EMBO/sd-character-level-ner huggingface dataset:
Induction of Syk deletion in the absence of CD19 and FoxO1 A, B B lymphocytes derived from mb1‐CreERT2;Sykfl/fl (first panel), CD19−/− (second panel), mb1‐CreERT2;Sykfl/fl;CD19−/− (third panel) and mb1‐CreERT2;Sykfl/fl;CD19−/−; FoxOfl/fl (fourth panel) mice were analyzed by flow cytometry after Tam treatment (see Materials and Methods).Figure 7A, B B lymphocytes derived from mb1‐CreERT2;Sykfl/fl (first panel), CD19−/− (second panel), mb1‐CreERT2;Sykfl/fl;CD19−/− (third panel) and mb1‐CreERT2;Sykfl/fl;CD19−/−; FoxOfl/fl (fourth panel) mice were analyzed by flow cytometry after Tam treatment (see Materials and Methods). (A) Flow cytometric analysis of BM‐derived (A) or splenic (B) B cells. IgM versus IgD dot plots representing B220+CD43−BM‐derived B cells are depicted in (A). The Fr. D, E, and F are distributed as indicated. A CD19 versus B220 dot plot is depicted in (B).C, D Statistical analysis of absolute B‐cell numbers derived from the (C) the BM, Fr F and (D) splenic M B cells of mb1‐CreERT2;Sykfl/fl (filled circles), mb1‐CreERT2;CD19−/− (open circles), mb1‐CreERT2;Sykfl/fl;CD19−/− (open squares) and mb1‐CreERT2;Sykfl/fl;CD19−/−;FoxOfl/fl (open diamonds). An asterisk (*) marks statistically significant differences (P 0.05); two asterisks (**) indicate a P 0.01. P‐values were obtained by a two‐tailed Student's t‐test. Shown are data from three to four mice per group. E, F Intracellular flow cytometric analysis of (E) Syk and (F) FoxO1 in mb1‐CreERT2;Sykfl/fl;CD19−/− (dashed line) and mb1‐CreERT2;Sykfl/fl;CD19−/−;FoxOfl/fl (solid line) B cells. In (E), B cells from mb1‐CreERT2mice served as a positive control for the Sykstaining (dotted line). In (F), T cells from mb1‐CreERT2;Sykfl/fl;CD19−/−;FoxOfl/flmice served as a positive control for FoxO1 expression showing that FoxO1 deletion was B cell specific.
Fig.Figure 1T-DNA insertion site in the PLDα6 gene and the complementation construct introduced into the T-DNA insertion mutant. Boxes denote exons and lines introns. PLDα6 transcript in WT, pldα6 and complementation line (PLDα6-COM). Leaf samples from 4-leaf stage rice (Dongjin background) were collected and the expression levels of PLDα6 were analyzed by normalizing to that of GAPDH. Values are means ± SD (n = 3 biological repeats). Seedling phenotype under GA treatment. After germination, Five-day-old seedlings with the same growth stage were transferred to 0.5 MS liquid media without or with different concentrations of GA3. PA from soybean was added to the media at a final concentration of 20 μM. Pictures were taken 7 days after transfer. The horizontal red line separates different plants and the vertical red scale bar represents 2 cm. Seedling length of WT, pldα6, COM, and PA-treated plants grown on 0, 0.1, 1 and 10 μM GA3 for 7 days. Values are means ± SD (n = 15 plants) from one representative of three independent experiments. E Fresh weight of 10 seedlings of WT, pldα6, COM, and PA-treated plants grown on 0, 0.1, 1 and 10 μM GA3 for 7 days. Values are means ± SD (n = 15 plants) from one representative of three independent experiments.
Figure 4A Scatter plots of pairwise comparisons of transcript expression in U2OS cells. Plots describe log2 fold change in transcript expression in mock and polyclonal MLLT6 knockout cells treated with and without IFN-γ as indicated. Horizontal axis shows log2 fold change in transcript expression and vertical axis represents statistical significance (log10 of p-value, n=3) with candidates nominated for further validation (red). B Correlation plots describing log2 fold change of transcripts in RNA sequencing (x axis) and Nanostring (y axis) measurements in mock cells +/- IFN-γ (left) and MLLT6 knockout cells with IFN-γ (right). Transcripts previously described to have a role in tumor immunity (red) and PD-L1 (blue) are represented on the plots as indicated. C Changes (log2 fold) in transcript expression (left) of select immune-related genes in U2OS mock or MLLT6 knockout cells treated with or without IFN-γ and total protein levels measured by immunoblotting (right). Numbers indicate band intensities normalized to GAPDH.
Figure 6(A-D) Panels (A) to (D) show the gene function prediction performance of the single-plant network versus sampled SRA networks for GO categories related to abiotic stimulus responses, development, biotic stimulus responses and hormone responses, respectively. Categories are shown in the context of the GO hierarchy and colored according to how well the single-plant network performs in comparison with 500 sampled SRA networks (see Materials and Methods). Solid arrows represent direct parent-child relationships in GO, dashed arrows represent indirect relationships. Grey nodes depict untested GO categories. White nodes depict GO categories for which there was insufficient information to score the performance of the single-plant network versus the sampled networks, i.e. categories for which the single-plant network and more than half of the sampled networks did not give rise to any predictions at q ≤ 10e-2.
Virion maturation requires acidic compartments.Figure 6(A) H1-Hela cells were infected at an MOI of 50 pfu/cell, and half of the samples were treated with NH4Cl. Cells were labeled with 35S-Methionine from 3 h.p.i. until collection at 5 or 6 h.p.i., and lysates were then separated on a 15-30% sucrose gradients. Fractions were then collected and the counts per minute (CPM) were analyzed for each fraction. Representative gradients from three independent experiments are shown. . (B) The three fractions representing the 150S peak in each experiment were pooled for plaque assay analysis. Data shown are the averages from three independent experiments. ( (C) Three fractions representing the 150S and 75S peaks were pooled and run on SDS-PAGE, and the 35S-Methionine labeled bands were visualized. The bands are labeled according to expected relative migration pattern, and VP2 is identified by its absence in the 75S peak. The ratio of VP0 to VP3 bands was analyzed from four independent experiments and plotted in arbitrary units. * p<0.05, ** p<0.01, *** p<0.0001.
Figure 7A Schematic diagram illustrating the generation of PdiS359A/S359A knockin (KI) mice by CRISPR/Cas9 mediated genome editing. B Sanger sequencing results of Pdi+/+ (WT) and PdiS359A/S359A (KI) mice. C (Left) Detection of Xbp1 mRNA splicing in mice liver after injection of Tm (50 ng/g, i.p.) for indicated times. Each lane represents independent animals. (Right) Quantification of Xbp1 mRNA splicing levels at indicated times. Data information: All data were shown as mean ± SEM from five biological replicates. , *p < 0.05, ***p < 0.001 (two-tailed Student's t-test). Detection of Xbp1 mRNA splicing in primary hepatocytes isolated from WT and KI mice treated with 0.2 μM Tg for 2 h (D) Detection of Xbp1 mRNA splicing in primary hepatocytes isolated from WT and KI mice treated with 0.5 μg/ml Tm for 2 h (E). Detection of Bip (F), Pdi (G), Erdj4 (H) mRNA levels by real-time PCR in the liver of the same animals after 24 h of Tm injection. Data information: All data were shown as mean ± SEM from five biological replicates. , *p < 0.05, ***p < 0.001 (two-way ANOVA, the post hoc Tukey's HSD test). Detection of Edem1 (I), Il6 (J), Chop (K) mRNA levels by real-time PCR in the liver of the same animals after 24 h of Tm injection. Data information: All data were shown as mean ± SEM from five biological replicates. , *p < 0.05, ***p < 0.001 (two-way ANOVA, the post hoc Tukey's HSD test). L Detection of serum alanine transaminase (ALT) levels of the same animals as in (C) after 24 h of Tm injection. Data information: All data were shown as mean ± SEM from five biological replicates. , *p < 0.05, ***p < 0.001 (two-way ANOVA, the post hoc Tukey's HSD test). M Hematoxylin and eosin staining of liver tissue the same animals as in (C) after 24 h of Tm injection (magnification × 400). Immune cell infiltration was indicated with a black arrow. Scale bar = 50 μm. Representative images were shown from five animals per group analyzed. N (Left) TUNEL assay of the same liver as in (M). Scale bar = 40 μm; Zoom box, scale bar = 5 μm. (Right) Quantification of apoptotic cells. 10 random fields were counted for each TUNEL-stained tissue sample. Data information: All data were shown as mean ± SEM from five biological replicates. , *p < 0.05, ***p < 0.001 (two-tailed Student's t-test). O Proposed model for the role of p-PDI. (Left) Under basal conditions, PDI is responsible for the catalysis of oxidative protein folding, and IRE1α is maintained by BiP chaperone in a monomeric inactive state. (Middle) Upon ER stress, Fam20C kinase is retained in the ER for rapid phosphorylation of PDI. Phosphorylated Ser357 induces an open conformation of PDI and turns it from a 'foldase' to a 'holdase', which is critical for preventing protein misfolding in the ER. Meanwhile, BiP dissociates and IRE1α is activated for XBP1 splicing. p-PDI directly binds to IRE1α to control the amplitude of IRE1α activity for adaptive UPR signaling. (Right) In PDI S357A mutant, protein aggregates form under ER stress due to incapable phosphorylation of PDI, and sustained ER stress induces hyperactivation of IRE1α, which leads to terminal UPR and apoptosis.
Fig.Figure 3Mouse brown adipocytes were differentiated from the freshly isolated cells in the stromal vascular fractions (SVF) of the interscapular BAT. (A) Upper: Oil red staining of the cells during differentiation. Lower: Relative mRNA levels of brown adipocyte markers were measured by qPCR. Mouse brown adipocytes were differentiated from the freshly isolated cells in the stromal vascular fractions (SVF) of the interscapular BAT. (B) Glycolytic intermediates were analyzed by GC/MS, after cultured with medium containing 10 mM [U-13C]glucose for 2 hours. Mouse brown adipocytes were differentiated from the freshly isolated cells in the stromal vascular fractions (SVF) of the interscapular BAT. (C) TCA cycle intermediates were analyzed by GC/MS, after cultured with medium containing 10 mM [U-13C]glucose for 2 hours.
Figure 4A Representative immunofluorescence images of foci induced by short CPT treatment (50 nM, 1 h) in U2OS cells treated with indicated siRNAs showing the effect of MMS22L depletion on the RAD51 foci. Scale bar: 5 μm.B Boxplot quantification of representative experiment in (A) (n = 3; nnuclei ≥ 78). Boxes indicate the 25-75 percentile and whiskers the 10-90 percentile; horizontal lines mark  the medians. Nuclei with ≥ 25 γH2AX foci (marker of replication stress sites) were analyzed. Statistical analysis: Mann-Whitney U test; *** P ≤ 0.0001. C, D Electron micrographs of a representative reversed fork (C) and a fork with extended ssDNA region at the junction (D) (arrow). P, parental duplex; R, regressed arm.E Frequency of reversed replication forks detected by EM. Similar results were obtained in one independent experiment; n = 2.F Distribution of ssDNA length at replication forks isolated from U2OS cells transfected with the indicated siRNAs and treated with 25 nM CPT for 1h. In brackets, the total number of analyzed molecules is given. Statistical analysis according to Mann-Whitney U test; ** P ≤ 0.01, n = 2.
Pull-down experiments to test the contribution of individual MICU2 residues to the interaction with MICU1 in the apo and Ca2+-bound states.Figure 3MICU2_WT (A), MICU2_K172A (B) were pulled-down by GST-MICU1 both in the absence and presence of 2 mM Ca2+. MICU2_EF1mut was not pulled-down by GST-MICU1 in the presence of 2 mM Ca2+ (C), while MICU2_EF2mut was pulled-down by GST-MICU1 both in the absence and presence of 2 mM Ca2+ (D). MICU2_R352A was not pulled-down by GST-MICU1 in the absence of 2 mM Ca2+ (E), while MICU2_E329A was pulled-down by GST-MICU1 both in the absence and presence of 2 mM Ca2+ (F). MICU2_D185A was partially pulled-down by GST-MICU1 in the presence of Ca2+, while they showed a strong interaction in the absence of 2 mM Ca2+. MICU2_E196K was not pulled-down by GST-MICU1 in the presence of 2 mM Ca2+, while a strong interaction was observed in the absence of 2 mM Ca2+.
a) ROS production determined by flow cytometry using the fluoroprobe dihydrorhodamine 123 (DHR) in LPS-primed BMMs stimulated with PA-BSA (0.figf3(a) ROS production determined by flow cytometry using the fluoroprobe dihydrorhodamine 123 (DHR) in LPS-primed BMMs stimulated with PA-BSA (0.5 mM) in the absence or presence of ROS inhibitor APDC (50 μM). MFI, mean fluorescence intensity. (b) ELISA for IL-1β on LPS-primed BMMs stimulated with PA-BSA in the absence of or presence of increasing concentrations of APDC. (c) immunoblotting for caspase-1 p10 and IL-1β p17 on LPS-primed BMMs stimulated with PA-BSA in the absence of or presence of increasing concentrations of APDC. (d) ELISA for IL-1β. Resting or LPS-primed BMMs were stimulated with PA-BSA (0.5 mM) in the absence or presence of the ROS inhibitors APDC (10 or 50 μM) and N-acetyl-cysteine (NAC, 5 or 25 mM) or the NAPDH oxidase inhibitor diphenyleneiodonium (DPI, 5 or 25 μM). Values are expressed as mean ± s.d. In a, one of two independent experiments is shown. In each of b-d, the results are representative of three independent experiments.
Figure 9A Mice received 10 min of training in an environment with two identical objects and received a retention test 24 h later in which one object was replaced with a novel one. LAMTOR1 shRNA-injected mice exhibited a significant deficit 24 h after training, which was reversed by ML-SI1 treatment (N = 7-18 mice). B % freezing for different experimental groups in context memory (N = 7-19 mice). Data information: Data with error bars are represented as means ± SEM. Statistical significance was assessed by two-way ANOVA with Tukey's post-test. *P < 0.05, ***P < 0.001, as compared to shSc; #P < 0.05, ###P < 0.001, as compared to shLAMTOR1. C Model illustrating the proposed role of LAMTOR1-mediated inhibition of lysosomal Ca2+ release via TRPML1 in the regulation of lysosome motility in dendrites and synaptic plasticity. More lysosomes with lower pH move faster and longer distances in LAMTOR1 KD neurons, as compared to control neurons. LAMTOR1 interaction with TRPML1 inhibits its Ca2+ release and LAMTOR1 KD increased its Ca2+ release and dynein-dependent dendritic lysosome trafficking (①). LAMTOR1 KD-induced TRPML1 Ca2+ release also activates CaN, efficiently dephosphorylating GluA1 and targeting internalized AMPARs to lysosomes for degradation, thereby regulating synaptic plasticity (②). Figs 3B, 5A, and 8C were created with BioRender.com.
B.Figure 1(A) Antibacterial activity of NGJ1, T6SS-1- and T6SS-2- strains against target bacteria. (B) Schematic representation of two different T6SS apparatus encoding gene clusters (named as T6SS-1 and T6SS-2) of NGJ1. The locus id (as per Burkholderia genome database) of upstream and downstream genes of the T6SS locus is provided. The gene disrupted in the respective T6SS mutants is marked by triangle (△). (C, D) Immuno-blots showing secretion profile of Hcp-1 (associated with T6SS-1) and Hcp-2 (associated with T6SS-2) proteins, using protein specific peptide antibody in different NGJ1 strains. The T6SS-1- mutant was defective in Hcp-1 secretion (due to polar effect of plasmid integration, synthesis was also prevented) but proficient in Hcp-2 secretion. On the other hand, the T6SS-2- mutant was defective in secretion of Hcp-2 (although the protein was synthesized) but proficient in secretion of Hcp-1.
Mammalian translation factor eIF3F is upregulated in symptomatic SCA8 BAC mice and can regulate RAN translation A qRT-PCR showing Eif3f expression levels are increased 2-fold in SCA8 cerebellar white matter compared to SCA8 cerebellar grey matter (n=3, **p<0.Figure 5qRT-PCR showing Eif3f expression levels are increased 2-fold in SCA8 cerebellar white matter compared to SCA8 cerebellar grey matter (n=3, **p<0.01; mean ± SEM; unpaired t test). B Schematic diagram showing constructs used for eIF3F knockdown experiments. All constructs have a tag in each reading frame. Red M indicates methionine (ATG) initiated reading frames. C Dot blot detection of polySer expressionin using α-FLAG antibody showing decrease in RAN polySer but not ATG-polySer when HEK293 cells are co-transfected with eIF3F siRNA. D Quantification of polySer detection (n=5, * p<0.01; n.s. no significance; mean ± SEM; unpaired t test). E Detection of polyAla expression using α-HA antibody showing decrease in RAN polyAla but not ATG-polyAla when HEK293 cells are co-transfected with eIF3F siRNA. F Detection of polyGP expression using α-FLAG antibody showing a decrease in RAN polyGP when HEK293 cells are co-transfected with eIF3F siRNA.. G Quantification of polyAla and polyGP detection (n=5, * p<0.01; n.s. no significance; mean ± SEM; unpaired t test).
Figure 1Chromatin-enriched fractions of HeLa cells exposed to the indicated genotoxic agents for 1 h were subjected to immunoblot analysis using SUMO2/3 antibody. except that cells were preextracted in 0.2% Triton X-100, fixed and immunostained with SUMO2/3 antibody. Representative images are shown. Scale bar, 5 μm. Relative DPC levels in cells treated were quantified using a KCl/SDS precipitation assay (mean±SD; n=3 independent experiments). HeLa cells were treated with formaldehyde for 1 h, and where indicated propagated for an additional h in the absence of formaldehyde (recovery). Cells were then fractionated into soluble and chromatin-enriched fractions and immunoblotted with SUMO2/3 and SUMO1 antibodies. using HeLa cells transfected with non-targeting control (CTRL) or SPRTN siRNAs. Experimental set-up for global proteomic analysis of formaldehyde-induced SUMOylation changes. Mass spectrometry-based analysis of formaldehyde-induced SUMOylation changes. His10-SUMO2 conjugates from HeLa/His10-SUMO2 cells subjected or not to formaldehyde were purified on Ni-NTA under stringent conditions and analyzed by mass spectrometry. All proteins displaying significant upregulation of SUMOylation in response to formaldehyde treatment were subjected to network analysis using the STRING database, at the default interaction confidence setting of 0.4. Proteins not connected to the network were omitted. SUMO2 conjugates from HeLa/His10-SUMO2 cells subjected or not to formaldehyde or heat stress for 1 h were purified and immunoblotted with indicated antibodies. SUMO target proteins displaying at least 2.5-fold upregulation of SUMOylation after formaldehyde treatment were mapped to the human proteome, which was annotated with Gene Ontology (GO) terms. Enrichment analysis was performed to find terms significantly enriched for formaldehyde-induced SUMOylated proteins, using Fisher Exact testing with multiple-hypothesis correction to achieve a final q-value of <0.02. A relative score was derived from a combination of the enrichment ratio and the q-value. A full list of all enriched terms is available GOBP, GO Biological Processes; GOCC, GO Cellular Compartments; GOMF, GO Molecular Functions.
Fig.Figure 5A, B. NIH-3T3 cells were either untransfected or transfected with FLAG-SMAD1 together with increasing (doubling) amounts of Opto-ACVR1WT or Opto-ACVR1R206H (5-20 ng). Post transfection, cells were either kept in the dark or exposed to blue light for 1 h. In (B) cells were treated in the presence or absence of 1 µM LDN-193189. Data information: In all panels, Western blots of whole cell lysates were probed with antibodies against pSMAD1/5 (that detected both endogenous and pFLAG-SMAD1), SMAD1 (which detects both endogenous and FLAG-SMAD1), HA (which detects the Opto-receptors), pSMAD3 (which detects pGFP-SMAD3), SMAD3 (which detects GFP-SMAD3) and either Tubulin or Actin as the loading controls. Lane numbers are given below each blot for clarity. C. NIH-3T3 cells were either untransfected or transfected with either FLAG-SMAD1 or GFP-SMAD3 together with Opto-ACVR1B*. Post transfection, cells were either kept in the dark or exposed to blue light for 1 h, in the presence or absence of 50 µM SB-505124 or 1 µM LDN-193189. Data information: In all panels, Western blots of whole cell lysates were probed with antibodies against pSMAD1/5 (that detected both endogenous and pFLAG-SMAD1), SMAD1 (which detects both endogenous and FLAG-SMAD1), HA (which detects the Opto-receptors), pSMAD3 (which detects pGFP-SMAD3), SMAD3 (which detects GFP-SMAD3) and either Tubulin or Actin as the loading controls. Lane numbers are given below each blot for clarity.
Figure 4(A) Western blot analysis of whole cell lysate (input) and Flag-specific immunoprecipitation (IP) from 293T cells co-transfected with expression plasmids encoding V5-NLRP3 and Flag ZsGreen-PB1-F2 from indicated viral strains for 24 h Representative blot of three independent repeats is shown. (B) Quantification of the NLRP3 IP band was normalized to the respective NLRP3 input band (n=3 independent biological replicates, normalized to the respective input). Mean values +- SD are indicated. (C) Alignment of PB1-F2 sequence from two avian IAV strains (VN (H5N1) and Sha (H7N9)) and two mammalian/human IAV strains (PR8 (H1N1) and Wyo (H3N2)) (Combet et al., 2000). * above the sequence shows a conserved amino acid between the four strains while ↑ on the bottom shows a different amino acid for PR8 in comparison to the other strains. (D) Schematic diagrams showing the truncated versions of Flag ZsGreen PB1 F2 used in Figure 6E (not to scale). (E) 293T cells co transfected with expression plasmids encoding different truncation mutants for Flag Zsgreen-PB1-F2 from VN (H5N1) and V5-NLRP3 were processed as described in (A). Representative blot of three independent repeats is shown.
Mutagenesis and functional assays identify determinants of catalysis.Figure 5(A) Sites for targeted mutagenesis on the flap domain and active site Lys88 residue. The central ribbon model provides context for the conserved loop (left) and epitopes distant from the active site in the flap domain (right). Residues subjected to mutagenesis are shown as stick models, with dotted envelopes in the central panel for emphasis. Docked pRab8a (yellow ribbons, right) highlights its proximity to mutagenized residues in the flap domain. The sequences of the conserved loop motif are shown below the left panel for several PPMs with known 3-D structures (PPM1H, PPM1K, PPM1B, PPM1B). (B) In vitro malachite green assay time course of recombinant PPM1H mutants against 16 μM pThr72 phosphorylated Rab8a protein (GTPγS, left), 32 μM pThr72 phosphorylated Rab8a peptide (middle), or 32 μM pThr73 phosphorylated Rab10 peptide (right). The experiments were repeated 4 times. The error bars represent SE of mean of the technical replicates. (C) 3 μg of recombinant wild-type or indicated mutant PPM1H proteins were resolved on 4-12% Bis-Tris gradient gel and stained with Instant Blue Coomassie. (D) HEK293 cells overexpressing indicated constructs were treated and analysed as described in Figure 4E. (E) Activity of 25 nM PPM1H flap domain mutants against 16 μM pThr72 phosphorylated Rab8a protein (GTPγS) using the malachite green assay time course. The experiments were repeated 3 times. The error bars represent SE of mean of the technical replicates. Quality of purified proteins (right panel) is shown using the protocol in (C). (F) HEK293 cells overexpressing indicated constructs were treated and analysed as described in (D), with pRab10 analysed in the upper panel, and pRab8a in the lower panel.
Figure 2The Figure shows a hierarchical clustering of nitrate-phosphoproteins with differential abundance at 5- or 20-min in response to nitrate. Phosphoproteins were clustered using the Euclidean distance method with average linkage. The resulting clusters are shown as a heat map, where vertical bars and numbers to the right of the map denote the group (composed of all terminal nodes in the hierarchical tree) of phosphoprotein (correlation > 0.9) with a selected profile in early (5-min) or late (20-min) nitrate-response (log 2 fold change for each phosphoproteins upon nitrate treatment are referred to KCl-treated samples). Functional Gene Ontology (GO) categories significantly enriched (hypergeometric test with FDR, p < 0.05) in each cluster are highlighted to the right of the group.