EMBO/sd-character-level-ner huggingface dataset:
Figure 1(A) A scheme of the experimental design. Triangles represent the time points (in minutes) of cell harvesting for RNA purification, after EGF stimulation. (GF, growth factor). (B) MCF10A cells were stimulated with EGF (10 ng/ml) for up to eight hours. At the indicated time points, cells were harvested, RNA was isolated and used to determine expression levels of specific lncRNAs and mRNAs, using the SurePrint microarray platform (from Agilent). The heatmaps depict lncRNAs (left) and mRNAs (right) for which a >1.5-fold (lncRNAs) or a >2-fold (mRNA) change in expression was observed in at least one of the time points, compared to un-stimulated cells (time zero). Note that each row of the heatmap represents an individual lncRNAs/mRNAs, and values (see color scale at the bottom) indicate the log2 ratio of RNA levels between each time point and time zero.
Figure 3Hydrolysis of fluorogenic substrates (Ac-PAL-AMC) for LMP2-activity of human (upper panel) or mouse (lower panel) 20S immunoproteasome at various concentrations of PRN1126 or ONX 0914. Data are presented as the means of fluorescence ± s.d. from quadruplicate assays. The experiments were repeated three times with similar results. Altered electrophoretic mobility of IP subunits by covalent modification with ONX 0914. Ficoll enriched lymphocytes from C57BL/6 mice were treated with DMSO or 300 nM ONX 0914 for 2 h in vitro. SDS-PAGE and immunoblotting against indicated proteins was performed. Shown are representative western blots out of three independent experiments with similar outcome.
Figure 2(A) Sb-Ccm1 protein is transported efficiently to S. cerevisiae mitochondria. Total cell extracts (total), cytosolic fractions (cyto) or mitochondrial fractions (mito) were hybridized with different antibodies to detect the proteins. G6PDH (glucose-6-phosphate dehydrogenase) and Cox2 served as cytosolic and mitochondrial markers, respectively. Both Sc-Ccm1 and Sb-Ccm1 were fused with c-Myc at the C-terminus and expressed from a single-copy plasmid. The Myc-tagged Ccm1 proteins consistently appeared as double bands, which were probably due to some unknown protein modifications. (B) The level of 15S rRNA is reduced in Sc+Sb-CCM1 cells. RNA isolated from Sc+Sb-CCM1 (labeled as Sb) or Sc+Sc-CCM1 (labeled as Sc) cells was hybridized with gene-specific probes using Northern blots. (C) Sb-Ccm1 interacts weakly with S. cerevisiae 15S rRNA. The level of Ccm1-bound 15S rRNA was measured using quantitative PCR analysis following mitochondrial RNA immunoprecipitation of S. cerevisiae cells expressing Sc-CCM1-13Myc or Sb-CCM1-13Myc. Data was normalized to the control group without the Myc tag to obtain the relative fold enrichment. Graph was plotted using data from three independent repeats for each strain and p value was calculated by unpaired, two-sided student t-test. Error bars indicate S.D. ***, p value < 0.001. (D) Overexpressing Sb-CCM1 rescues the respiration defect of Sc+Sb-CCM1 cells. Cell cultures were serially-diluted and spotted onto YPD (glucose) or YPG (glycerol) plates. The plates were then incubated at 28°C until colonies were easily observed. (E) The steady-state level of mtDNA-encoded proteins is reduced in Sc+Sb-CCM1 cells. Immunoblotting for the mitochondrial proteins in Sc+Sb-CCM1 (labeled as Sb) or Sc+Sc-CCM1 (labeled as Sc) cells. Cox1, Cox2 and Cox3 are mtDNA-encoded complex IV subunits. Tom20 is a nucleus-encoded mitochondrial protein.
S.Figure 1 (A) Negative stain electron micrograph showing the tendency of S. cerevisiae prApe1 (sc-prApe1) dodecamers to form higher-order assemblies or chains (black arrowheads). (B) Negative stain electron micrograph of S. cerevisiae mApe1 (sc-mApe1) showing monodisperse dodecameric particles (circles) as well as hexameric particles (white arrowheads). (C) Bar plot showing the occurrence of single, double, triple or multiple aggregates of sc-prApe1 in comparison with sc-mApe1. Insets show single, double, triple or multiple dodadecamers. Data information: n=3,186 particles were counted in the case of sc-prApe1 and n=3,107 particles were counted in the case of sc-mApe1. Particles were picked from 20 different micrographs. ***P < 2.2x10-16 (Pearson's Chi-squared test).  (D) Negative stain electron micrograph of GraFixed sc-mApe1 showing dodecameric particles (circles). (E) Class averages of sc-mApe1 dodecamers including views of two-fold and three-fold symmetry. (F) Fourier shell correlation of the EM reconstruction of the sc-mApe1 dodecamer indicates a resolution of 24 Å. (G) C. thermophilum mApe1 crystal structure fitted into electron microscopy (EM) map of sc-mApe1 dodecamer. Each of the 12 chains is depicted by a different color. A black sphere represents the N-terminus where the propeptide would emanate from the dodecamer. From left to right: two-fold axis view from the edge, three-fold axis view from the facet and three-fold axis view from the vertex.
AosR is crucial for cellular-redox homeostasisa.Figure 7Indicated bacterial strains were subjected to oxidative stress with 50 µM CHP for 6 h and free thiol content was quantified. Data presented is representative of one of two biological replicates, each performed in triplicates (n=3). Y-axis represents µM thiol /µg of WCL (mean ± SD). Metabolite levels of reduced mycothiol, MSH ergothioneine, ERG were measured in Rv, Rv∆aosR, and Rv∆aosR::aosR in normal and CHP stressed cells using LC-MRM MS/MS analysis. The absolute values obtained under normal conditions were normalized to 100% for each strain and the relative values were calculated for samples processed from CHP treated cells. The data indicates relative levels of metabolites (mean percent ± SD) of four replicates (n=4). Metabolite levels of reduced NADH were measured in Rv, Rv∆aosR, and Rv∆aosR::aosR in normal and CHP stressed cells using LC-MRM MS/MS analysis. The absolute values obtained under normal conditions were normalized to 100% for each strain and the relative values were calculated for samples processed from CHP treated cells. The data indicates relative levels of metabolites (mean percent ± SD) of four replicates (n=4). To evaluate the contribution of AosR on the antioxidant capacity; Rv, Rv∆aosR and Rv∆aosR::aosR cells were treated with 5 mM H2O2 and the ratiometric response (mean ±SD; n=3) was measured at indicated time points. Schematic depicting the pleiotropic role of AosR. Oxidative environment induces disulfide bond formation in AosR and transcription of mec-cysO-cysM through an auxiliary promoter resulting in enhanced L-cysteine biosynthesis. This further induces genome-wide transcriptome changes. The latter set of changes in indirectly regulated genes can be mitigated by the addition of L-cysteine in the media. qRT-PCR measurements for the log2 fold induction (mean ±SD, n=3) for indicated genes in Rv∆aosR and Rv∆aosR treated with 1mM L-cysteine as compared with Rv. All strains were treated with 50 μM CHP. Data were normalized with respect to 16s rRNA and is representative of one of two biological replicates.
Figure 2Representative IF staining of Prom1 using specific antibody clone 13A4 targeting extracellular loop (green) on the stem cell and transit amplifying cell regions of lower incisor CLE at P7. Sample is counterstained with DAPI (blue). Dotted lines, basement membrane. SCs, stem cells; TACs, transit amplifying cells; Ams, ameloblasts. 3D reconstruction showing the association of Prom1 (green) with AcTub-labeled (red) primary cilia in stem cell and transit amplifying cell regions. Note that the expression of Prom1 is not limited primary cilium but also to microvilli. A representative example of Prom1 association with one primary cilium at the stem cell to transit amplifying cell transition region. Green channel transparency was set up to 70%. Representative IF staining of Prom1 using antibodies directed either its extracellular loop (clone 13A4, green) or cytoplasmic C-terminal end (Biorbyt, Orb129549, red) on transit amplifying cell regions of the WT vs. Prom1 KO mice. Samples are counterstained with DAPI (blue). Note the lack of Prom1 labeling in Prom1 KO mice. The mRNA profiling on shRNA-meditated Prom1 knockdown (3 different shRNAs were used, marked as NO.1, 2 and 3) in cultured CLESCs. qRT- PCR results are in arbitrary values after normalization for GapDH. protein profiling on shRNA-meditated Prom1 knockdown (3 different shRNAs were used, marked as NO.1, 2 and 3) in cultured CLESCs. The immunoblotting was performed with anti-Prom1 C-terminal antibody. β-actin was used as loading control (F). Molecular mass markers are indicated. Representative IF staining of Prom1 (red) and Sox2 (green) in CLESCs in vitro. Sample is counterstained with DAPI (blue). Arrows indicate colocalisation of Prom1 with low-medium level of Sox2 in the cell nuclei. Subcellular fractionation of CLESCs into cytoplasm/membrane (C+M) and nuclear (N) fractions. Materials were analyzed by immunoblotting for Prom1 (H) and Arl13b (I). As controls, membranes were blotted for anti-Lamin A/C (nuclei markers) and GapDH (cytoplasm marker). Molecular mass markers are indicated. Note the presence of full-length Prom1 as well as Arl13b in nuclear fraction.
a) Growth of L.figf4(a) Growth of L. monocytogenes in S2 cells (S2) or S2 cells expressing PGRP-LE (S2-LE) transfected with double-stranded RNA (RNAi) specific for Atg5, Rel or Dif and dl (Dif-dl) and infected for 1.5 h with wild-type or Δhly L. monocytogenes, followed by 6 h of incubation in medium containing CuSO4 and gentamicin, quantified by plate assay of colony-forming units. *, P 0.001 (t-test). Data are representative of two independent experiments (error bars, s.d.). (b,c) Fluorescence confocal microscopy of the localization of PGRP-LE together with wild-type L. monocytogenes in S2 cells engineered to express YFP-PGRP-LE (YFP-LE) and infected for 0.5 h with wild-type L. monocytogenes (b) or Δhly L. monocytogenes (c), followed by 1 h of incubation in gentamicin-containing medium, then DAPI staining. Merge, YFP-PGRP-LE (green) and DAPI (magenta). Filled arrowheads indicate L. monocytogenes; open arrowhead indicates accumulation of YFP-PGRP-LE around the bacteria. Scale bars, 5 μm. Data are representative of three experiments.
Validation of AGR2-TMED2 interaction and its impact on AGR2 dimerization.Figure 3A) Co-immunoprecipitation of AGR2 with TMED2 under basal and tunicamycin induced ER stress in intestinal epithelial cells treated in situ. *indicates Immunoglobulin heavy and light chains. B) Structural association of TMED2 and AGR2. The six TMED2 orientations (pink, dark pink, purple, blue, yellow and green) best positioned to stabilize the AGR2 dimer (colored tan with N-termini and dimer interface alpha helix in red). C) Changes in AGR2 dimer and monomer ratio in TMED2 overexpressing cells. DSP-stabilized AGR2 was analyzed under non-reducing (top blot) or reducing conditions (bottom blot). D=dimeric, M=monomeric AGR2. D) Specific interactions between residues in AGR2 dimer (tan) and best oriented TMED2 structure (green). Glu51, Arg74 and Arg97 of TMED2 form ionic interactions with Lys66, Glu59 and Asp97 of AGR2. His28 of TMED2 interacts with Tyr111 of AGR2 through π-π-interactions. E): Co-immunoprecipitation of AGR2 wild-type (WT, left panel) or AGR2 AA mutant (right panel) with TMED2 in HEK293T cells. Immunoprecipitation was performed with mouse anti-Flag antibody to pull-down the ectopic protein tag. F) Western blot analysis (left panel) and quantification (right panel) of AGR2 intracellular expression upon TMED2 overexpression. Data are representative of 4 independent experiments. Tubulin (TUB) was used as a loading control. G) HEK293T cells were transfected with control (ev) or TMED2 plasmid and then used in the ERMIT assay with AGR2 WT (n=3). H) Western blot analysis (left panel) and quantification (right panel) of AGR2 intracellular expression in total cell lysate (TCL) upon TMED2 silencing. Data are representative of 4 independent experiments. Tubulin (TUB) was used as a loading control. I) HEK293T cells were silencing for TMED2 and then used in the ERMIT assay with AGR2 WT (n=3).
Autophagy in sav mutants.Figure 9(A) EM scan of a 7‐day‐old sav3 mutant ommatidium and zoom‐in on autophagosomes containing undigested debris (arrowhead). Scale bar: 1 μm for panel on the left and 0.2 μm for zoom‐in panel on the right. (B) Graph of the quantification of autophagic vesicles (AV) per photoreceptor found in EM sections of 1‐week‐old sav3 clones. Significant accumulation of AV is found in mutant cells. ***P0.001 in one‐tailed t‐test. N=45 pigmented cells versus 42 non‐pigmented cells. (C) Model for the relationship between Atro, Ft/Hippo and neuronal homeostasis. Atro wt overexpression, and more effectively polyQ Atro overexpression, causes a downregulation of ft transcription and of other genes important for photoreceptor neuron function and homeostasis. This results in downregulation of Ft/Hippo signalling, which affects neurodegeneration. Healthy Ft/Hippo signalling contributes to neuronal homeostasis through effective autophagy. Atro may also exert Ft‐independent effects through other factors (like Tetraspanins) and polyQ Atro also affects neurons through polyQ‐specific factors (like misfolding and aggregation), which put additional pressure on the autophagic system. Other polyQ proteins may not feed into the Ft/Hippo pathway, as in the case of Htt‐ex1‐93Q in flies.
DIA1(R1204X)-TG mice show progressive hearing loss and a reduction of HC.Figure 8(A), Age-related click and pure tone-burst (8, 16, 24, 32 kHz) ABR thresholds (dB SPL, mean SE) in DIA1(R1204X)-TG (5 weeks, n = 6; 10 weeks, n = 16; 25 weeks, n = 42) and control mice (5 weeks, n = 6; 10 weeks, n = 16; 25 weeks, n = 33). Note the progressive hearing loss of DIA1(R1204X)-TG mice starting at high frequency sounds from 10 weeks. **P= 0.0039 (24 kHz) and **P< 0.0001 (32 kHz) at 10 weeks and **P< 0.0001 (click), **P< 0.0001 (8 kHz), **P= 0.0001 (16 kHz), **P< 0.0001 (24 kHz), and **P< 0.0001 (32 kHz) at 25 weeks by Bonferroni's post hoc test following two-way ANOVA. (B) Representative SEM images of the organ of Corti at the basal turn obtained from DIA1(R1204X)-TG and control mice at 5, 10, and 25 weeks of age. White and red circles show IHC and OHC losses, respectively. Note OHC-dominant HC loss. Scale bars: 5 µm. (C) The percentages of remaining OHC and IHC in each turn in DIA1(R1204X)-TG and control mice at 25 weeks of age (n = 5). Schematic illustration shows the regions of the cochlea divided into three parts: Ap; apical turn, Md; middle turn, Bs; basal turn. **P= 0.0017 (OHC at Md), **P< 0.0001 (OHC at Bs), *P= 0.0117 (IHC at Ap), *P= 0.0330 (IHC at Md), and **P= 0.0003 (IHC at Bs) by Bonferroni's post hoc test following two-way ANOVA. (D) DPOAE (f2 frequency at 8, 12, 16, 20 kHz) amplitude (dB SPL, mean SE) in DIA1(R1204X)-TG (n = 37) and control mice (n = 30) at 25 weeks of age. *P= 0.0127, **P= 0.0029 (at 12 kHz), and **P= 0.0013 (at 16 kHz) by Bonferroni's post hoc test following two-way ANOVA. (E) High magnification SEM images of OHCs and IHCs at the basal turn of cochlea obtained from DIA1(R1204X)-TG and control mice at 25 weeks of age. Note the sparse and short (arrow) stereocilia of OHC, and sparse and fused (arrowheads) stereocilia of IHC in DIA1(R1204X)-TG mice. Scale bars: 1 µm in OHCs and 5 µm in IHCs.
Proteasomal degradation of acetylated histones following replication stress.Figure 6(A) Western blots of HeLa cells treated with proteasome inhibitor MG132 one hour before UV irradiation and lysed at different time points after UV (16 J/m2). Blots were stained with α-acetyl-lysine (top panel), α-histone H4 (middle panel) and α-tubulin (bottom panel) Quantifications of (B) histone H4 level normalized against tubulin levels. Average of 6 experiments and error bars represent SEM. Representative blots are shown in panel A Quantifications o (C) acetylation levels, normalized against tubulin levels. Average of 6 experiments and error bars represent SEM. Representative blots are shown in panel A (D) Fractionation experiments comparing histone and acetylation levels in chromatin and soluble fractions at the indicated time points after UV irradiation (16J/m2). HP1y was used as loading control for chromatin fraction, tubulin for soluble fraction (E) Quantification of histone acetylation and H2B levels in chromatin fraction. Non-irradiated samples were set as 1. N≥4, error bars indicate SEM (F) Representative western blots of fractionated cell extracts of HeLa cells at indicated time after UV irradiation (16J/m2) using histone modification specific antibodies. Loading controls for H3K9 and H3K27 are depicted in figure F (G) Quantification of the signal of the indicated histone modification specific antibodies stanings in the chromatin fraction. N≥4 and error bars represent SEM. Significant differences were calculated with T-test and are indicated with * (p<0.1), ** (p<0.05) and *** (p<0.01)
Ykt6 is a direct Atg1 kinase target.Figure 1A) Alignment of yeast and human Ykt6 protein sequence. The SNARE domain is labelled in yellow, phosphorylation sites are marked in green and red. Red phosphorylation sites match the Atg1 kinase consensus. B) Constructs used in in vitro kinase assay shown in C). C) GST, GST-Atg19 C-terminus (control), GST-Ykt6∆ac (1-194 aa), GST-SNARE∆ac (142-194 aa), GST-T-peptide, GST-SS-peptide and alanine (A) mutants were purified from E. coli and in vitro phosphorylated with immunoprecipitated Atg1-TAP bound to IgG magnetic beads. One representative experiment out of three is shown.
Mitochondrial fission occurs before dendritic injury and without AIF or cytochrome c release from mitochondria.Figure 2Mitochondrial fission occurs before dendritic injury and without AIF or cytochrome c release from mitochondria. Neurons were co‐transfected with vectors encoding (A) Mito‐DsRed2 and (B) MyrPalm‐mCFP plus (C) AIF‐GFP and the effects of SNOC were recorded using 3D time‐lapse imaging. Projection images zoomed in onto the dendritic arbor of one neuron (representative of n=42). Arrows indicate dendritic spines. The inset shows the soma at appropriate grayscale levels. The apparent brightening of AIF‐GFP fluorescence in the nucleus at 5 h was due to nuclear and cell body shrinkage rather than nuclear translocation of AIF. Scale bars, 10 μm. See also Supplementary Video 2. (D) 3D time‐lapse image reconstructions of mitochondria and cytochrome c in a dendritic arbor at indicated time points following SNOC addition. Scale bar, 10 μm. Five mitochondria were selected and motion tracked throughout the imaging series. (E) Traces show the ratio of mean GFP to DsRed2 emission intensity from each mitochondrion. Each trace color corresponds to the same colored mitochondrion. (representative of n=9) (F-G) Immunocytochemistry for staining anti‐AIF antibodies (red) anti‐NeuN antibodies (green) and nuclei labeled with Hoechst 33342 dye (blue) after (F) aged SNOC or (G) SNOC (150 μM; 4 h) treatment. Volume rendered 3D reconstruction of confocal image stacks are shown above, and a single confocal plane in grayscale below. Grid, 3.3 μm for (F) and 1.9 μm for (G), scale bars, 5 μm. (H) Ratio of nuclear to somal AIF signal was measured as mean fluorescence intensity in the center of nuclei divided by the mean fluorescence intensity in the cell body, outlined by the NeuN staining (excluding the nucleus). The bar diagram summarizes the mean±s.e.m. of 282 and 262 neurons measured in n=19 and 20 image stacks in three independent experiments for aged SNOC and SNOC, respectively (P∼0.99 not significant by Student's t‐test). (I) DEVDase activity. Purified cortical neurons were exposed to either 200 μM SNOC or 1 μM staurosporine, as positive control. The rate of zDEVD‐AMC caspase substrate cleavage per minute was monitored and expressed in arbitrary fluorescence units. Data show mean±s.e.m. for four independent experiments (*significance at P0.01 by ANOVA).
Fractionated neutral lipid from stimulated macrophages activates iNKT cells: A: Neutral (Fractions 1-3) and polar lipids (Fractions 4-6) were isolated from J774.Figure 4Neutral (Fractions 1-3) and polar lipids (Fractions 4-6) were isolated from J774.2 macrophages stimulated with thapsigargin, tunicamycin or DMSO and fractionated using solid-phase extraction (SPE) cartridges. Bar graph represents NKT cell activation by CD1d protein bound neutral lipids fractions isolated from thapsigargin- (black bars) or tunicamycin-treated (red bars) J774.2 cells. Polar lipid fractions induce very little IL-2 secretion by 2C12. In total, n=2 biological replicates were performed (two technical replicates per biological replicate). Graphs show mean ± SEM of each biological replicate. Graph represents enhancement of loading with neutral lipid fractions isolated from thapsigargin-treated J774.2 at acidic pH and reduced mCD1d loading at neutral pH. Graphs show mean ± SEM from n=2 biological replicates (two technical replicates per biological replicate). Neutral (Fractions 1-3) and polar lipids (Fractions 4-5), isolated from murine bone marrow derived macrophages stimulated with thapsigargin or DMSO and fractionated using solid-phase extraction (SPE) cartridges, were co-cultured with purified NKT1 subsets. Bar graphs represent IFN-γ release by NKT1 cells induced by CD1d protein bound neutral lipids fractions isolated from thapsigargin- (black bars) or DMSO-treated (white bars) primary murine macrophages. Polar lipid fractions induce a minimal amount of IFN-γ secretion by the NKT1 sublineage. In total, n=2 biological replicates were performed (two technical replicates per biological replicate). Graphs shows mean ± SEM of each biological replicate. Neutral (Fractions 1-3) and polar lipids (Fractions 4-5) were isolated from primary macrophages derived from human peripheral blood stimulated with thapsigargin or DMSO and fractionated using solid-phase extraction (SPE) cartridges. Bar graph represents IFN-γ release by NKT1 cells by CD1d protein bound neutral lipids fractions isolated from thapsigargin- (black bars) or DMSO-treated (white bars) human primary macrophages. Polar lipid fractions induce only minimal amounts of IFN-γ secretion by the NKT1 sublineage. In total, n=2 biological replicates were performed (two technical replicates per biological replicate). Graphs shows mean ± SEM of each biological replicate.