EMBO/sd-character-level-ner huggingface dataset:
Figure 1(A) ROS burst in wild type (WT) and 35S::GLV1 and 35S::GLV2 after elicitation with 100 nM flg22. The kinetic of ROS production is shown over 40 min -/+ SE, n=8 biological replicates. RLU = Relative light units. Similar results were obtained in 3 independent experiments. (B) 5-day-old seedlings of WT, 35S::GLV1 16-1 and 35S::GLV2 8-5 were treated with 100 nM flg22 for 7 days before measuring fresh weight. Shown is the mean of relative fresh weight compared to the MS medium control -/+ SD, n=8 biological replicates (one-way ANOVA, Dunnett post-hoc test, a-b p<0.05). Similar results were obtained in 3 independent experiments. (C) Mean colony-forming units (cfu) of Pto DC3000 COR- 4 days after spray infection of WT, 35S::GLV1 16-1 and 35S::GLV2 8-5; n=12 biological replicates from 3 pooled experiments -/+ SD (one-way ANOVA, Dunnett post-hoc test; a-b, p<0.01). (D) 5-day-old seedlings of Col-0Cas9 WT and CRISPR glv were treated with 100 nM flg22 for 7 days before measuring fresh weight. Shown is the mean of relative fresh weight compared to the MS medium control -/+ SD, n=40 biological replicates from 5 pooled experiments (Students T-test, ***p<0.001). (E) Ethylene production was measured in Col-0Cas9 WT and CRISPR glv 3.5 h after elicitation with 500 nM flg22. Shown is the mean of n=12 biological replicates from 3 pooled experiments -/+ SD (one-way ANOVA, Tukey post-hoc test, a-b, p<0.001; a-c, p<0.001; b-c, p<0.001). Ethylene production was normalized to the average of WT responses to flg22 set as 1. (F) 5-day-old Col-0Cas9 WT and CRISPR glv seedlings were treated with 100 nM flg22 or co-treated with 1 μM GLV2 for 7 days before measuring fresh weight. Shown is the mean of relative fresh weight compared to the MS medium control -/+ SD, n=30-32 biological replicates from 2 pooled experiments (one-way ANOVA, Tukey post-hoc test, a-b p<0.001). (G) Ethylene production was measured in Col-0Cas9 WT and CRISPR glv 3.5 h after elicitation with 500 nM flg22 or co-treatment with 1 µM GLV2. Ethylene production was normalized to the average of WT responses to flg22 set as 1. Shown is the mean of n=11-12 biological replicates from 3 pooled experiments -/+ SD (one-way ANOVA, Tukey post-hoc test, a-b p<0.01). (H) Mean cfu of Pto DC3000 COR- 4 days after spray inoculation of Col-0Cas9 WT and CRISPR glv; n=12 biological replicates from 3 pooled experiments-/+ SD (Students T-Test, *p<0.05).
Human C/EBPβ also confers resistance to inhibitory effect of IFN-γ and IL-4 on human iTreg generation A Flow cytometry of intracellular FOXP3 staining (right) or isotype control (left) in human CD4+CD25-CD45RA+ naïve T cells transduced with control retrovirus (Min) or retrovirus encoding hC/EBPβ and cultured for 2 d under conditions as indicated.Figure 6Flow cytometry of intracellular FOXP3 staining (right) or isotype control (left) in human CD4+CD25-CD45RA+ naïve T cells transduced with control retrovirus (Min) or retrovirus encoding hC/EBPβ and cultured for 2 d under conditions as indicated. Representative experiments are shown in the left panel and pooled data with mean±s.e.m are shown on the right. Histograms are gated for CD4+NGFR+. Statistical analysis was performed using unpaired two-tailed t test. (*p<0.05; ns, not significant) Flow cytometry of CFSE-labeled human CD4+CD25- T cells (responder) stimulated 4 d alone or together with NGFR+-sorted human CD4+CD25-CD45RA+ naïve T cells (suppressor) transduced with control retrovirus (Min) or retrovirus encoding hC/EBPβ and cultured for 5 d in the presence of hTGF-β, anti-hIFN-γ and anti-hIL-4 Abs. Histograms are gated for CFSE+. The ratio of responder to suppressor is 1:0.2 (top) or 1:0.4(bottom). Data are representative of two independent experiments with consistent results
Figure 6(A) Primary CD4 T cells infected with HIV-1 (Transmitted/Founder strains CH058 and CH077, and the primary strain vKB18) were incubated with the indicated bNAbs and surface levels were analyzed by flow cytometry. The numbers indicate the % of bNAb+ cells among infected (Gag+) cells. The percentages of infected cells were 27%, 30% and 6% for CH058, CH077 and vKB18, respectively. One representative experiment is shown. (B) CH058-infected CD4 T cells were cultured with the indicated WT or E430G mutant antibody and NHS. After 24h, the C3 surface levels on infected (Gag+) cells were determined by flow cytometry. One representative experiment is shown. (C) CH058-, CH077- and vKB18-infected primary CD4 T cells were incubated with the indicated antibodies and NHS. After 24h, C3 surface levels on infected cells were analyzed by flow cytometry. Results are expressed as MFI of staining on infected (Gag+) cells. Each dot represents a donor of CD4 T cells. n=6 donors of CD4 T cells. (D) Primary CD4 T cells infected with the indicated HIV-1 strains (CH058, CH077 or vKB18) were cultured with the indicated WT or E430G mutant antibodies and NHS or HIHS to determine complement-mediated cytotoxicity. After 24h, the percentage of infected (Gag+) cells in each condition was determined by flow cytometry. For each antibody, the % of CDC was calculated at 24h as the % of disappearance of Gag+ cells in NHS compared to HIHS. Each dot represents a single donor of CD4 T cells. n=6 donors of CD4 T cells.
Figure 6(A) A549 and H1299 cells were co-transfected with QKI-5 and ORF-only of TGFβR1, and then subjected to western blot assays for detection of QKI-5 and TGFβR1 expression. (B) After serum-starvation for 24 h, the above-treated A549 and H1299 cells were stimulated with or without TGF-β1 (5 or 10 ng/ml) for 24 h. The expression of p-SMAD3, SMAD3 and EMT markers (N-cadherin and Snail) was determined using western blot. β-actin was used as an internal control. (C-F) In the presence or absence of TGF-β1, the migratory and invasive capabilities of above-treated A549 and H1299 cells were evaluated by Transwell assays Migrated and invasive cells were stained and counted under a light microscope. (G and H) qRT-PCR analysis of TGFβR1 mRNA in 61 LUAD tissues and matched paracarcinoma tissues (G) and T/N mRNA expression of TGFβR1 in 61 paired LUAD tissues (H). T, LUAD tissues; N, paracarcinoma tissues. Y axis represents the log10 transformed fold change of mRNA expression of TGFβR1. Central dotted line of the violin plot represents median, and upper and lower dotted line represents quartile (G). (I) Correlation between QKI-5 and TGFβR1 mRNA expression in 61 paired LUAD tissues. X and y axes represent the log10 transformed fold change of T/N expression ratios of QKI-5 and TGFβR1 mRNA, respectively. (J) Kaplan-Meier survival curves of LUAD patients (n=865) with high or low expression levels of TGFβR1. The log-rank test was used to compare the difference between two groups.
a) TUNEL staining of apoptotic cells in penumbral brain slices in vivo (scale bar, 100 μm) and (c) in cultured neurons when H3R was inhibited with thioperamide (scale bar, 50 μm) after tMCAO and OGD/R.f2(a) TUNEL staining of apoptotic cells in penumbral brain slices in vivo (scale bar, 100 μm) when H3R was inhibited with thioperamide (scale bar, 50 μm) after tMCAO. TUNEL-positive cells are green, and all cells are stained with 4',6-diamidino-2-phenylindole (blue). (b,d) H3R was inhibited by thioperamide, and cleaved caspase-3 protein expression is shown by western blot in WT and H3R−/−mice after tMCAO (b) (n=6-7 per condition; **P0.01, ***P0.001 with analysis of variances (ANOVAs) followed by Bonferroni/Dunn post hoc test) (c) TUNEL staining of apoptotic cells in cultured neurons when H3R was inhibited with thioperamide (scale bar, 50 μm) after OGD/R. TUNEL-positive cells are green, and all cells are stained with 4',6-diamidino-2-phenylindole (blue). (b,d) H3R was inhibited by thioperamide, and cleaved caspase-3 protein expression is shown by western blot in WT and H3R−/−mice in cultured neurons subjected to OGD/R (d) (n=8 per condition; *P0.05, ***P0.001 with ANOVAs followed by the Bonferroni/Dunn post hoc test). Data are presented as mean±s.e.m. Full-size blots are shown in Supplementary Fig. 10.
Figure 2(A) smFISH images showing selected viral mRNAs in reactivating neurons probed for ICP27 (immediate-early), UL30 (early), UL36 (true-late) or cellular markers, Ntrk (neurons), or Col3a1 (fibroblasts). Nuclear DNA was visualized using DAPI (blue). To induce reactivation, latently-infected SCG-derived cultures were simultaneously treated with LY294002 (20 μM) and WAY-150138 (20 μg/mL) and cultured for 48 hrs prior to processing for smFISH. Scale bar, 10 μm. (B) The percentage of cells positive for a viral mRNA or for GFP fluorescence was quantified at different time points and displayed as bar graphs with mean ± SEM. Latently-infected cultures were untreated (latent) or treated with LY294002 and WAY-150138 for 18, 48, or 72 hrs. Each dot corresponds to the value from each biological replicate. Data information: P values equal to or less than 0.05 were considered significant, asterisks denote statistical significance (***, p<0.001; ****, p<0.0001). P values are calculated using two-tailed unpaired Student's t test. (C-D) Reactivating neurons probed simultaneously for two separate viral productive cycle mRNAs. Signal overlap is evident as white or yellow nuclear foci in the merge. Bar, 10 μm. In (D), the percentage of positive nuclei with overlapping UL30 and UL36 smFISH signals were quantified as the mean ± SEM (n=3 biological replicates). Data information: P values equal to or less than 0.05 were considered significant, asterisks denote statistical significance (***, p<0.001; ****, p<0.0001). P values are calculated using two-tailed unpaired Student's t test.
Figure 1(A) The simplified biochemical network comprises amino acid biosynthetic and respiratory pathways in central carbon metabolism and a specialized metabolic pathway, aliphatic glucosinolate biosynthesis, studied in this paper. (B) Each pathway in central carbon and specialized metabolism vary in gene number. (C Y1H identified TFs that interact with promoters of genes in each pathway in central carbon and in the specialized metabolic pathway. D) Y1H identified TFs that interact with promoters of genes in each pathway in central carbon and in the specialized metabolic pathway. (E) Majority of TFs bind to promoters from two or more metabolic pathways.
Figure 6(A) Insulin stimulates the IR-Akt pathway activating Akt which phosphorylates Afadin at S1795. This allows the docking of HDAC6, which subsequently inhibits IR-Akt signaling through regulation of total IR protein in a deacetylase-dependent manner. (B) Adipocytes lacking Afadin have increased IR availability due to the loss of HDAC6-mediated inhibition, consequently enhancing insulin signaling and action.
53BP1 self-assembles at nucleation centers in a concentration-dependent manner.Figure 5Cry2-mCherry-53BP1 1203-1972 W1495A optoDroplets undergo fusion events upon light induction. Fusing optodroplets are highlighted by green arrowheads and in the magnified insets. Concentration-dependent optoDroplet formation exemplified by two neighboring cells that differ in their Cry2-mCherry-53BP1 1203-1972 W1495A expression level. Cells were imaged at 15 seconds intervals. Quantifications of optoDroplet formation in lowly expressing cells (mClow) and highly expressing cells (mChigh) from single-cell QIBC analysis of 3 independent experiments are shown with mean (solid line) and standard deviation from the mean (dashed lines) indicated. Cry2-mEGFP-53BP1 1203-1972 W1495A assembles into FokI-induced DNA repair compartments within seconds after light induction. FokI was induced for 1h where indicated before cells were light-activated and imaged at 15 seconds intervals. Red arrowheads mark the FokI-induced lesion with mCherry accumulation.
a, Diagram of Apg12 C-terminal mutants.figf3a, Diagram of Apg12 C-terminal mutants. b, Δapg12 cells were transformed with the mutant plasmids and their lysates were immunoblotted with anti-HA antibody. c, Autophagic activity was measured as described for Fig. 1d. d, Transport of pro-API to the vacuole was examined by immunoblotting with anti-API antiserum. The positions of pro-API and mature API are indicated.
Expression of hTauAT causes Tau phosphorylation and mis-sorting in aged mouse hippocampi.Figure 1A. The diagram shows the domain structure of wild type human Tau and the location of the substitution A152T (hTauAT). Tau domains are broadly divided into the N- terminal "projection domain" (amino acids M1-Y197) and the C-terminal "assembly domain" (amino acids S198-L441). The C-terminal assembly domain includes three or four pseudo-repeats (~31 residues each, R1-R4), which together with their proline-rich flanking regions (P1 and P2) constitute the microtubule-binding region. Repeat R2 (exon10) and the two near-N-terminal inserts (N1 and N2, exons 2 and 3) may be absent due to alternative splicing. B. Blots showing expression of hTauAT (hTau, Mr ~67 kDa) and/or endogenous mouse Tau (mTau, Mr ~45-55 kDa) in the hippocampus of 10 month old heterozygous (+/-) and homozygous (+/+) hTauAT mice in comparison to age matched control (Ctrl) detected by pan-Tau antibody K9JA. Actin at 42 kDa serves as loading control. C. Quantification of (B). Ratio of hTau / mTau indicating a higher hTauAT -expression in homozygous mice (~1.8) compared to heterozygous mice (~1.0). Each bar represents an average of n=4 animals, error bars represent SEM. D. Quantification of mRNA levels in hippocampi of heterozygous and homozygous hTauAT 16 month old mice (n = 4 per group). E. Distribution of hTauAT visualized by the human Tau specific antibody HT7 in the hippocampal CA3-region of hetero- and homozygous hTauATmice at the age of 14 month. Notice the mis-sorted hTauAT in cell bodies and dendrites (arrowheads) of pyramidal neurons of the CA3 region and immunreactivity of the axons (mossy fibers, asterisks). By contrast the control shows no immunoreactivity. F. Elevated hTauAT -expression causes Tau-pathology inside area CA3 of the hippocampus. Note the increase in Tau phosphorylation probed with the antibody against pT217, a site upstream of the repeat domain. Arrowheads point to areas with phosphorylated and mislocalized Tau in cell somata of pyramidal neurons in area CA3. G. Tauopathy detected by antibody AT180 specific for phosphorylation sites pT231+pSer235 (arrowheads denote somato-dendritic mislocalized tau). H. Antibody PHF-1 (phosphorylation sites pSer396+pSer404) illustrates pathological phosphorylation of Tau due to hTauAT expression especially in stratum lucidum (asterisks) of area CA3 and pyramidal neuroncell bodies (arrows). I. Tau phosphorylation detected by antibody AT8 (pS202+pT205) in stratum pyramidale of area CA3 (arrowhead). Note that immunoreactivity increases with expression level.
Matrin3 splicing function requires PRI and RRMs A Schematic representation of Matrin3 with its domains in the wild‐type construct (WT) and the mutants used in overexpression, mPRI which has corresponding domain deleted in each one of the mutants, mPRI where the PRI motif is mutated from GILGPPP to GAAAPPA, ΔZ1 where the first zinc finger is deleted, ΔRRM where both the RRM domains are deleted and ΔZ2 where the second zinc finger is deleted.Figure 6A Schematic representation of Matrin3 with its domains in the wild‐type construct (WT) and the mutants used in overexpression, mPRI which has corresponding domain deleted in each one of the mutants, mPRI where the PRI motif is mutated from GILGPPP to GAAAPPA, ΔZ1 where the first zinc finger is deleted, ΔRRM where both the RRM domains are deleted and ΔZ2 where the second zinc finger is deleted. B Western blot of the overexpressed Matrin3 and its mutants probed with anti‐FLAG and anti‐actin antibodies. C, D RT-PCR analysis using primers specific to the ST7 exon 11 (C) and ABI2 exon 8 (D) splicing reporters, respectively, of samples where FLAG‐tagged Matrin3 and its mutants have been overexpressed. Quantification of at least three replicates for each condition is shown as a histogram of the percentage of exon inclusion. *P 0.01 compared with control sample (−). E Western blot of overexpressed FLAG‐tagged Matrin3 and PTB, in control siRNA and PTB/nPTB siRNA‐transfected cells, probed with anti‐FLAG to detect overexpressed proteins, anti‐PTB to detect the knockdown levels and anti‐actin to confirm protein loading. A titration of control sample (lane 1-12.5%, lane 2-25%, lane 3-50% and lane 4-100%) is also included to assess the levels of knockdown. The band present above the PTB doublet in lane 6 is FLAG‐tagged PTB. F, G RT-PCR analysis using primers specific to the ST7 exon 11 (F) and ABI2 exon 8 (G) splicing reporters, respectively, of samples where Matrin3 or PTB expression vectors were co‐transfected, in control siRNA and PTB/nPTB siRNA‐transfected cells. Quantification of at least three replicates for each condition is shown as a histogram of the percentage of exon inclusion. *P 0.01 when compared with control sample in each condition (‐).
Figure 7A. EYFP-Parkin translocation requires Ca2+. PC6 cells plated in separated compartments of the same dish maintained during the experiment in Krebs containing 1 mM Ca2+ or in Ca2+ free Krebs containing 5 μM BAPTA-AM, were treated with A&O (both 10 μM). Images (left panels) demonstrate that EYFP-Parkin translocation was significantly weaker at zero Ca2+ at 195 min after A&O treatment (compared with 15 min; note the spatial heterogeneity values shown in photos). Spatial heterogeneity analysis (right panel) demonstrated that Parkin translocation was significantly decelerated in zero Ca2+ conditions (n = 25-27 cells; difference between the curves P < 0.0001, two-way ANOVA; lines show mean, dashed area shows SEM) B. EYFP-Parkin translocation accelerates in the presence of the L-type Ca2+ channel agonist Bay K 8644 (5 μM; n = 19-21 cells; difference between the curves p < 0.0001, two-way ANOVA; lines show mean, dashed area shows SEM) Treatment of neurons for 10 min with 100 μM glutamate/10 μM glycine led to excessive Ca2+ entry into the cytosol, visualised by increased cytosolic GCaMP7b fluorescence Treatment of neurons for 10 min with 100 μM glutamate/10 μM glycine led to excessive Ca2+ entry into the cytosol Glutamate-induced Ca2+ entry was not affected by Miro1 WT or Miro1 EF overexpression; n = 31-48 neurons per group; ns, Kruskal-Wallis test) E, F Glutamate treatment (100 μM glutamate/10 μM glycine for 10 min) led to the appearance of a few Parkin-positive strong puncta co-localising with mitochondrial marker (white arrow), imaged 2.5 h after treatment (E). Further analysis demonstrated that the expression of Miro1-EF mutant decreased and Miro1 WT increased the number of neurons with EYFP-Parkin-positive mitochondria following glutamate treatment (F; n = 6-10; **P < 0.01 and ****P < 0.0001, one-way ANOVA) G. Glutamate treatment (100 μM glutamate/10 μM glycine for 10 min) was associated with an increased number of mitochondrial co-localisation with the autophagosome marker EGFP-LC3B in neuronal soma at 5-6 h later, which was not observed in Miro1-EF-expressing neurons (n = 8 dishes; *P < 0.05, one-way ANOVA) H. Glutamate treatment (30 μM glutamate and 1 μM glycine for 10 min) was also associated with a significant loss of mitochondrial density in axons at 6 h later, which was reversed by Miro1-EF but not by Miro1 WT (n = 60-90 axons from 6-9 dishes; **P < 0.01, ***P < 0.001 and ****P < 0.0001, Kruskal-Wallis test) I. Glutamate treatment (100 μM glutamate and 10 μM glycine for 10 min) led to a decline, at 6-8 h, in cytosolic ATP measured using ATP/ADP sensor Perceval HR, which was reversed by Miro1-EF but not by Miro1 WT (n = 59-60 neurons from 6 dishes; **P < 0.01, Welch"s A J. Glutamate (100 μM glutamate and 10 μM glycine for 10 min) induced excitotoxicity (24 h after treatment) that was partially reversed by Miro1-EF but not by Miro1 WT. Data is presented as a percentage of control neuron count (n = 10 dishes; ***P < 0.001, repeated measures ANOVA).
Reduced TNFR1 protein levels in S mice and genetic linkage study of this trait.Figure 2TNFR1 protein levels in serum and tissues of different mouse groups (n = 4-13). TNFR1 protein levels were measured by ELISA and compared to B, the levels of which were set as 100% for each tissue. From left to right: B mice (black), TNFR1+/- mice (grey), (BxS) F1 mice (grey), S mice (grey); B.S-chr6-BB (black), B.S-chr6-SS (green) and B.S-chr6-BS (grey). TNFR1 expression in splenic neutrophils of wild-type, TNFR1 KO and SPRET mice determined by FACS. Neutrophils were defined by flow cytometry (FACS) as SSChighCD11b+Ly6GhighLy6Cmid and TNFR1+ cells from different strains were plotted in a histogram. In the insert, the median of TNFR1-PE signals were plotted and this shows that SPRET mice have an intermediate TNFR1 expression compared to WT mice. TNFR1 mRNA levels measured by qPCR in organs of B (n = 3) and S (n = 4). Affymetrix microarray data from hepatocytes (Affy Mo Gene 1.0 ST Array). Backcross experiments in mice to determine genetic linkage between TNF resistance (left) and TNFR1 liver protein levels (right). QTL mapping of lethal response (n = 178) was based on the data previously obtained and discussed by Staelens et al (2002), but the data were re-analysed in the current study using R/qtl software. TNFR1 protein level in the liver of N2 mice (n = 214). For both TNF resistance and TNFR1 levels, the LOD scores show a QTL on chr2 (TNF resistance 15 cM, LOD = 2.25 and TNFR1 levels 5 cM, LOD = 1.31) and chr6 (TNF resistance 46 cM, LOD = 3.11 and TNFR1 levels 70 cM, LOD = 4.15). Horizontal lines in the figures represent LOD values that are suggestive (dots), significant (stripes) and highly significant (full line).
AAV2.Figure 1Vector mediated eGFP expression after intravitreal injection of novel capsid variants compared to controls in 2-month-old mice. (A) In vivo cSLO examinations of the mouse retina at 1- and 2-weeks post injection (WPI) of 2E9 total vg (in 1 µl) of sc-CMV-eGFP packaged with parental serotype AAV2 , state-of-art AAV2.7m8, novel AAV2.GL and AAV2.NN capsid variants. (B) Confocal scans of retinal cross sections immunolabelled for eGFP and DAPI. Acquisition settings were kept constant for all samples. Scale bar: 100 μm. ONL, outer nuclear layer; INL, inner nuclear layer. (C) Confocal tile-scans of retinal cross sections immunolabelled for eGFP and Hoechst. Scale bar: 500 μm (D-E) Immunolabelled cross sections of mouse retina infected with AAV2.GL and AAV2.NN respectively, with focus on the ONL and INL, stained for eGFP, recoverin and DAPI. Scale bar: 50 μm ONL, outer nuclear layer; INL, inner nuclear layer. (F) qRT-PCR data comparing fold change in eGFP transcript levels from retinal samples infected with capsid variants AAV2.7m8, AAV2.GL and AAV2.NN compared to AAV2.
a) Flow cytometry of T cells transduced with lentivirus expressing GFP and nontargeting control shRNA (Scr) or Lamp2a mRNA–specific shRNA (shL2A) (left), and immunoblot analysis of LAMP-2A in sorted GFP+ T cells (right).figf2(a) Flow cytometry of T cells transduced with lentivirus expressing GFP and nontargeting control shRNA (Scr) or Lamp2a mRNA-specific shRNA (shL2A) (left), and immunoblot analysis of LAMP-2A in sorted GFP+ T cells (right). Numbers above bracketed lines (left) indicate percent GFP+ cells. (b) Enzyme-linked immunosorbent assay of IL-2 and interferon-γ (IFN-γ) in TH1 cells transduced as in a and then left unstimulated (Rest) or stimulated for 24 h with anti-CD3 and anti-CD28 (Act). *P = 0.038 and **P = 0.031 (t-test). (c) Proliferation in TH1 cells transduced as in a and left unstimulated or stimulated for 24 h as in b, assessed by incorporation of the thymidine analog BrdU and presented as absorbance at 495 nm (A495). *P = 0.013 (t-test). (d) Quantification of annexin V-positive (AnnV+) cells among resting or stimulated TH1 cells transduced as in a.