EMBO/sd-character-level-ner huggingface dataset:
(C) Representative pictures of myocardial immunofluorescence staining for the mitosis marker phospho-histone H3 (pH3), troponin T and DAPI in mice as indicated. Enlarged pH3 positive cardiomyocyte nuclei are shown separately. The arrows indicate pH3 positive cardiomyocyte nuclei. Scale bar: 25µm. The quantification of pH3 positive (+) cardiomyocytes per microscopic field is shown as bar graph (C´); *p=0.046.
(B) RNF26-mediated polyubiquitination of MITA depends on the enzymatic activity of RNF26. The 293 cells (5×106) were transfected with Flag-MITA (5 µg) and HA-Ub (1 µg) together with RNF26 or its mutants (1 µg each). IP under denatured conditions and ubiquitination assay was performed as described in (A).
Quantification of HA-APPsα and HA-APPsβ expression in total hippocampal homogenates and of soluble HA-APPs after UC normalized to β-tubulin. Note the comparable expression in AAV-APPsα and AAV-APPsβ injected mice. Age: 5-6 months. N=Number of animals. Data represent mean ± SEM. Data were analyzed by unpaired two-tailed Student's t test. nsp>0.05.
PCA and cluster analysis of the colonies based on initial flow cytometry gating revealed (F) cells lacking expression of analyzed lineage markers (Lin-).
Double immunolabeling of PDS5B (green, F-J) and SYCP3 (red) in Pds5AB cKO spread spermatocytes at the indicated stages.
(C) UbL domains targeted YFP substrates to degradation in the presence of an initiation region. The Y-axis plots abundance of the UbL-YFP-tail proteins normalized by a translation control, which is the same UbL-YFP protein without the tail. The ratio is equal to one for domains that are not recognized by the proteasome, and smaller than one if the UbL is recognized by the proteasome and the UbL-YFP-tail protein is degraded. YFP fluorescence is also corrected for plasmid copy number using RFP fluorescence. The median of YFP/RFP ratio for each construct was calculated from 10,000 cells collected in one flow cytometry run and reflected the abundance of YFP substrates in yeast cells as described in Methods. Data points represent mean values determined from at least three repeat experiments; error bars indicate s.e.m.
(I) Western blot showing protein depletion efficiency. Cre expression was induced with tamoxifen for 24 hours in proliferating Smc3 and Cdca5 conditional knockout immortalized fibroblasts.
A Immunoblot analysis of TRAF6-mediated ubiquitination of wild type, and mutant FOXP3 molecules. 293T cell lines were transfected with a normal HA-tagged Foxp3 construct, another encoding a ubiquitination resistant mutant in which all lysine residues were replaced by arginines (20R), or one of 20 single lysine-containing constructs with only the indicated lysine residue available for modification. These cell lines also received expression vectors encoding TRAF6 and ubiquitin molecules labeled with Myc and FLAG tags, respectively. Negative controls did not receive TRAF6 or expressed FOXP3 alone. Labeled FOXP3 proteins were pulled down from cell lysates (anti-HA) and ubiquitinated species were visualized by immunoblotting for FLAG.
(B) Stereological quantification of the number of cortical GFAP-positive astrocytes in female APP23p40+/+ (n=8) and APP23p40-/- (n=8) mice (left). Right: representative images of GFAP staining, scale bar = 200 µm. Mean ± s.e.m., statistical analysis: two-tailed unpaired t-test, p=0.1579.
qPCR analyses showing the expression (fold induction) of UPR signature genes compared to DMSO in human macrophage cell lines U937 stimulated either with thapsigargin, tunicamycin or DMSO for 4 or 8 hours. Graphs show mean ± SEM from n=2 biological replicates (two technical replicates per biological replicate).
Mean fluorescence intensity (MFI) comparing surface TLR2 expression on healthy/naïve (SC-naïve; blue; n = 7) and COVID-19 patient (SC-conv; red; n = 10) derived macrophages. Students t-test with Welsh corrections was used to calculate statistical differences as indicated.
(A) Expression of RagC WT, 3A, or 3E was induced by overnight addition of tetracycline in HeLa cells followed by cell size analysis using a particle size counter (Horizontal lines, box limits and whiskers represent the median, the 25th percentile ot the 75th percentile and the minimum to the maximum respectively. * p<0.05, # no significant difference, One-way ANOVA with Bonferroni multiple comparisons, n=4)
C Hypocotyl length of seedlings of AtWT, (C) transgenic 35S:GFP-ΔNt-HFR1 (35S:ΔNt-HFR1), two lines of 35S:PIF7-CFP (35S:PIF7 #1 and #2), and 35S:GFP-ΔNt-HFR1 35S:PIF7-CFP double transgenic (35S:ΔNt-HFR1 x 35S:PIF7 #1 and #2) seedlings grown under different R:FR. Seedlings were grown in W (R:FR > 1.5) for 7 days or for 2 days in W and then transferred to two W+FR treatments (R:FR 0.06 or 0.02) for 5 additional days. Values of hypocotyl length are the means ± SE of three independent biological replicates (at least 10 seedlings per replica). D Aspect of representative 7-day-old W-grown seedlings shown in C. Scale bar is 1 cm.
(B) Cells were stimulated with 10 μg/mL of MDP for 24 h, and NOD2 and IκBα proteins were measured in the cytosolic fractions by western blot analysis. The fold increase relative to unstimulated cells and normalized to tubulin of one representative experiment out of three is reported.
(I) Cumulative population doublings of KP and KPΔNF1 cells after transduction with sgTom- or sgPsat1-containing vectors (n = 4 biological replicates).
A, B S. cerevisiae wt, hsp42Δ, btn2Δ, and hsp42Δ btn2Δ cells expressing GFP‐VHL (green, A) or GFP‐luciferase‐DM‐NLS (green, B) were grown at 30°C and shifted to 37°C for 90 min in the presence of MG132, and protein localizations were recorded. DNA was stained by DAPI (blue), and the nuclear envelope was stained by Nsp1immunofluorescence microscopy (red). Solubilities of GFP‐VHL and GFP‐luciferase‐DM‐NLS were determined after stress application by Western blot using GFP‐specific antibodies. Solubility of actin was determined as a control. T, total fraction; S, soluble fraction; P, pellet fraction. Scale bars, 2 μm.
(E-L) Atg16L1-replaced wild-type or FIP200-KO MEFs were infected with Salmonella (MOI = 10) for 1 h and subjected to immunocytochemistry for Atg16L1 and Ub (E and F) or LC3 and Ub (I and J). The percentages of Atg16L1-positive (G and H) or LC3-positive per Ub-positive bacteria were enumerated (K and L). At least 50 Salmonella were counted. The average ± SD is shown for three independent experiments. Statistical analysis was performed by Student's t test. *, P < 0.05; NS, not significant. Bar, 5 µm.
Once internalized in donor cells (A) recombinant humanα-synucleinfibrils are mainly targeted to lysosomalvesicles (and found to a lesser extend in early and recycling endosomes). Lysosomalvesicles containing α-synucleinfibrils transfer from donor to (B) acceptor cells inside TNTs. Once in the acceptor cells, α-synucleinfibrils induce the formation of de novo α-synucleinaggregates (seeding). We propose that (A) α-synucleinfibrils accumulated inside lysosomes would impair their function leading to cellular stress (e.g., oxidative stress). In turn this would induce TNTs formation allowing spread to (B) healthy cells in order to lower the burden of α-synucleinfibrils inside lysosomes. α-synucleinfibrils would then escape lysosomes possibly by inducing LMP. Once free in the cytoplasm they would recruit soluble α-synuclein and induce de novo formation of α-synucleinaggregates. As a consequence α-synucleinfibrils would propagate from stressed to unstressed cells through TNTs contributing to the intercellular spreading of the pathology. Because endogenous α-synuclein when aggregated is targeted to lysosomesin vivo, we propose a similar mechanism occurring for endogenously formed aggregates (C).
Peak values of Ca2+ transients in MEFs transfected with ligase-active MARCH5(WT), MULAN(WT), and Parkin(W403A) [Parkin(WA)] or dominant-negative forms of MARCH5(H43W) [MARCH(HW)], MULAN(C339A) [MULAN(CA)], Parkin(C431A) [Parkin(CA)] (E) Error bars represent ±SD from six independent experiments. Data information: For graph the P values was determined by a Mann-Whitney U test. ns = not significant, *P < 0.05
(I) Lysates from HeLa cells stably expressing GFP-WIPI2B WT and GFP-WIPI2B FTTG mutant were incubated with agarose beads coated with PI(5) P and PI(3) P, eluted with SDS-PAGE sample buffer, and recovered proteins were assessed by western blotting using antibodies against GFP. See also Figure S6.
(c,d) Mean ± s.e.m. of the number (c) and size (d) of primary and secondary neurospheres from Ctrl, FIP200GFAP cKO, 2cKO and Trp53GFAP cKO mice at P28 (n = 3 mice for each).
Time-lapse images of agarose-embedded cells were taken at RT with supply of fresh PM Glc+ura medium throughout one budding cycle. Only (F) shows fixed cells immunostained for Sed5p. All cells produced the β'-COPVN•Dsl3pVC BiFC pair. Most strains contained plasmids encoding different organelle markers as indicated below. BiFC signals are pseudocolored green, organelle markers are pseudocolored red. Scale bar 5 µm.A. RFPHDEL, ER marker
(A) Widefield images of rhodamine‐PE labelled GUVs 30 min GUVs cluster only in the presence of the Atg5-Atg12/Atg16 and Atg5/Atg16 complexes.
Relationship between stress response and penetrance. Single-cell UPRE-RFP levels were measured in ~60 different mutant strains that we had identified as penetrance mutants with intermediate penetrance with defects in actin or vacuole morphology. Cells were binned into equal-sized bins, from low to high stress response, assessed as outlier or inlier, and clustered based on their penetrance profile (composed from the fraction of outliers in each stress-response bin). Each line plot represents a penetrance profile. * denotes the cluster with a profile most similar to wild-type. Insert pie charts show the proportion of mutant strains in each cluster.
E Left panel: Bar graph showing average of master segments counted after incubation of HUVEC cells with conditioned medium from LM1, HR1, 4T1 (Nfib high-expression and the respective Nfib low-expression or KO controls) and SUM159PT gNFIB shCTRL, SUM159PT gNFIB sh1 ERO1A, SUM159PT gNFIB sh2 ERO1A cells. Human VEGF121(0.4 ng/μl) was added to SUM159PT gNFIB sh1 and sh2 ERO1A cells, n = 2 technical replicates, means ± s.d., two-tailed Student's t-test. Right panel: Representative images showing tube formation four hours after addition of conditioned medium from HR1 gCtrl or g4Nfib.Scale bar 100 μm.
(d) Viable OT-II and Irgm1−/− OT-II CD4+ T cells 72 h after restimulation with OVA peptide in the presence of T cell-depleted APC samples.
(A) Top bar: gradient of population admixture of the indicated HKC strains of Black versus White individuals, on the basis of the SNP genotype analysis shown in Figure 1B, with the simplest assumption of two ancestries (K = 2). Gradient ranges from black to white colors for Black and White individually with no population admixture, respectively. Individuals with increasing proportions of White population admixture are represented with degrading shades of grey. Bottom: Heatmap of differentially expressed genes (characterized transcripts) in HKC strains from Black versus White individuals ordered according to genome admixture calculations. 164 genes with significantly different expression levels between the two groups (log2(Foldchange)|>0.58 and P value <0.05) were identified (Dataset EV1). Modified Z scores of the individual genes, as median-centered log2 Intensity values divided by standard deviation, are shown by color gradient variations. Unsupervised hierarchical clustering of genes was based on their modified Z scores, using Manhattan distance as distance metric and complete linkage method (Shirkhorshidi et al., 2015). 4 main groups of co-expressed genes were identified (purple triangles).
Schema of the in vivo analyses of tumor growth in mice (n=10/group) injected with FaDu cells. After tumor appearance, mice were injected intratumoral with high titer viruses encoding for control or antimiR-9 sequences. After two injections of virus mice were treated with Cetuximab (IP injections) and RT (4Gy dose) as indicated and then sacrificed 36 days after injections.
C. Representative images of transwell migration and invasion assays in Hela PTEN-/- cells and H4 cells stably transfected with PTENε, PTENε Y210L, or an empty control (magnification, 200x). The scale bars represent 50 μm.
(C) Myosin II was activated upon nutrient deprivation in MCF7 cells. MCF7 cells were cultured in serum containing DMEM medium (nutrient‐rich condition) or in Earle's balanced salt solution (EBSS; starved condition) for 2 h. The myosin II activity was significantly downregulated after EBSS‐starved cells were replenished with fresh DMEM medium containing 10% FBS for 2 h. The myosin II activity was quantified and expressed as a fold changes compared with the DMEM controls. Each value represents mean±s.e. of triplicates.
(H) qRT-PCR analysis of endogenous mRNA levels of QKI-5, QKI-6, and QKI-7 in human bronchial epithelial cell line BEAS-2B, LUAD cell lines A549 and H1299.
Overlap between 91 candidates identified in (E) and (C) identify 38 overlapping genes leading to mitochondrial elongation (hypertubulation in CTL-1, CTL-2 and rescued in OPA1S545R fibroblasts) and 53 genes that specifically rescue mitochondrial fragmentation in OPA1S545R fibroblasts.
G) The qRT-PCR results show oecircFNTA efficacy in J82 cells compared with PWPI vector (left panel) and oecircFNTA or oelinearFNTA in J82 cells (right panel).
B In vitro PARP1 activity assay in the presence of increasing concentrations (10, 25 and 50 μM) of purified macrodomains of human macroH2A1.1, macroH2A1.1 G224E mutant and macroH2A2. PARP1 activity is assessed by its auto-PARylation detected by an anti-PAR immunoblot. The naphtol blue staining shows the increasing amounts of purified macrodomain added to each reaction. A representative blot of one of three independent experiments is shown.
(b) WT, dnm1Δ and dnm1Δ mgm1Δ mutants were grown in SL medium as described in Methods, and samples (10 OD600 units) were taken at each time point. Protein extracts were prepared and equal protein amounts (20 μg) were subjected to SDS-polyacrylamide gel electrophoresis and immunoblotting with anti-GFP antibody.
(G) Representative images of primary intestinal organoids cultured at indicated conditions and timepoints. Scale bar: 100 μm. The organoids were co-cultured in a mixed medium of the supernatant with/without mIslr from CT26 cells and regular organoid culture solution at the ratio of 1:4. Quantification of organoids diameter at indicated timepoints. n>150 organoids at each timepoint.
B) Left panels: representative images of ORO staining on ACM and HC C-MSC in AM supplemented or not with 20μg/ml 13HODE, 5mmol/L NAC or both. Right panel: image quantification (n=13 biological replicates; Two-Way Anova). Data information: mean ± SEM. * p<0.05, ** p<0.01, *** p<0.001.
(G, H) HeLa CCl2 cells infected with either sh-control luciferase (shGL) or shβ-catenin lentiviruses were co-immunostained with anti-β-catenin and anti-Ect2 antibodies. Localization of β-catenin and Ect2 in the midbody of telophase cells was observed using a confocal microscope (G) and the signal intensity of Ect2 was monitored in the midbody of each knockdown telophase cell (H). Arrows indicate total β-catenin and Ect2 located at the midbody in telophase HeLa CCL2 cells (G). A dot density graph with a box plot shows the signal intensity of midbody-localized Ect2 in telophase cells. The box plot represents: central blue band, Median; box, Interquartile range; upper whisker, Maximum observation below upper fence; lower whisker, Minimum observation above lower fence; dot, Outlier. Approximately 70 cells from three independent experiments were measured. ***, p<0.001 (unpaired two-tailed t test); Scale bars, 10 μm. The value of the relative neck thickness is shown in the lower left corner of the merged images (G).
(F-H) Similar experiments as in (A-C) were performed in W1118 and TBPH-/- adult flies. The data from three independent experiments indicated the means ± S.E.M., **, p<0.01, one-way ANOVA. W1118 and TBPH-/- larvae were blotted using antibodies against phosphorylated p70S6K (T398), phosphorylated 4E-BP1 (T37/46) and tubulin antibodies.
The model outlining the key elements of the AAR and growth factor signaling pathways working in concert to regulate melanoma cell adaptation to asparagine restriction. Asparagine restriction activates GCN2, which phosphorylates and inactivates eIF2α, leading to the suppression of cap-dependent translation and the enhancement of cap-independent translation. Among transcripts showing increased translation are ATF4, multiple RTKs, and VEGFA. ATF4 orchestrates an expansive adaptive program to amino acid/asparagine restriction via inducing the expression of a large array of amino acid biosynthetic enzymes and transport proteins. Activated in response to enhanced RTK expression, MAPK signaling promotes mTORC1 activity and the consequent ATF4 translation through a concerted regulation of two distinct mechanisms- (i) suppression of the tumor suppressor tuberous sclerosis 2 (TSC2) activity, a negative regulator of mTORC1 function, and (ii) alleviation of GSK3-β-mediated c-MYC degradation, leading to the induction of c-MYC transcriptional target SLC7A5. SLC7A5 in turn enhances the uptake of EAA, providing critical cues for mTORC1 activation.
(C) Quantitative analysis of the fluorescence signal targeted to mitochondria in the panel (B).
Adult flies were fed with regular sucrose diet (RS) or high sucrose diet (HS), and survival percentage of adult flies of the indicated genotypes were examined. Kaplan-Meier survival analysis, n ≥ 40 for each group.
(F) qRT-PCR analysis with total RNA isolated from HT-29 cells transfected with control siRNA or IRGM siRNA or doubly transfected with IRGM siRNA and STAT1 siRNA or IRGM siRNA and STAT2 siRNA as indicated.
A. Asynchronous MEFs expressing Flag-tagged REV3L from the endogenous locus were lysed and REV3L was immunoprecipitated using anti-Flag (M2) antibodies. Co-immunoprecipitated proteins were analyzed by immunoblotting using the indicated antibodies. *: IgG light chain. This experiment was repeated 2-4 times.
(B) Cell immunostaining for HOPX in control, hFOXM1-FL and mutants in mice primary neural progenitors 3 days following lentiviral infection. White arrows represent GFP HOPX double-positive cells. Scale bar, 50μm. (C) Graph shows the percentage of HOPX+ cells among GFP+ cells (Control n=3; hFOXM1-FL n=6; hFLΔexon9 n=5; one-way ANOVA, F (2, 12) = 29.13, control vs. hFOXM1, P=0.0002; control vs. hFOXM1Δexon9, P=0.9080). (D) Cell immunostaining for HOPX in control, mFoxm1-FL and mutants in mice primary neural progenitors 3 days following lentiviral infection. White arrows represent GFP HOPX double-positive cells. Scale bar, 50μm. (E) (E) Graph shows the percentage of HOPX+ cells among GFP+ cells (Control n=4; mFoxm1-FL n=6; mFL+h exon9 n=4; one-way ANOVA, F (2, 11) = 8.163, control vs. mFoxm1, P= 0.4747, control vs. mFoxm1+h exon9, P= 0.0066). (
F Effects of NR and NAM supplementation (24 hours) on the NAD levels in DC fibroblasts. All values are presented as mean ± SD of four replicates. One-Way ANOVA was performed on DC cells in indicated conditions.
(B) After knockdown of Tollip, SH-SY5Y cells were treated with 5 μM antimycin A/10 μM oligomycin B (AO) or 25 μM Antimycin A (AA) alone for 2 hours, then fixed and stained for PDH E2/E3bp (green), TOM20 (red) and nuclei (blue). Images were captured using widefield immunofluorescence microscopy. Zoom insets represent 4x magnification and arrowheads indicate TOM20-positive MDVs.
(B) Quantification of progressive anterior-to-posterior PC loss. PC densities from 30-d npc1+/+ and 30-, 50-, and 70-d npc1−/− mice were quantified from five sections from two mice each. Error bars show standard deviation.
Quantification of apoptotic corpse in the meiotic compartment of CED-1::GF relative to the total number of meiotic cells, fed bacteria transformed with either empty-vector (con) or vector expressing dsRNA against frh-1 or isp-1 and left untreated or treated with gamma radiation 125 Gy (A Data information: Data are presented as mean ± SEM. For each panel at least N=3 and 10 worms for replicate and condition. * P < 0.05, *** P < 0.001 and **** P < 0.0001 vs untreated; # P < 0.05, ## <0.01, ### P < 0.001 and #### P < 0.0001 vs con. Not significant if nothing is specified. 2-way ANOVA Tukey"s multiple comparisons
WT B6 or B6 Signr3−/− (KO) mice were orally gavaged with NCK56, NCK2187, or SlpA on days −3 and −1, and 3% DSS was given in the drinking water. Mice were gavaged with bacteria or purified SlpA every other day for an additional three times and monitored for disease progression.G UniFrac analyses were used to calculate distances between the microbial communities of the different samples (day 10), and three‐dimensional scatter plots were generated by using PCoA. Light gray: WT + DSS; green: WT + DSS + NCK56; brown: WT + DSS + NCK2187; blue: WT + DSS + SlpA; aqua: KO + DSS; red: KO + DSS + NCK56; yellow: KO + DSS + NCK2187; purple: KO + DSS + SlpA. n = 4-6 mice/group.
(E and F) Wild-type primary B cells were treated with vehicle control (-), 5 µg/mL anti-kappa antibody or 1 µM LatA for the indicated time. Cells were lysed and analysed by SDS-PAGE followed by immunoblotting with phospho-PLCγ2 (pPLCγ2) and total ERK as loading control. The intensity of phosphorylated proteins, normalized to ERK, was referred to the respective unstimulated sample, set as 1.
C Gene Set Enrichment Analysis of RAD51-overexpression vs control HGSOC cell-lines (n=4). The top ten enriched pathways in RAD51-overexpression cell lines are listed on the right, ranked by the lowest false-discovery rate (FDR). The corresponding Enrichment Score (ES) is shown. See Table EV1 for details on all data points. Colouring denotes the top two enriched pathways. GO - Gene Ontology.
Representative immunofluorescence images of sections from ear skin tumors formed by SCC13 cells co-injected with EV or dnActRIB fibroblasts stained for K14 (left) or PDGFRα (right) (green) and mDia2 (red), counterstained with Hoechst (blue). Bar graph shows the percentage of mDia2-positive area. N=3.
(C) Dose response curve. H2K 2B4 cells were treated with serial dilutions of 11 hit compounds for 24 h and fixed, stained and imaged as described above. The average values and standard deviations of the mean fluorescent intensities of technical triplicates were calculated and a non-linear regression curve was fitted for the 3 compounds showing the highest increase in α-dystroglycan glycosylation (IIH6 immunostaining). Representative images of cells treated with 5 µM of the respective compounds as well as EC50 values are shown. HPFPTzone: (5Z)-5-[(3-ethoxy-4-hydroxy-phenyl) methylene]-3-(4-fluorophenyl)-2-thioxo-thiazolidin-4-one. 4BPPNit: 4-(4-bromophenyl)-6-ethylsulfanyl-2-oxo-3,4-dihydro-1H-pyridine-5-carbonitrile. DPPTzone: (5E)-5-[(2,5-dimethyl-1-phenyl-pyrrol-3-yl) methylene]-3-phenyl-2-thioxo-thiazolidin-4-one. Regression curve and EC50 values were calculated using GraphPad Prism 7 software. In images shown, green represents glycosylated α-dystroglycan and blue Hoechst staining. Scale bar, 50 µm.
A The number of differentially expressed olfrs from RNA sequencing in the MOE of the NC and miR-200b/a KD mice.
High-dimensional UMAP analysis shows a dynamic distribution of distinct myeloid clusters in eWAT during aging and the development of obesity. Three timepoints were analysed (8, 12, and 16 weeks) with annotated, colored clusters based on their expression of 13 different markers (n = 3 for each timepoint). Illustration of global topological relationships between clusters of different myeloid cell populations in the eWAT based on the result in D.
IL-1β concentration (pg/ml) in supernatants of SC-naïve individuals (n = 7) which were pre-treated for 24h with FCCP (1µg/ml) and H2O2 (10µg/ml). Macrophages were then incubated with LPS or S-Protein (4h) and nigericin (2h) as indicated in the figure. Control cells were left unstimulated or stimulated with LPS or S-protein without pre-treatment with FCSP or H2O2 (n = 6). For statistical analysis, Brown-Forsythe and Welsh ANOVA test was used.