EMBO/sd-character-level-ner huggingface dataset:
Figure 1(A) Expression in E. coli and purification of human GS (hGS). Protein was separated by SDS-PAGE and stained with Coomassie Blue dye. M: marker Precision Plus Protein Dual Color Standard (Biorad). From right Lanes 1-4: E. coli Bl21(DE3) cells containing the expression vector without (lanes 1 and 2) and with (lanes 3 and 4) the coding sequence of hGS. Samples were taken immediately before (lanes 1 and 3) and 3,5 h later (lanes 2 and 4) the induction of expression with isopropil-β-D-1-tiogalattopiranoside (IPTG) 0.7mM. The same number of bacteria was analyzed in each sample. Lane 5: isolated and purified inclusion bodies (4 μg). Adiacent boxed lane: Western Blotting analysis of GS. Kinetic study of the reaction catalyzed by recombinant hGS. The reaction, started by adding ATP, was linear for at least 10 min at 25°C (B). Kinetic study of the reaction catalyzed by recombinant hGS. Lineweaver-Burk plot reporting the hGS activity at the indicated concentrations of glutamate in the absence (), or in the presence of glufosinate (C) Symbols- (): 0.025 glufosinate; or 0.050 mM of glufosinate; or 0.065 mM of glufosinate; 0.075 mM of glufosinate. Kinetic study of the reaction catalyzed by recombinant hGS. Lineweaver-Burk plot reporting the hGS activity at the indicated concentrations of glutamate in the absence (), or in the presence of MSO Symbols- (): 2 mM MSO ; () 3 mM of MSO ; () 4 mM of MSO ; () 5 mM of MSO Kinetic study of the reaction catalyzed by recombinant hGS. The insets represent the secondary plot of the slopes of Lineweaver-Burk plot obtained at the indicated concentrations of glufosinate (C) or MSO (D) used for determining the inhibitor constant Ki. GS inhibition (%) in presence of increasing concentrations of glufosinate () or MSO () results from the average of at least three independent experiments (E). The control value for uninhibited hGS activity is 0.51 ±0.08 μmol/(min x mg protein). (F-K) Comparative analysis of GS structures from several organisms. Lateral (F) and top view (G) of the entire GS decameric (a dimer of pentamers) structure are reported in pink cartoon representation. (H) Cartoon representation showing the top view of the monomer-monomer interface hosting cofactors, substrates or inhibitors (Dataset EV1) participating to or inhibiting the conversion of glutamate to glutamine. In particular ADP (cyan), Phosphate ions (brown), MSO phosphate (P3S, yellow), phosphoaminiphosphonic acid-adenylate ester (ANP, white), glutamate (magenta), citrate (black) and the imidazopyridine inhibitor ((4-(6-bromo-3-(butylamino)imidazol(1,2-a)pyridin-2-yl) phenoxy) acetic acid, green) are reported in sticks representation, whereas Mn2+ ions are reported in pale blue spheres and Mg2+ in pale green spheres (involved in the coordination of the imidazopyridine inhibitor). (I) Superimposition of all the sampled 17 crystallized structures (Dataset EV1). (J) 2D representation of glutamate, P3S, glufosinate and glyphosate. (K) Zoomed view of the crystallized P3S binding region and the docked glufosinate and glutamate binding regions within the superimposed human (white cartoon) and Z.mays (pink cartoon) GS structures.
Figure 5A Scheme for activation of the integrated stress response (ISR) and effects on UPF1-dependent decay. B Density plots of changes in relative mRNA abundance as determined by RNA-seq following treatment of HEK-293 cells with 1 µM thapsigargin for 6hr (left) or 9hr (right). Genes were categorized as up-regulated by siUPF1total only or siUPF1LL only under basal conditions. Statistical significance was determined by K-W test, with Dunn's correction for multiple comparisons. C RNA-seq analysis of HEK-293 cells identifies populations of genes that decreased in abundance with thapsigargin treatment and were rescued by UPF1LL-specific knockdown. Indicated are genes that increased in abundance at least 1.4-fold (FDR < 0.05) with UPF1LL-specific knockdown under normal conditions. D RT-qPCR analysis of indicated transcripts following transfection of HEK-293 cells with indicated siRNAs and treatment with 1 µM thapsigargin for 6hr. Relative fold changes are in reference to vehicle-treated, NT siRNA. Black dots represent individual data points and error bars indicate mean±SD (n = 3 biological replicates). Dashed lines indicate log2(fold change) of ±0.5. PTC+ indicates the use of primers specific to transcript isoforms with validated poison exons
Figure 1Growth curves of the MCF7 (left) and T47D cells (right) upon gene silencing with siRNAs. siNC: non-targeting siRNA as a negative control, siHN-1: siRNA sequence 1 for HNRNPC, siHN-2: siRNA sequence 2 for HNRNPC, siLMNA: siRNA for LMNA as another negative control. Each sample has 3 replicates. Error bars represent mean ± SD. Relative protein levels of HNRNPC upon siRNA-mediated silencing, quantified from Western blots of 3 replicates. The error bars represent the ± SD. Growth curves of the Tet-on Crispr-Cas9-MCF7 (left) and Tet-on Crispr-Cas9-T47D cells (right). Expression of Cas9 was induced with 5uM doxycycline after transfection of the sgRNAs. sgNC: non-targeting sgRNA as a negative control, sgHN-1: sgRNA sequence 1 for HNRNPC, sgHN-2: sgRNA sequence 2 for HNRNPC, sgLMNA: sgRNA for LMNA as another negative control. Each sample has 3 replicates. Error bars represent mean ± SD. Relative protein levels of HNRNPC upon CRISPR-mediated gene knock-down, quantified from Western blots of 3 replicates. The error bars represent the ± SD. Anchorage independent cell growth assays of the HNRNPC-deficient MCF7 (top) and T47D (bottom) cells. Relative protein levels of HNRNPC upon shRNA-mediated silencing, quantified from Western blots of 3 replicates. The error bars represent the ± SD. Images, weights, and growth records of the xenograft tumor models in female NSG mice established from the MCF7 cells with lentivirus-mediated stable gene knock-down. Each group has 6 mice. The error bars represent the ± SEM. Images, weights, and growth records of the MCF7-derived xenograft tumors, which were subjected to periodic siRNA injection, starting from 2 weeks after transplantation of the cells. Each group has 4 mice. The error bars represent the ± SEM.
Figure 5(A) Mediator genes that possibly transmit genomic information to size phenotype are chosen by selecting genes whose RNA or protein level exhibits a triangular association among genotype, gene expression and wing size traits. (B) Selection of mediator RNAs and the linked eQTLs. RNAs co-associated with SNPs and wing size traits are further tested whether the SNPs are also associated with wing size traits (absCS and relCS) for each sex. The dotted lines indicate a significance threshold (p=0.05). (C and D) RNAi of CG34015 in the wing P-compartment significantly reduces the size in both sexes. The images are taken from female wings. Statistical significance was evaluated by Wilcoxon test. (E) Genome-wide association of CG34015 expression. Manhattan plot shows a strong association of CG34015 RNA with X chromosome in both sexes. P-values are plotted against ordered SNPs along the genome. eQTLs associated with CG34015 are designated as red dots. (F) Zoomed plot around the CG34015 gene region on X chromosome. eQTLs linked to CG34015 are mostly located upstream of the CG34015 gene region. The red arrow indicates the transcriptional direction. (G) SNP variation of CG34015-linked eQTLs. SNP states at each eQTL are depicted for each line using different colors (grey dots: reference SNP, blue dots: alternate SNP with a negative effect on RNA level). Lines are ordered from left to right as CG34015 RNA levels increase. Note that the eQTLs are highly linked with each other. (H and I) Segregation of CG34015 RNA levels and wing size (absCS) by the variation of an eQTL (X_16344746_SNP). P-values are shown. (J) Association of the eQTL (X_16344746_SNP) with population-wide, wing size variation among 143 DGRP lines. P-value and R2 are shown for each sex.
Figure 3(A) CasTA2.1 upregulates a genomically inserted GFP reporter. (B) Chromosomal gene targets activated by CasTA2.1 with bars showing the activation fold change and dots showing basal expression of each gene. Expression was quantified using RT-qPCR. (C) CasTA2.1 can mediate CRISPRi with appropriate gRNA designs by positioning different gRNAs relative to the target gene. A non-specific gRNA is used as the negative control. (D) Demonstration of multiplexed CRISPRa and CRISPRi using CasTA2.1 on a reporter containing GFP and mScarlet. Parental cells had low basal GFP and high basal mScarlet expression.
Figure 5(A) Plot of the most enriched TF binding motifs in regions that showed gained ATAC-seq signal at ChromHMM states 1, 8, 9 and 11 (predictive for enhancers). Color coding represents different TF classes. The size of the spots is proportional to the percentage of target sequences with a given motif. (B) H3K27ac (left) and DNA methylation (right) at NFAT binding sites. CLL cells showed both an H3K27ac enrichment and DNA-hypomethylation at NFAT target sites, suggesting a higher activity of TFs from the NFAT family in CLL. (C) Same as panel A but for lost ATAC-seq signal. (D) ATAC footprints for E2A, EBF and CTCF motifs from the Homer analysis. The E2A motif footprint (binding site of E protein family TFs like TCF4) displayed an increased binding signal in CLL while sites with the EBF and CTCF motif lost the ATAC signal. (E) Expression of the genes nearest to target enhancers with constitutively bound ("stable") CTCF vs. enhancers that lost CTCF in CLL. Loss of CTCF binding correlated with reduced gene expression. (F) Correlation matrix of simultaneously open regions computed from the scATAC-seq data. For all loci the pair-wise correlation coefficients were calculated and plotted to visualize how different loci were wired to each other. As an example, the TCF4 locus on chromosome 1 is depicted. (G) Enhancer-promoter rewiring at the NFKB2 locus. A switch of interactions between the NFKB2 promoter and two different enhancers in CLL (red line) vs. NBCs (grey line) was observed. Based on the CTCF ChIP-seq analysis both intronic enhancers at the NFKB2 and the FBXL15 gene show (nearby) constitutively bound CTCF, even though the targets of the two enhancers are switched.
Figure 5(A) Representative images of γH2AX positive nuclei in lungs of 6-month STZ induced diabetic mice, transduced with respective RAGE virions as described in Methods. The lungs were harvested 6-weeks after viral transduction. The empty vector served as control. Red represents γH2AX foci green cytoplasmic staining represents α-tubulin; blue nuclear staining represents DAPI (Scale 10µm). No primary antibody control served as a negative control (from control lung) of the staining. (B) Mean percentage of nuclei positive for the DNA-DSBs marker γH2AX in transduced lungs of 6-month STZ diabetic mice as described in Figure 5a (mean ± SEM; : p<0.01; N=8). (C) Representative images of lungs from 6-month STZ induced diabetic mice, transduced with respective RAGE virions as described in Methods. Lungs were stained for cellular senescence-associated β-galactosidase as described in Figure 5a and visualized by bright field and polarized light, where the accumulated senescent areas are recognized by its blue staining (Scale 40µm). (D) Quantitative analysis of persistent DNA damage associated inflammatory marker IL-6, in transduced lungs from 6-month STZ-diabetic mice, as determined by mean fluorescence intensity of respective group lungs (mean ± SD,: p<0.05, : p<0.001; N=8). (E) Quantitative analysis of Masson's Trichrome stain for lungs of 6-month STZ induced diabetic mice with respective RAGE virions as described in Figure 5F (mean ± SD, : p<0.001; N=8). (F) Representative images of Masson's Trichrome stain for lungs transduced with the respective RAGE expressing virions as described in Figure 5a and visualized by bright field and polarized light, where the accumulated ECM areas are recognized by its blue staining (Scale 40µm). The dotted lines represent the respective zoomed window. (G) Pressure-volume curves of diabetic and non-diabetic mice were determined using the FlexiVent system 6 weeks after transduction using respective the RAGE virions, as described in Figure 5a. The curves represent group averages (N=8). The lungs studied in Figure 5a, b, c and d are identical (mean ± SD; Vector alone vs. RAGE-EE; : p<0.05; N=8).
Fig.Figure 2A Experimental timelines for apyrase administration and behavioral study. B The effects of apyrase (40, 80 and 160 U/ml) infusion into cerebral ventricle on social interaction time (n = 8, 10, 10, 10 mice. Interaction F3, 34 = 3.917, P = 0.0167; Target F1, 34 = 0.0004, P = 0.9834; Drug F3, 34 = 2.295, P = 0.0954; For target, Apyrase 80 U/ml vs control, P = 0.0008, Two-way ANOVA with Tukey's post-test). C The effects of apyrase (40, 80 and 160 U/ml) infusion into cerebral ventricle on sucrose preference (n = 8, 10, 9, 11 mice. Treatment F3, 34 = 2.87, P = 0.0505; Apyrase 80 U/ml vs control, P = 0.007, one-way ANOVA with Fisher's LSD test). D Experimental timelines for apyrase/ATP administration and behavioral tests. E The effect of apyrase (40 U/ml) infusion into hippocampal on the social interaction time (n = 14, 20 mice. Interaction F1, 32 = 7.0754, P = 0.0121; Target F1, 32 = 0.68447, P = 0.4142; Drug F1, 32 = 18.211, P = 0.0002; Apyrase vs control, P < 0.0001, Student's t test). F The effect of apyrase (40 U/ml) infusion into hippocampal on the sucrose preference (n = 10, 16 mice. Apyrase vs control, P = 0.0003, Student's t test). G The social avoidance behavior in mice with ATP intra-hippocampal infusion after apyrase exposure (n = 14, 10, 10 mice. Interaction F2, 31 = 2.051, P = 0.1457; Target F1, 31 = 2.666, P = 0.1127; Drug F2, 31 = 3.711, P = 0.0359; For Target, Apyrase-ACSF vs Vehicle, P = 0.0387; Apyrase-ATP vs Apyrase-ACSF, P = 0.0099, Two-way ANOVA with Tukey's post-test). H The sucrose preference in mice with ATP intra-hippocampal infusion after apyrase exposure (n = 10, 8, 8 mice. Treatment F2, 23 = 3.32, P = 0.0540; Apyrase-ACSF vs Vehicle, P =0.0466, one-way ANOVA with Fisher's LSD test).
Figure 5A. An anti-KV10.1 antibody directed against an extracellular epitope labels a structure related to the centrosome (pericentrin-positive) and positive for Arl13b with an appearance suggestive of the primary cilium in HFF (human foreskin fibroblast) cells. Cells were serum starved for 24 or 48h, fixed, blocked (10% BSA) and then stained without permeabilization with anti-KV10.1 antibody recognizing an extracellular epitope. Immunostaining for pericentrin was performed after subsequent permeabilization and additional block. Scale bars: 10 µm (pericentrin), 20 µm (Arl13b). B. Anti-KV10.1 antibodies recognize ciliary structures in hTERT-RPE1 cells. After serum starvation for 48h, serum was reintroduced for 4h to induce KV10.1 expression and ciliary resorption. Cells were then fixed and stained with a mixture of anti-KV10.1 mouse monoclonals and a rabbit monoclonal anti acetylated α-tubulin. Scale bar: 2 µm. C. Specificity of the monoclonal anti KV10.1 (mAb 62) as revealed by staining MEFs from wild type (left) or knockout animals (right) Scale bar: 50 µm: D. Images from primary cilia of MEFs from KV10.1 knockout mice obtained using mAb62 and a rabbit polyclonal anti acetylated a-tubulin antibody. No recognizable structures were stained by mAb62. Scale bar: 1 µm.E. In contrast, mAb62 did stain ciliary-related structures (acetylated-tubulin-positive) in wild type mice. Scale bar: 1 µm.
Figure 7Heat map plots of the mRNA level changes of chaperones, ubiquitin-proteasome pathway, and autophagy genes (gene list in Table EV1F) in the indicated strains (data from Dataset EV1 and Dataset EV10). Selected genes for growth analysis in B are highlighted in red. Summary of the growth results of the indicated strains. -: no effect on growth. +: growth inhibition. ++: strong growth inhibition. Genes that are preferentially required in ρ0 puf3∆ cells are highlighted in red. Deletion of HSC82 selectively impairs the proliferation of ρ0 puf3∆ cells. Cells were cultured from 0.05 OD in SCD, and OD600 was measured every 2 hours. Data are mean ± SD from three biological replicates. Deletion of HSC82 selectively decreases ∆ψm in ρ0 puf3∆ cells. Cells were cultured to mid-log phase and stained with 125 nM TMRM for FACS analysis. The red ] highlight two cell lines for comparison. Cartoon illustration of the progressive activation of adaptive responses to convergently maintain ∆ψm in OXPHOS mutants.
Figure 1(A) Phenotypes of wild type (Col), kup9 mutants (kup9-1 and kup9-2) and the kup9-1/ProKUP9:KUP9 complementation lines (COM1 and COM2). Seeds were germinated and grown on low-K+ (LK, 50 μM) or high-K+ (HK, 5 mM) medium for 7 d. Scale bar, 1 cm. (B) Primary root length of the plants tested Data are means ± SE (n = 25, individual plants). Student's t-test (**P < 0.01) was used to analyze statistical significance, and "#" represents the control. (C) Diagram of KUP9 gene structure. The T-DNA insertion site in the kup9-1 mutant and the mutant site in the kup9-2 mutant are indicated by the triangle and arrow, respectively. The filled boxes and lines represent exons and introns, respectively. (D) RT-qPCR analysis of KUP9 transcript levels in various materials. Data are means ± SE (n = 3, biological replicates; each replicate contains 120 individual plants). (E) and (F) K+ content of the various plant materials tested in (A). Data are means ± SE (n = 3, biological replicates; each replicate contains 120-150 individual plants).
Figure 2Micropeptide CIP2A-BP was immune-stained with anti-CIP2A-BP antibody in MCF-10A and MDA-MB-231 cells either mock treated or treated with TGF-β. Micropeptide CIP2A-BP levels were determined by western blot with anti-CIP2A-BP antibody in MCF-10A and TNBC cells either mock treated or treated with TGF-β. MCF-10A cells were first transfected with anti-LINC00665 translation-blocking antisense oligo, then micropeptide CIP2A-BP was detected. Micropeptide CIP2A-BP levels were detected by western blot with anti-CIP2A-BP antibody in the indicated non-metastatic TNBC tissues (non-metastasis) and metastatic TNBC tissues (metastasis). Micropeptide CIP2A-BP expression in MCF-10A cells was used as the positive control. Kaplan-Meier overall survival curves for TNBC patients with high or low micropeptide CIP2A-BP expression in Suzhou cohort (discovery set, n = 112) and Guangzhou cohort (validation set, n = 105).
Figure 2(A) Fresh weight quantification of Arabidopsis and tobacco seedlings. Seedlings were pretreated with mock (water), Tyr-Asp (100 μM), Tyr and Asp (100 μM), Ser-Leu (100 μM), or Gly-Pro (100 μM) for 1 h prior an oxidative stress (catechin or H2O2). Each treatment was compared to its respective control, corresponding to plants grown in one 24-well plate (represented by the adjoined bars). Data information: Data are mean ± SEM of n=10-12 (seedlings). Unpaired two-tailed Student t-test was performed to compare treatments with control. Scale bar: 10 cm. (B) Catechin- (left upper panel) and (catechin-) Tyr-Asp-treated tobacco plants (right upper panel) grown for four days in liquid media. H2O2- (left upper panel) and (H2O2-) Tyr-Asp-treated tobacco plants (right upper panel) grown for two days in liquid media. All plants were 2-weeks-old before starting the stress treatment. Data information: Data are mean ± SEM of n=10-12 (seedlings). (C) Leaf series prepared from tobacco seedlings pretreated (1 h) with either mock or Tyr-Asp and exposed to catechin for four days. Data information: Data are mean ± SEM of n=10-12 (seedlings).
Figure 6A. A human brain random cDNA library was screened in a Y2H assay using C9orf72S as bait. A clone coding for aa46-205 of Rab1a, comprising most of the GTP-binding G-box domain and the C-terminal CC-motif was found to interact with C9orf72S. B. Cell lysates of HEK293 cells co-transfected with Myc-Rab1a and either empty vector, FLAG-C9orf72S or FLAG-C9orf72L were subjected to immunoprecipitation with anti-FLAG antibody. Bound protein was eluted from beads using excess FLAG peptide. Immune eluates were probed for FLAG-C9orf72 and Myc-Rab1a on immunoblots. The input levels of FLAG-C9orf72 and Myc-Rab1a in the transfected cells are shown (Inputs).* indicates remaining Myc-Rab1a signal after reprobing for FLAG-C9orf72. C. Cell lysates of HEK293 cells transfected with Myc-C9orf72S or Myc-C9orf72L were subjected to immunoprecipitation with anti-Myc antibody. The resulting immune pellet was probed for endogenous Rab1a. D. 35S-radiolabelled recombinant Myc-Rab1a protein loaded with vehicle, GDP or GMP-PNP was added to GST, GST-C9orf72S and GST-C9orf72L immobilized on glutathione-coated beads. 35S-radiolabelled recombinant Myc-Rab1a protein was visualized by autoradiography (top panel). Coomassie-stained GST, GST-C9orf72S and GST-C9orf72L in the pull-down samples are shown (bottom panel). The identity of the Coomassie protein bands was confirmed by mass spectrometry (# = E. coli DnaK Chaperonin; * = E. coli 60kD Chaperonin; Appendix Fig S3). Relative binding of Rab1a to C9orf72 was quantified from 3 independent experiments (Mean ± SEM; one-way ANOVA with Fisher's LSD test; ns, not significant; * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001). E. Increasing volumes of 35S-radiolabelled recombinant Myc-C9orf72L protein were incubated with equal amounts of GST-Rab1a immobilized on glutathione-coated beads in an equilibrium binding experiment. 8 µl of 35S-radiolabelled recombinant Myc-C9orf72L protein was incubated with GST as a negative control. Bound 35S-radiolabelled Myc-C9orf72L protein was visualized by phosphoimager. Coomassie stained GST-Rab1a and GST in the pull down samples are shown. Densitometry analysis of the amount of 35S-radiolabelled recombinant Myc-C9orf72L protein bound to GST-Rab1a in the different binding reactions was used to fit an equilibrium binding hyperbola (R2=0.94).
Unifying induction of MYCN and Wnt inhibitors in HGSOC and KD samples.Figure 6Comparative analysis of microarray data revealed consistent up-regulation of MYCN, the canonical Wnt inhibitors KREMEN2 and DKK3 as well as differentiation marker FOXJ1, AR and PGR in HGSOC cancer organoids as well as triple KD FT organoids. Differential expression was determined either by single-color microarray for the cancer samples (8 replicates) or dual-color microarray for the knockdowns (2 replicates) and is significant for all genes with p < 0.05. Transcriptional induction of MYCN is confirmed by qPCR for three patients between cancer organoids and normal FT organoids as well as triple KD organoids grown in OCM vs. FTM conditions. Depicted is the mean ±SEM of the normalized MYCN mRNA expression level of three biological replicates (n=3). ** p < 0.01, *** p < 0.001, two-sided Student`s t-test. Microarray data revealed that expression levels of Wnt target genes are downregulated in triple KD compared to WT control FT organoids. Differential expression determined by dual-color microarray for 2 biological replicates was significant for all genes with p ≤ 0.005. FOXJ1 expression was upregulated in HGSOC organoids in FTM compared to OCM as shown by qPCR analysis of 3 different patient samples. Data represent the mean ±SEM of technical triplicates.
Let‐7 and miR‐125 suppress hundreds of transcripts by binding their 3′ UTR and ORFsMicroarray analysis following introduction of let‐7 and miR‐125 in Dgcr8Δ/Δ GPCs.Figure 5Microarray analysis following introduction of let‐7 and miR‐125 in Dgcr8Δ/Δ GPCs. Upregulated transcripts are shown in red, downregulated transcripts in blue (adjusted P‐value 0.05). Data shown is for average of 3 replicates. Analysis of seed matches in the promoter, 5′ UTR, ORF and 3′ UTR of downregulated and upregulated transcripts. Data are presented as the mean number of seeds matches per kb of sequence for the listed groups of altered genes described in (A). P‐values calculated by the Wilcoxon rank‐sum test and Bonferroni corrected are shown for P 0.01 (***P‐value 1e−40, **P‐value 1.5e−8, *P‐value 1e−3). Box plots showing levels in Dgcr8Δ/Δ GPCs of probes upregulated, unchanged and downregulated upon addition of let‐7 and miR‐125. P‐values were calculated using the Fisher's exact test (***represents P 2.2e−16).