented gene. Colored bars represent Z-scores of log2 transformed values.
Alterations in lipid composition of endolysosomal membranes from LSD cells.Figure 2(A) Cholesterol levels were measured in the indicated endolysosomal membrane samples containing equal amount of proteins and expressed as ng of cholesterol per μg of protein (see Materials and methods section for details). (B) Filipin staining showing cholesterol accumulation in the endolysosomal compartment of MSD and MPS‐IIIA MEFs (arrowheads and enlarged images). (C, D) Total lipids extracted from the indicated endolysosomal membrane samples (30 μg of proteins) were either (C) subjected to a phosphate assay to quantify the bulk of phospholipids or (D) separated by TLC. Phospholipids and cholesterol on TLC plates were revealed by molybdenum blue staining. CHOL, cholesterol; LBPA lysobisphosphatidic acid; PC, phosphatidylcoline; PE phosphatidylethanolamine; PI phosphatidyinositol; SM sphingomyelin. (A, C) Values represent the mean±s.e.m. values of three independent experiments. *P0.05, Student's t‐test: (A, C): WT versus MSD and WT versus MPS‐IIIA. Scale bar, 10 μm (B).
SARS-CoV-2 infects human organoid-derived 2D small airway cultures.Figure 1A, B) Infectious virus titers can be observed by virus titrations on VeroE6 cells of apical washes at 2, 24, 48, and 72h after infection at MOI 0.01 (A) or 0.1 (B) with SARS-CoV-2 (red). The dotted line indicates the lower limit of detection. Error bars represent SEM. N=4. H p.i. = hours post infection. Immunofluorescent staining of SARS-CoV-2 infected differentiated small airway cultures. Nucleoprotein (NP) stains viral capsid (red), which colocalized with the ciliated cell marker AcTUB Phalloidin was included to stain actin Scale bars indicate 50 μm. Immunofluorescent staining of SARS-CoV-2 infected differentiated small airway cultures. Nucleoprotein (NP) stains viral capsid (red), which colocalized with the ciliated cell marker AcTUB club cell marker CC10 Scale bars indicate 50 μm. Immunofluorescent staining of SARS-CoV-2 infected differentiated small airway cultures. Nucleoprotein (NP) stains viral capsid (red), which colocalized with the ciliated cell marker AcTUB Goblet cells are identified by MUC5AC Scale bars indicate 50 μm.
a, Vacuole in a fibre of the soleus muscle.figf3a, Vacuole in a fibre of the soleus muscle. b, Ultrastructure of a vacuole in a cardiomyocyte. Scale bars: a, 1 µm; b, 1.5 µm. c, Typical original twitch recordings of developed force of heart muscles from control (thin line) and LAMP-2-deficient (thick line) mice. d, Average values of developed force. Preparations of LAMP-2 deficient mice had a significantly lower baseline developed force at a stimulation frequency of 4 Hz and 10 Hz; P  0.01.
Figure 6(A) Confocal images of the polytene chromatin show strong co-localization of HP1c and Su(H), with enlarged images are also shown. Arrowheads in (A''') indicate HP1c (red) and Su(H) (green) signals, while yellow arrowheads indicate co-localizations. Scale bars, 20 μm. (B) The relative binding of HP1c with Notch target genes when Su(H) KD using ChIP-qPCR analysis (n=3 technical replicates, mean ± s.d.). Data are evaluated with two-tailed Student's t-test (*p < 0.05, **p < 0.01). (C) The relative binding of HP1c on Notch target genes when NICD OE using ChIP-qPCR analysis. (n=3 technical replicates, mean ± s.d.). Data are evaluated with two-tailed Student's t-test (***p < 0.001). ChIP-qPCR results show the association of Flag-tagged HP1c with Notch target genes. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin (equivalent to 1). HA antibody is used as a control. Error bars indicate SEM. n=3 technical replicates. Data are evaluated with two-tailed Student's t-test (*p < 0.05, **p < 0.01, ***p < 0.001). ChIP-qPCR results show the association of Flag-tagged chromo-domain depleted HP1c mutant (HP1c △CD) with Notch target genes. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin (equivalent to 1). HA antibody is used as a control. Error bars indicate SEM. n=3 technical replicates. Data are evaluated with two-tailed Student's t-test (*p < 0.05, **p < 0.01, ***p < 0.001).