EMBO/sd-character-level-ner huggingface dataset:
AML-EV-contained miR-1246 suppresses protein synthesis in LT-HSC via the mTOR pathway.Figure 4qRT-PCR gene expression analysis showing the fold change of Pi3K/mTOR associated gene panel in KSL cells sorted from: (A) Molm-14-xenografted mice qRT-PCR gene expression analysis showing the fold change of Pi3K/mTOR associated gene panel in KSL cells sorted from: IF injected mice with Molm-14-EV relative to their controls and normalized to Gapdh endogenous control. Flow cytometric analysis showing the histograms (C) Flow cytometric analysis showing the MFI of intracellular pS6KRP in LT-HSC in: (D) Molm-14-xenograft (red, n=9) vs. non-engrafted control (black, n=7), Flow cytometric analysis showing the MFI of intracellular pS6KRP in LT-HSC in: IF injection of EV from Molm-14, U-937, HL-60 (red, n=7,3,3) or human CD34 cells (blue, n=4) normalized to the control contralateral femur with subtraction of background fluorescence. Flow cytometric assessment of protein synthesis showing the MFI of: (F) OPP, or (G) pS6RP in NIH-3T3 cells 72 hours after transfection with the indicated miRNA mimics. The results were calculated relative to control (miR-scramble) with the background fluorescence subtracted and performed with at least three independent experiments, in technical replicates. Flow cytometric analysis showing the MFI of Raptor in NIH-3T3 cells 72 hours after transfection with the indicated miRNA mimics. The data were calculated relative to miR-scramble and presented as means bars ± SEM and the background fluorescence was subtracted. Performed with at least three independent experiments, in technical replicates. Dual Luciferase reporter assay. NIH-3T3 was transfected with the miRNA mimics. Three hours later the cells were transfected with the Raptor 3′UTR cloned into the psiCheck-2 vector for a total of 48 hours. Data are presented as %RLU (relative luciferase units) of the miR-scramble control as means ± SEM from at least three independent experiments, performed in technical replicates. Cell-cycle flow cytometric analysis using Ki67/Hoechst-33342 staining of the percentage of LT-HSC in the G0 phase after nucleofection of cKit+ cells using the AmaxaTM P3 Primary Cell 4D-Nucleoeftcor kit, (J) Cells were nucleofected with miR-Scramble (CTRL) or miR-1246 mimic (n=3) for 72 hours, Cell-cycle flow cytometric analysis using Ki67/Hoechst-33342 staining of the percentage of LT-HSC in the G0 phase after nucleofection of cKit+ cells using the AmaxaTM P3 Primary Cell 4D-Nucleoeftcor kit, Cells were nucleofected with Anti-miR-Scramble (CTRL) or Anti-miR-1246 (n=5) and 1-hour later co-treatment with Molm-14 EVs for 72 hours.
Figure 3(A) Clcn3unc/unc/Clcn4-/- and Clcn3-/- mice died within 3-4 weeks after birth. Approximately 20% of the animals survived in either line (Clcn3unc/unc/Clcn4-/-, n = 136 and Clcn3-/-, n = 187). All Clcn3-/-/Clcn4-/- mice (n= 4) died within 1-2 weeks after birth. (B) Nissl-stained paraffin sections show progressive neuronal cell loss (arrows) that begins in hippocampal CA1 region of P14 Clcn3unc/unc/Clcn4-/- mice and results in a complete loss of the hippocampus at P28. Neurodegeneration progresses slower in Clcn3-/- mice Stobrawa et al, 2001() (scale bar: 200 µm). (C) Semi-thin sections of P28 retinae revealed degeneration of photoreceptor cells in the outer nuclear layer and outer and inner segment of Clcn3unc/unc/Clcn4-/-, but not of Clcn3unc/unc or Clcn4-/- mice (scale bar: 50 µm). RPE, retinal pigment epithelium; OS, photoreceptor outer segments; IS, photoreceptor inner segments; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer.
Figure 7A Changes in exon usage between shNTC and shH2A.B L1236 HL cells. Highlighted in red are RP genes. The significance (-log10 (P value)) is plotted against the fold change of exon usage (log2FC). B Differential exon usage of the CD44 gene. C. Differential exon usage of the RPL17 gene.
Figure 7(A) GSEA on RNA-sequencing data from the two comparisons (LL37 vs control, and LL37 vs LL37+RAPA) both show enrichment for chemokine signaling pathway in LL37 group. Significance was calculated by permutation test. (B) The mRNA expression levels of mouse chemokines (Cxcl11, Cxcl12, Ccl2 and Ccl3) in skin lesions (n=6 for each group). (C) The mRNA expression levels of rosacea-associated chemokines in primary human keratinocytes treated with LL37 ± RAPA in vitro. Data represent the mean ± SEM (from 3 replicates). (D) GSEA on RNA-sequencing data from rosacea skin lesions versus HS skin samples shows enrichment for chemokine signaling pathway in rosacea. Significance was calculated by permutation test. (E) Heatmap of upregulated chemokines and cytokines in rosacea skin lesions determined by RNA-sequencing. All results are representative of at least 3 independent experiments. Data represent the mean ± SEM. *P < 0.05, **P < 0.01. 1-way ANOVA with Bonferroni's post hoc test was used.
Figure 4A-C) Correlation of proteins with ELISA-measured t-tau concentration across samples within the Sweden (A), Magdeburg (B) and Berlin (C) cohorts. Proteins with a q-value below 0.05 are labeled in yellow. Proteins of the 40 protein signature are colored in red for those with higher abundance in AD CSF and in blue for those with higher abundance in non-AD CSF. D) Three cohort summary of proteins significantly correlating with ELISA-measured t-tau. Protein names given for the 29 proteins out of the 40 protein signature with significant (p < 0.05) correlation in each of the three cohorts. Pyruvat kinase PKM (PKM) was quantified in two isoforms and UniProt IDs are given in parenthesis. E) Overlap of proteins significantly differing by AD status with proteins significantly correlating to ELISA-measured t-tau. F) Annotation enrichment in the AD versus non-AD fold change dimension. Terms with positive enrichment means are enriched in AD CSF over non-AD CSF. Conversely, terms with enrichment means below zero are enriched in non-AD compared with AD CSF. Annotations filtered for significance of enrichment (p-value < 0.0.05) and term size (10-100 proteins per term) in all three cohorts. Protein abundance distribution of CSF showing the abundances of AD-modulated CSF proteins. Proteins of our 40 protein signature are highlighted in red (elevated abundance in AD) and blue (elevated abundance in non-AD). Proteins linked to glucose metabolism are highlighted in purple and labelled. Protein abundance distribution of brain showing the abundances of AD-modulated CSF proteins. Proteins of our 40 protein signature are highlighted in red (elevated abundance in AD) and blue (elevated abundance in non-AD). Proteins linked to glucose metabolism are highlighted in purple and labelled.
Figure 5Schematic Representation of CRISPR/Cas9-based Genome-Wide Screen under Doxorubicin treatment. Genes significantly enriched or dropped out after a 14-day treatment with Doxorubicin were identified by plotting as a Log2 fold change compared to untreated. Ranking was determined based on the Log2 fold score (Left). The top ten Doxorubicin sensitizers are indicated on the right with their respective fold change (Fc) in Log2. 293T cells were transfected with Flag-REV7 and GFP-SHLD1 (Left) or Flag-SHLD2 and GFP-SHLD1 (Right) expression vectors as indicated. 24h post-transfection cells were treated with DMSO or with 10uM of ATM inhibitor KU-60019 for 1 h prior to irradiation. 1h post-irradiation (10 Gy) nuclear extracts were prepared and REV7 or SHLD2 complexes were immunoprecipitated using anti-Flag (M2) Resin and then analyzed by immunoblotting using GFP and REV7 antibodies. U2OS EJ5-GFP cells were transfected with either siCTRL, siSHLD1 #1 or siSHLD1 #2. At 24hr post-transfection, cells were transfected with the I-SceI expression plasmid, and the GFP+ population was analyzed 48 hr post-plasmid transfection. The percentage of GFP+ cells was determined for each individual condition and subsequently normalized to the non-targeting condition (siCTRL). CH12F3-2 cells stably expressing either shCTRL, shSHLD1#1 or shSHLD1#2 were stimulated with a cocktail of cytokines (CIT) to induce class switching to IgA. The percentage of IgA+ cells was monitored 24 and 48h post-stimulation by staining with an anti-IgA antibody followed by flow cytometry analysis. U2OS DR-GFP cells were transfected with the indicated siRNAs. At 24hr post-transfection, cells were transfected with the I-SceI expression plasmid, and the GFP+ population was analyzed 48 hr post-plasmid transfection. The percentage of GFP+ cells was determined for each individual condition and subsequently normalized to the non-targeting condition (siCTRL).
The N-terminal domain of m152 directs the interaction with STING (A) Schematic representation of wildtype m152, wildtype CD4, CD4-m152 chimeric constructs, m152 glycosylation mutants, and m152 stalk mutants used in this study.Figure 3(A) Schematic representation of wildtype m152, wildtype CD4, CD4-m152 chimeric constructs, m152 glycosylation mutants, and m152 stalk mutants used in this study. CD4-m152 chimeras (1-6): The relevant domain of m152 was replaced singly or in combination with the respective domain of murine CD4. m152 glycosylation mutant (7): Asparagine (N) at position 83, 230 and 263 were mutated to Glutamine (Q). m152 stalk mutants (8-9): m152-Δstalk has a deletion of amino acids 300-326 (8), and for m152-GSstalk a glycine-serine linker ((G4S)5) was inserted to replace the stalk region (9). SP = signal peptide, NTD = N-terminal domain, TM = transmembrane domain, CTD = C-terminal domain. Branched symbols represent the three glycosylation sites of m152. (B) Cherry-STING, IFNβ-Luc, pRL-TK, cGAS-GFP (stimulated) or IRES-GFP (unstimulated) and indicated expression plasmids as shown in (A) were transiently expressed in 293T cells and a dual luciferase assay was performed. Data is combined from two out of three independent experiments and shown as mean ± SD. Data information: Student's t-test (unpaired, two-tailed), n.s. not significant, **p<0.01, ***p<0.001, ****p<0.0001 (C) 293T cells were co-transfected with expression plasmids for Cherry-STING and indicated chimeras as shown in (A). An anti-V5 IP was performed and samples were analyzed by IB with indicated antibodies. IB shown is representative of three independent experiments. (D) Cherry-STING, IFNβ-Luc, pRL-TK and either CD4, m152, m152-Δstalk or m152-GSstalk were transiently expressed in 293T cells. For stimulation, samples were co-transfected with cGAS-GFP, and unstimulated samples with IRES-GFP. Lysates were analyzed as described in (B). Data is combined from two out of three independent experiments and shown as mean ± SD. Data information: Student's t-test (unpaired, two-tailed), n.s. not significant, **p<0.01, ***p<0.001, ****p<0.0001 (E) 293T cells were co-transfected with expression plasmids for Cherry-STING and either CD4, m152, m152-Δstalk or m152-GSstalk. An anti-V5 IP was performed and samples were analyzed by IB with indicated antibodies. Immunoblot shown is representative of three independent experiments.
Jumpy blocks autophagic degradation.Figure 2(A) Proteolysis of long‐lived proteins in C2C12 myoblasts. C2C12 cells were transfected with control (scramble) or Jumpy siRNA (siJumpy), labelled overnight in media containing [3H] leucine, washed, incubated for 2 h in complete media (containing cold leucine) and incubated for 4 h in full or starvation media. Leucine release was calculated from radioactivity in the tricarboxylic acid‐soluble form relative to total cell radioactivity. Results shown represent mean±s.e.m. for combined data from three independent experiments. (B) Quantitation of p62 protein levels. C2C12 cells were transfected for 48 h with control (sc) or Jumpy siRNA and p62 levels analysed by immunoblotting, quantitated by densitometry and represented as a percentage of control. Data are mean±s.e.m. (n=4 independent experiments). (C) C2C12 cells were transfected for 48 h with control (sc) or Jumpy siRNA, incubated for 4 h with or without 100 nM Bafilomycin A1 in full media, lysed, probed for endogenous p62 and actin by immunoblotting and percentage of p62 were quantitated (mean±s.e.m., n=3). (D) C2C12 cells were transfected for 48 h with control (sc) (lanes 1, 2) or Beclin siRNA (lanes 3, 4) followed by a second transfection for 48 h with same siRNA and control (lanes 1, 3) or Jumpy siRNA (lanes 2, 4). Cells were lysed, probed for p62, Beclin and actin by immunoblotting and percentage of p62 were quantitated (mean±s.e.m., n=5). (E-M) C2C12 cells were transfected for 48 h with GFP, GFP‐Jumpy (Jumpy), GFP‐MTM1 (MTM1) or GFP‐MTMR2 (MTMR2), fixed and immunostained with anti‐p62 antibody (red). p62 puncta were quantitated by confocal fluorescence microscopy. Representative confocal images of GFP (E), GFP‐Jumpy (F), GFP‐MTM1 (G), GFP‐MTMR2 (H) transfected cells, immunostained for p62 (I), (J), (K) and (L), respectively. Scale bars, 5 μm. White lines represent outline of the cells. Quantitation of p62 puncta per cell (M). Bars, SEM (n=3 independent experiments, with an average of 30 cells per experiments). (N, O) C2C12 cells were transfected for 48 h with GFP, GFP‐Jumpy (Jumpy), GFP‐MTM1 (MTM1) or GFP‐MTMR2 (MTMR2), lysed, analysed for p62, GFP and actin by immunoblotting (N) and percentage of p62 were quantitated (mean±s.e.m., n=3) (O). (P, Q) C2C12 cells were transfected for 48 h with GFP or GFP‐Jumpy (Jumpy), incubated with or without 100 nM Bafilomycin A1 (BafA1) for 4 h, lysed, analysed for p62, GFP and actin by immunoblotting (P) and percentage of p62 were quantitated (mean±s.e.m., n=4) (Q). *P0.05, ***P0.001, †ns (t‐test).
Evaluation of enzymatic activity for the wild-type and mutated promiscuous enzyme, YihS.Figure 2YihS V314L + R315S mutant enzyme activity on D-mannose and D-lyxose. LC-MS was used to analyze YihS activity at saturating substrate concentrations to compare turnover rates on each substrate. Product formation was followed over time at a constant enzyme concentration. Turnover rates were calculated using linear regression (n=3 replicates for each enzyme, Dataset EV4). The error bars represent standard deviation (n=3) of the peak area. Turnover ratios of substrate conversion of D-lyxose / D-mannose are shown for the wild type YihS and mutant YihS enzymes. A ratio < 1 indicates a higher turnover rate on D-mannose compared to D-lyxose. Error bars represent standard error (n=3) calculated from the linear regression analysis.
Intrinsic type I IFN pathways are specifically suppressed in proliferating PCa cells from bone (A-C) Bulk RNA-seq analysis of DE genes (DEG) significantly suppressed (FDR < 0.Figure 2(A Bulk RNA-seq analysis of DE genes (DEG) significantly suppressed (FDR < 0.05; generalized linear model extraction (GLM) by edgeR) in proliferating RM1 bone-derived (BD) cells compared to parental RM1 and lung metastases. Tumor-intrinsic type I IFN signaling is suppressed in proliferating bone lesions (n = 1) compared to parental RM1 (n = 2) cells as shown by (A) camera analysis of Hallmark gene set responses associated with bone metastasis (bar length is the -log10FDR; false discovery rate). Color indicates mean log2fold change (FC) of all genes in gene set. Bar width is relative gene set size. Dashed line shows FDR = 0.05; (B) barcode plot showing enrichment of the Hallmark IFN-α response gene set in genes suppressed in bone metastases, using the signed log-likelihood ratio statistic (EdgeR). Bars indicate value of the statistic for each gene in the gene set; (C) log2FC values of key downregulated IRGs, with genes retained in dormant cells and classical downstream IFN signaling targets indicated. Altered IRGs directly involved in IFN-α/β production (light blue) compared to downstream IFN targets (dark blue) are segregated. p values represented as * < 0.05, ** < 0.005 and *** < 0.0005; GLM by edgeR. (D) Schematic of proliferating RM1 cell isolation from bone and lung metastases following IC injection for preliminary RNA-seq and orthogonal validation by qRT-PCR. (E) Heatmap of mean normalized voom expression of IRGs suppressed in RM1 bone metastases (n = 1) compared to lung metastases (n = 1) and parental cells (n = 2). (F) qRT-PCR validation of Irf7 and Irf9 downregulation in RM1 cells from bone metastases (RMI BD) compared to parental RM1 cells, lung metastases (RM1 lung) and naïve bone marrow (BM) (n = 3 mice per group). p values represented as ** < 0.005 and *** < 0.0005 (Student's t test). (G) qRT-PCR of Irf7 and Irf9 downregulation in parental RM1 cells, RM1 cells from bone metastases (RM1 BD) in WT and Ifnar1 deficient (-/-) mice, with naïve BM from WT and Ifnar1 (-/-) animals for reference. (n = 3 mice per group; n = 1 for -/- BM control). p values represented as * < 0.05 and ** < 0.005 (Student's t test).
Figure 3a) Network of proteins commonly altered or targeted during SARS-CoV-2 infection. Proteins displaying significantly altered changes in thermal stability were used as seeds for building the network. SARS-CoV-2 infection regulated phosphosites and their predicted upstream kinases as well as protein-protein interactions between viral baits and host prey proteins were used to propagate the network. After network propagation, clustering was performed to split the network into modules (see materials and methods). GO term enrichments for each module are shown bottom right. Protein thermal stability data is from this study, whereas phosphorylation/kinase regulation (Bouhaddou et al, 2020) and viral-human protein-protein interaction (Gordon et al, 2020b) data was acquired from previous work and used to propagate the TPP hits that seed this network. All TPP stability hits per module are displayed. b) Two statistical tests were then performed on the generated network from A. First, to assess whether the modules are enriched in TPP hits (Fischer test) and second, to determine if they received a significant amount of starting signal (KS test). Dashed red line represents significance threshold (p-adj <0.05). c) Heatmap showing the time point at which all TPP hits within each module defined in A. Green tile indicates significant changes in thermal stability.
Figure 1A) BW5147 reporter cells stably expressing human FcγR-ζ chain chimeras or BW5147 parental cells (grey/dashed) were stained with FcγR specific conjugated mAbs as indicated and measured for surface expression of FcγRs via flow cytometry. Bar graphs show means of three independent experiments performed in technical duplicates. Error bars = SD. B) FCS coating of an ELISA microtiter plate allows for suspension of subsequently added IgG. Plate bound IgG was quantified via ELISA. Bar graph shows means of one experiment performed in technical triplicates. Error bars = SD. C) Immobilized IC, immobilized IgG and IgG opsonized cells represent qualitatively similar ligands for FcγRs. Response curves of human FcγRs activated by opsonized cells (293T cells stably expressing CD20 + Rituximab [Rtx]), immobilized IC (rec. soluble CD20 + Rtx) and immobilized IgG (Rtx). sICs formed using monovalent antigen (rec. soluble CD20 + Rtx) do not activate human FcγRs. X-Axis shows sample concentration determined by antibody molarity. Y-Axis shows FcγR activation determined by reporter cell mouse IL-2 production (OD 450nm). Two independent experiments performed in technical duplicates. Error bars = SD. D) Schematic of used assay setups. BW5147 reporter cells expressing chimeric human FcγR receptors express endogenous CD69 or secrete mouse IL-2 in response to FcγR activation by clustered IgG. sICs are generated using mAbs and multivalent antigens. sIC suspension requires pre-blocking of an ELISA plate using PBS supplemented with 10% FCS (FCS coat, grey-dashed).
and holoElongator models.Figure 5A. GST-pull down assays of purified GST-tagged Elp3 SAM domain (aa72-389) and KAT domain (aa348-557) with untagged full length Elp1 and Elp451-2706. GSH resin and proteins were used as input controls. Lower panel shows 5% of the input and upper panel shows bound fractions. All samples were analyzed by SDS-PAGE and stained by Coomassie. Identities of respective proteins are indicated on the right. B. same as A, but using only GST-tagged Elp3 SAM domain with combinations and variants of Elp1, Elp2, and Elp456. C. same as A using GST-tagged full length Elp1 and truncations and deletions of Elp456. D. same as A using GST-tagged C-terminally truncated Elp1 in combination with truncated versions of Elp4.
Figure 1Alignment of amino acid sequences potentially encoded by the proximal 3′UTR of AGO1 mRNA. Conserved residues are shown in grey background; sequence of the peptide used to raise an antibody against the putative readthrough product (Ago1x) is shown. *, position of in-frame stop codons. Schematic of the construct used in luciferase-based readthrough assays. Last 696 nucleotides of coding sequence of AGO1 and 99 nucleotides of the ISR were cloned upstream and in-frame with firefly luciferase (FLuc). Demonstration of translational readthrough of AGO1 using luciferase-based reporter assay. Plasmid containing in-frame AGO1-TGA (or GCA)-ISR-FLuc were transfected in HEK293 cells and translational readthrough was detected as FLuc activity normalized to the activity of co-transfected renilla luciferase (RLuc). FLuc mRNA levels determined by RT-PCR are shown below. ***, P = 0.0001. Significance of the identity of the canonical stop codon in AGO1 translational readthrough. AGO1-ISR-FLuc constructs containing TGA or TAA or TAG were transfected in HEK293 cells and translational readthrough was quantified as described above. FLuc mRNA levels determined by RT-PCR are shown below. ***, P = 0.0002; **, P = 0.0004. First 57 nucleotides of ISR are sufficient to drive AGO1 translational readthrough. AGO1-TGA-FLuc constructs with different lengths of ISR (all in-frame with AGO1 and FLuc) were in vitro transcribed and in vitro translated using rabbit reticulocyte lysate. FLuc activity reflects readthrough activity. **, P = 0.012; *, P = 0.017. Demonstration of translational readthrough of AGO1 using fluorescence-based reporter assay. Plasmid containing in-frame AGO1-TGA (or GCA)-ISR-GFP were transfected in HEK293 cells and translational readthrough was detected as fluorescence. Scale bar, 50 um. The bar graph shows mean fluorescence intensities in cells transfected with indicated constructs. Fluorescence intensity was measured by flowcytometry. ***, P = 0.007.
Figure 2A Full-length polypeptides were translated in the presence of 2 μM HisTrx or HisTrx-SGTA using RNAs lacking a stop codon and either stabilised at the ribosome with cycloheximide (CHX) or released with puromycin (puro). Reactions were incubated with HisPur Cobalt resin, bound proteins eluted at high imidazole concentration, resolved by SDS-PAGE and visualised by phosphorimaging Filled circles indicate slower migrating species of radiolabelled products selectively enriched in CHX-treated samples. total translation reactions were treated with RNaseA and immunoprecipitated with anti-C99 antibody Following in vitro translation of the indicated proteins in the presence of 2 μM HisTrx-SGTA and recovery on HisPur Cobalt resin the eluted fractions were subjected to RNaseA treatment (C) -tRNA" and "+ tRNA" indicate tRNA-lacking and tRNA-bound protein species, respectively. D Following in vitro translation of the indicated proteins in the presence of 2 μM HisTrx-SGTA and recovery on HisPur Cobalt resin precipitation with hexadecyltrimethylammonium bromide (CTAB) (D) ;-tRNA" and "+ tRNA" indicate tRNA-lacking and tRNA-bound protein species, respectively.