EMBO/sd-character-level-ner huggingface dataset:
Localization of wild‐type Atg5 and Atg5 K160E, R171E to the PAS.Figure 8(A) Yeast cells of the indicated genotype expressing Atg5-3 × mCherry or Atg5 K160E, R171E-3 × mCherry were treated with rapamycin and imaged using a Deltavision microscope. Maximum projections of z‐stacks are shown. (B) Graph showing the percentage of mCherry‐positive cells that display Atg5-3 × mCherry dots. In total, 3 independent experiments were conducted and >400 cells were counted. (C) Graph showing the percentage of cells that display Atg16-3 × mCherry dots. In total, 3 independent experiments were conducted and >750 cells were counted. The graphs show the averages and the error bars representing the standard deviations. P‐values were calculated using Student's t‐test. Scale bars: 5 μm.
Identification of novel Beclin 1 interactors.Figure 1(A) Beclin 1 (BCN1) interactors identified by yeast two‐hybrid technology. Proteins binding to BCN1 are listed and their interacting domains are indicated by black bars. Numbers refer to amino‐acid positions. (B) Identification of TAB2 and TAB3 fragments interacting with BCN1 in the yeast two‐hybrid system. Blue lines depict the fragments of TAB2 or TAB3 that were found to interact with BCN1 (numbers on the right refer to the amount of yeast clones identified for each fragment). The minimal domain required for the interaction is referred to as Beclin‐binding domain (BBD). (C) Constructs derived from TAB2 and TAB3 used in this study. (D, E) Co‐immunoprecipitation of TAB2 or TAB3 with BCN1. The indicated constructs, namely HA‐tagged and T7‐tagged TAB2, and TAB3 constructs in (D) and (E), respectively, and Flag‐tagged Beclin 1 (Flag-BCN1) were transfected into HeLa cells alone or in combination. Twenty‐four hours later, TAB2 and TAB3 were immunoprecipitated with antibodies specific for HA (D) or T7 (E) and the precipitate was separated by SDS-PAGE and revealed with an antibody specific for Flag. (F) Immunoprecipitation of endogenous BCN1 with endogenous TAB2 or TAB3. HeLa cells were subjected to autophagy induction with starvation conditions, 1 μM rapamycin or 30 μM pifithrin α (PFTα) for the indicated time and then processed for TAB2 or TAB3 immunoprecipitation followed by the immunodetection of BCN1, TAK1, TAB2 and TAB3. Results in (E) and (F) are representative for three independent experiments.
Figure 1(A) Blood parameters of patient are depicted. (B) Patient's skin alterations are depicted. (C) T2-weighted MR images in coronal plane show abscess formation in the left lower pulmonary lobe (upper panel) and in spleen and left axilla (lower panel). (D) Histological sections of patient biopsies stained with hematoxylin and eosin (left panel) or with an actin antibody (right panel). Scale bars: skin: left 1000µm, right 100µm; lung: left 500µm, right 250µm; spleen: left 1000µm, right 75µm. (E) Whole Exome Sequencing (WES) and targeted Sanger sequencing identified compound heterozygous variants in OTULIN at position c.258 G>A, p.M86I on the paternal allele and at position c.500 G>C, p.W167S on the maternal allele. (F) The pedigree is depicted. The patient (II-2) is the second child of non-consanguineous parents.
Fig.Figure 3(A) Localization of Citrine-RBOHD (left) or Citrine-RBOHF (right) in endodermal (en) cells. Both proteins were expressed under the control of pCASP1, an endodermis-specific promoter. Representative pictures are shown, 2 positions from 10 roots for each transgenic line were inspected. Scale bar = 5 µm. (B) Lignin accumulation in WT and rbohD and rbohF single mutants and a double mutant with or without 2-hour 100 nM CIF2 peptide treatment. Arrowheads indicate excess lignification. Pictures are shown as overviews (maximum projection) or median sections. Lignin and cell walls are shown with magenta (stained with Basic Fuchsin) and gray (stained with Calcofluor White) respectively. Representative pictures are shown, 12 roots (overview) and 2 positions in 12 roots (median section) were inspected. Scale bars = 20 µm (lignin overviews), 5 µm (median sections). "inner" designates the stele-facing endodermal surface, "outer", the cortex-facing surface.
Suppressed accumulation of Ub in Atg7-/Nrf2-deficient livers.Figure 3.Suppressed accumulation of Ub in Atg7-/Nrf2-deficient livers. (A and B) Quantitative mass spectrometry analysis of P2UBA captured p62 (A), total (A), and isopeptide-linked (B) Ub from the soluble and insoluble fraction of autophagy-deficient livers (28 d after pIpC injection). Control littermates (F/F or F/F:Nrf2+/−; denoted +; open circles; n = 4), Nrf2 KO (F/F:Nrf2−/−; denoted −; closed circles; n = 4), Atg7 KO (F/F:Mx1 or F/F:Mx1:Nrf2+/−; denoted −; closed diamonds; n = 6), or Atg7-/Nrf2-DKO (F/F:Mx1:Nrf2−/−; closed triangles; n = 6) are shown. Each symbol represents one animal. **, P ≤ 0.01; ***, P ≤ 0.005.
Figure 1(A) Domain architecture and putative crosslinking model of ACTN4 antiparallel dimers. Actin-binding-domain (ABD), calponin-homology (CH1, CH2), spectrin-repeats (SR), calmodulin-like domain (CaM), EF-Hand domains. (B) Immunoblots from cytosolic (cyt) and nuclear (nuc) fractionations of RPE-1 cells in early-G1 showing the presence of endogenous ACTN4 in nuclear fractions. Proteins were detected with antibodies against (α) ACTN4, α-Tubulin or Histone 3 as indicated (C) Upper and middle panels: immunoblots showing precipitation of ACTN4 and β-actin from RPE-1 nuclear lysates in early-G1 in the presence or absence of biotin-phalloidin, as indicated. Lower panel: Immunoblot of cytosolic (cyt) and nuclear (nuc) fractions. Proteins were detected with antibodies against (α) ACTN4, β-Actin, α-Tubulin or Histone 3 as indicated. (D) Immmunofluorescence images of endogenous ACTN4 in non-dividing and post-mitotic NIH3T3 and RPE-1 cells in as indicated. Shown are maximum intensity projections. Cells were fixed in 4% PFA and stained for ACTN4 and nuclei (DAPI) as indicated. Scale bars 10 μm.
Figure 9CPEcells form a tight barrier between the peripheral blood and the brain. CPEcells are highly secretory and secrete exosomes from MVBs into the CSF. Systemic inflammation induces activation of the exosome machinery, resulting in an increased released of miRNA-containing exosomes into the CSF. Next, the exosomes cross the leaky ependymal cell layer that line the ventricle and are taken up by astrocytes and microglia, whereupon they transfer a pro-inflammatory message.
Figure 4A) Acetylation tracks for H3K9/14ac and H3K27ac ChIP-seq (on the left) and corresponding FPKM values by RNA-seq (on the right) for representative genes in YC, YT, OC and OT FAPs. B) Graphs showing RNA levels for the representative genes shown in (A) measured by qPCR. C) Graphs showing the H3K9/14 acetylation levels for the representative genes shown in (A) measured by ChIP-qPCR. D) Representative images of the phospho-NFkB staining in FAPs (pNFkB-p65 in red/CD90 in green/Laminin in grey/DAPI in blue) on tibialis anterior muscle transversal section of YC, YT, OC and OT mice; Scale bar = 25 μm; E) Box plot showing the percentage of pNFkB-p65 positive FAPs in the staining shown in (D) F) Representative images of the phospho-p38 staining in FAPs (pP38 in red/CD90 in green/Laminin in grey/DAPI in blue) on tibialis anterior muscle transversal sections of YC, YT, OC and OT mice. Scale bar = 25 μm; G) Box plot showing the percentage of pP38 positive FAPs in the staining shown in H) Representative images of the phospho-SMAD2/3 staining in FAPs (pSMAD2/3 in red/CD90 in green/Laminin in grey/DAPI in blue) on tibialis anterior muscle transversal sections of YC, YT, OC and OT mice. Scale bar = 25 μm; I) Box plot showing the percentage of pSMAD2/3 positive FAPs in the staining shown in (H).
ESCRT-independent functions of the Dsc complex in membrane proteostasis.Figure 1(A) Synthetic genetic array technology of S.cerevisiae was used to cross the ESCRT mutant (query mutation vps4::NatR) with all non-essential genes (xxx::KanR). The growth of double mutants (vps4::NatR xxx::KanR) was scored in two independent replicates for normal growth, synthetic sick or synthetic lethal phenotypes. 119 genes showed synthetic sickness in one replicate and synthetic lethality in the other replicate or synthetic lethality in both replicates. (B) Gene Ontology (GO) analysis for macromolecular complexes of the 119 genes that showed synthetic sickness in one replicate and synthetic lethality in the other replicate or synthetic lethality in both replicates A hypergeometric test was used to estimate if the mapped GO term is significantly enriched with the selected genes. Significant GO terms (p < 0.05) and their associated genes identified in the screen are shown with a cut-off for 15-fold enrichment over the genome frequency. (C) Equal amounts of WT cells and indicated single or double mutants in serial dilutions were incubated on agar plates at the indicated temperatures. The synthetic growth defects of vps4∆ tul1∆ or vps4∆ ubx3∆ double mutants at 26°C and 37°C were complemented with plasmids encoding VPS4 or TUL1 or UBX3. The dashed boxes indicate the non-complemented double mutants. (D) Cartoon presentation of Dsc complex members and evolutionary conserved functional domains with human candidate genes that show the highest homology in the indicated domains.
Sustained induction of HS memory-related gene expression after an acclimatizing HS depends on HSFA2.Figure 2A. Transcript levels of the indicated HS-inducible genes after ACC in Col-0 (blue bars) and hsfa2 (orange bars) as determined by quantitative Reverse Transcription-PCR (qRT-PCR). Transcript levels were normalized to TUB6 and the respective NHS harvested at the same time point ([GENE OF INTERESTACC x h/ TUB6ACC x h]/ [GENE OF INTERESTNHS x h/ TUB6NHS x h]). Note that the y-axis is on a log10 scale. Error bars show SE of at least three biological replicates. Asterisks show significant differences between the genotypes at the same time point based on Student´s t-test (*: p<0.05; **: p<0.01). B. Immunoblotting of HSP21 and HSP101 in Col-0 (C) and hsfa2 (h) at the indicated time points after ACC or NHS. TUBULIN was used as a loading control. M, marker.
Figure 6A. Schematic diagram of the OIR model (top) and representative images of P17 OIR retinas are shown (bottom). Neonatal mice were exposed to 75% oxygen from P7 to P12 to induce vessel loss. Then mice received intravitreal injections of PBS vehicle, mouse-specific anti-VEGF (as a positive control) delivered at 0.1 μg/μl, or F4L5.13v2 (a second-generation F4L5.13 modality, in which the N-terminal FZD4-specific diabody was replaced with a FZD4-specific Fab) at 50nM or 500nM target vitreous concentrations. Mice were returned to room air from P12 to P17 to induce maximum pathologic neovascularization at P17. At P17, retinas from injected mice were collected, dissected as flat‐mounts and stained with Isolectin B4. Scale bar = 500 µm. B. Quantification of the ratio of neovascular and avascular area on total area of vascularization. Data are presented as mean ± SEM, n=11-13 retinas per group. Significance was calculated by one-way ANOVA with Bonferroni's multiple comparisons test (indicated p-values are in comparison to Vehicle). E
Figure 3A Schematic diagram of humanINPP5E and mutants used in this study. D477N, phosphatase-inactive mutant. B N1E-115 cells stably expressing mSt-INPP5E (WT, D477N, ∆CAAX, or 295-644) treated with siControl or siINPP5E were cultured in growth medium with or without 125 nM Baf.A1 for 2 h, and then analyzed by immunoblot using anti-LC3 and anti-GAPDH antibodies. The mSt-INPP5Es used in this study were resistant to mouse siINPP5E because target sequence of the siRNA is different from the human correspondent sequence.C Quantitation of protein signal intensities from immunoblots in B, showing differences in LC3-II levels in the presence and absence of Baf.A1 following normalization to the control proteinGAPDH. Results represent means ± s.d. of three independent experiments. **, P < 0.01; *, P < 0.05. D NIE-115 cells stably expressing mSt-INPP5E (WT, D477N, or ΔCAAX) were cultured in growth medium. Cells were fixed and stained with anti-LAMP1 antibodies, and then analyzed by immunofluorescencemicroscopy. Insets show the boxed areas at high magnification. The number showing to the right side indicates the colocalization rate of LAMP1 with mSt-INPP5E. Bar, 10 µm.
Schematic representation of mDA neuron survival promoted by forced Nurr1 + Foxa2 gene delivery.Figure 6Nurr1 and Foxa2 are endogenously expressed in mDA neurons and promote cellsurvival in a cell-autonomous manner (left). Expression of Nurr1 and Foxa2 is reduced in mDA neurons during aging and degenerative processes. In addition, glia come to be polarized toward the harmful M1-type. DA neurons with reduced Nurr1/Foxa2 expression are more vulnerable to the toxic environment in the aged or degenerating midbrain (middle). Forced expression of Nurr1 + Foxa2 in mDA neurons promotes their survival. In addition, Nurr1 + Foxa2 transgene expression in glial cells (M2-type) creates a neuroprotective environment by suppressing the secretion of pro-inflammatory cytokines as well as promoting neurotrophic factor release (right).
Figure 4(A) Changes in transcript abundance 30 and 120 min after addition of antimycin A to exponentially growing cells without (EMM) or with arginine (EMM+R). Transcripts annotated as up- or down-regulated Core Environmental Stress Response (CESR) are indicated in red and blue, respectively. All data is normalised to EMM time point 0. (B) Graphs as in (A) showing expression profiles of transcripts encoding enzymes for arginine catabolism (blue) or arginine biosynthesis (red). (C) Heat map of expression changes for transcripts repressed over 2-fold at 30 min in cells supplemented with arginine (EMM +R) relative to cells without supplementation (EMM). Values for timepoints 0 and 30 min after antimycin A addition are shown for cells grown in EMM, EMM with arginine and ssp1 deletion mutant grown in EMM. The data are normalised to EMM timepoint 0. (D) Heat map of expression changes of the same genes as in (B) after one hour of inhibiting TOR kinase with torin1 or caffeine/rapamycin treatment relative to cell before treatment; cells were grown in rich media - YES (data from (Rodríguez-López et al., 2019)) (E) Overlaps between genes repressed with arginine supplementation and genes induced in tor2 thermo-sensitive mutant (p <E-27) (Matsuo et al., 2007), or repressed in strains deleted for the TOR inhibitors tsc1 and tsc2 (p <E-16) (Van Slegtenhorst et al., 2004). (F) Enriched and underrepresented amino acids in proteins encoded by 40 genes that are down-regulated in media with arginine. Analysis performed using online tool AnGeLi (Bitton et al, 2015).
Figure 4A-D Transcript levels were determined by qPCR and normalized to AtACT2. (A, C) Localized NCED2 and NCED3 expression in the wild-type senescing leaf. Half-senesced leaves were split into three parts, as shown in Fig. 1A, C. (B, D) NCED2 and NCED3 expression during leaf senescence in the vector control and CDF4- RNAi-1 plants. Transgenic plant leaves were analyzed from 7 to 56 d (d, days after planting). Four independent experiments were conducted. Values are given as mean ± SD, n=4. *p<0.05 by Student's t-test. E Observation of rosette leaf longevity from the 26-day-old vector control, pER8::CDF4, pER8::CDF4 & nced2, pER8::CDF4 & nced3 and pER8::CDF4 & nced2nced3 transgenic plants after 20 μM estradiol induction for two weeks. Phenotype of detached rosette leaves arranged from oldest to youngest. Scale bars indicate 1.5 cm. (F) SAG12 expression level in rosette leaves at various development stages. Three independent experiments were conducted. Values are given as mean ± SD, n=4. *p<0.05 by Student's t-test. (G) Chlorophyll concentration in rosette leaves at various development stages. Three independent experiments were conducted. Values are given as mean ± SD, n=4. *p<0.05 by Student's t-test.
Src‐dependent phosphorylation of FAK is required for Ret/FAK binding.Figure 3(A) FAK−/− cells stably re‐expressing FAK‐wt, FAK‐Y397F, FAK‐KD or FAK‐Y4F‐Y9F were immunoblotted using anti‐FAK, anti‐FAK Y397, anti‐FAK Y861, anti‐FAK Y925, anti‐Src, anti‐Ret and anti‐actin. FAK and Ret were immunoprecipitated from these cells and then samples were immunoblotted with anti‐Src, anti‐FAK or anti‐Ret. (B) Cells were fixed and stained with anti‐Ret (green) and either anti‐FAK (red in the upper panels) or anti‐PY416 Src (red in the lower panels). Merged images are shown. Solid arrows indicate Ret in adhesions, while broken arrows show Ret in puncta. Scale bars, 20 μM. (C) Quantification of percentage of cells that contained Ret in intracellular puncta is shown. Data are presented as mean±s.d. and the significance calculated using a Student's t‐test (n=3). FAK, focal adhesion kinase; Ret, rearranged during transfection.
Figure 4A. Gene regulatory network underlying mESC differentiation to the three germ layers. Gray node shades indicates the significance of the motif activity, edge thickness indicates the strength of the interaction. B. Scheme of the experimental approach to quantitatively capture gene expression changes during EpiSC differentiation towards the three germ layers. C. Gene regulatory network underlying EpiSC differentiation to the three germ layers. Green node shades indicates the significance of the motif activity, edge thickness indicates the strength of the interaction. Nodes circled in blue are shared between the mESC and EpiSC differentiation networks.
Mosaic epiblast labelling allows tracking of bottle-shaped cells and cell division.Figure 2(a) Z-projection of an E7.25 embryo mosaically labelled through OH-tamoxifen injection, imaged by two-photon microscopy. Scale bar: 50 μm. Data information: n embryos=54. (a') Mouse line strategy used to produce embryos ubiquitous for membrane tdTomato (grey) and mosaic for membrane GFP (mGFP, green) in the epiblast. (b) Z-projections of stills from live imaging recording of a bottle-shaped cell (indicated by a white arrow) that delaminates through the basal membrane at time 0min, 25 min, 50 min, 1h15min and 1h40min. (b') Top: Bottle shape events: GFP+ bottle shape cells over total GFP+ cells, expressed in percentage, in anterior versus posterior region of the epiblast. The posterior region includes the PS region, as it could not be precisely discriminated. Percentage of bottle shape cells exiting the epiblast among total number of GFP+ bottle shape cells, in anterior versus posterior epiblast. white dotted lines outline basal (top) and apical (bottom) borders of the epiblast. (c) Z projection (c) and 3D reconstruction (c') of stills from live imaging recording showing an apical division without basal contact, followed by daughter cells dispersion at 0min, 25 min, 50 min, 1h15min and 1h40min. white dotted lines outline basal (top) and apical (bottom) borders of the epiblast. (c") Left: Frequency of apical division (GFP+ apical division over total GFP+ cells, expressed in percentage) in anterior and posterior epiblast. Middle: Apical division with or without persistence of contact with the epiblast basal pole, normalized by the total number of GFP+ apical division, in anterior versus posterior epiblast. Right: Cell dispersion (defined as loss of basolateral contact between daughter cells after reinsertion in the epiblast layer) after apical division in anterior and posterior epiblast.
Figure 3A, HCT116 cells were treated with either vehicle alone (-), 1 µM MPA, 10 µM MPA or 5 nM ActD for 24 hrs and mRNA levels of p21 were quantified by real-time qPCR in 2 independent experiments carried out in triplicate and normalized to 28S rRNA. Data information: , data are presented as mean ± SD, relative to control. *p<0.05, **p<0.01, ***p<0.001, by two tail T-Student's test. B, Autoradiogram (upper panel) and Ethidium Bromide (EtBr)-stained agarose gel (lower panel) of 18S and 28S rRNA from HCT116 cells treated as indicated in the Methods section. C, Autoradiogram (upper panel) and EtBr-stained TBE-urea polyacrylamide gel (lower panel) of 5S, 5.8S rRNA and tRNAs in lysates HCT116 cells were treated for 24 hrs. Cell lysates were collected, subjected to ultracentrifugation, spiked with firefly luciferase mRNA and immunoprecipitated with anti-HDM2 rabbit antibody (IP HDM2) or the IgG control (IgG). D, Levels of HDM2, RPL5, RPL11, and the IP negative control mTOR in the whole cell extract (WCE), the post-ribosomal lysates (INPUT, left panel) and in the HDM2 immunoprecipitated fraction (right panel) were analyzed on Western blots. The results are representative of two independent experiments. Quantification of band intensity of HDM2, RPL5 and RPL11 of at least two independent experiments is shown (lower right panel). HCT116 cells were treated for 24 hrs. Cell lysates were collected, subjected to ultracentrifugation, spiked with firefly luciferase mRNA and immunoprecipitated with anti-HDM2 rabbit antibody (IP HDM2) or the IgG control (IgG). E, the Levels of immunoprecipitated 5S rRNA were determined by real time qPCR and normalized to the firefly luciferase mRNA. Data are normalized and presented as relative to untreated cells immunoprecipitated with anti-HDM2. The results are representative of three independent experiments carried out in triplicates. Data information: data are presented as mean ± SD, relative to control. *p<0.05, **p<0.01, ***p<0.001, by two tail T-Student's test. F, HCT116 cells were pretreated with vehicle alone (-), 1 µM, or 10 µM MPA for 24 hrs, and the levels of p53 and p21 were followed on Western blots in presence of 100 µg/mL cycloheximide (CHX) for the indicated time, in the continued presence of the treatment. GAPDH was used as a loading control. Quantification of band intensities is shown in the lower panels as relative to time 0 of CHX treatment. G, HCT116 cells were pretreated as in (F) and 10 µM of the proteasome inhibitor MG132 was added for 4 hrs when indicated. The levels of p53 and p21 were analyzed on Western blots. GAPDH was used as a loading control. Quantification of p21 band intensity of at least four independent experiments is shown in the lower panel. Data information: In panels data are presented as mean ± SD, relative to control. *p<0.05, **p<0.01, ***p<0.001, by two tail T-Student's test.
Figure 6(A) Schematic diagram showing the generation of Slc6a20a-mutant mice. The SLC6A20 antibody is denoted by "Y" above exon (Ex) 6. (B) Decreased levels of Slc6a20a, but not Slc6a20b, mRNAs in the brain of Slc6a20a+/- (HT) and Slc6a20a-/- (KO) mice (P21-28), as shown by qRT-PCR analysis. (n = 4, 3, 3 mice for WT, HT, and KO; *P < 0.05, **P < 0.01, ns, not significant, one-way ANOVA with Bonferroni's test). The error bars represent SEM. (C) Reduced expression of SLC6A20 proteins in the brains of Slc6a20a+/- and Slc6a20a-/- mice (P21 for HT and P24-28 for WT and KO), as indicated by immunoblot analysis of total brain lysates. β-actin and α-tubulin were used as loading controls. (n = 3 mice for WT, HT, and KO, *P < 0.05, Student's t-test). The error bars represent SEM. (D) Increased extracellular levels of glycine and proline in the brain of Slc6a20a+/- and Slc6a20a-/- mice, as shown by microdialysis analyses. Note that glycine levels are increased in both Slc6a20a+/- and Slc6a20a-/- mice, whereas proline levels are increased only in Slc6a20a-/- mice. (glycine, n = 19 mice for WT, 11 for HT, 8 for KO, **P < 0.01, Mann-Whitney U test; proline, n = 15 mice for WT, 11 for HT, 14 for KO, ***P < 0.001, ns, not significant, Mann-Whitney U test). The error bars represent SEM. (E) Increased NMDA/AMPA ratio at hippocampal SC-CA1 synapses in Slc6a20a+/- mice (P17-20), as indicated by the ratio of NMDAR-EPSCs to AMPAR-EPSCs. (n = 9 neurons from 3 mice for WT and 11 (4) for Slc6a20a HT mice, **P < 0.01, Mann Whitney U test). The error bars represent SEM. (F) Normal frequency and amplitude of mEPSCs in hippocampal CA1 pyramidal cells in Slc6a20a+/- mice (P17-20). (mEPSC, n = 14 neurons from 3 mice for WT and 16 (3) for HT; ns, not significant, Student's t-test). The error bars represent SEM.