EMBO/sd-character-level-ner huggingface dataset:
Figure 3BsAb were incubated at the indicated concentrations with PBMC of healthy donors together with LNCaP- expressing high and low amounts of PSMA, respectively. After three days, CD4+ and CD8+ T cell activation were determined by flow cytometry as described in the methods section. Mean values and standard deviations of triplicate measurements are indicated. The dotted line represents the baseline T cell activation in the absence of bispecific antibodies. BsAb were incubated at the indicated concentrations with PBMC of healthy donors together with LNCaP- expressing high and low amounts of PSMA, respectively. After three days, target cell depletion were determined by flow cytometry as described in the methods section. Mean values and standard deviations of triplicate measurements are indicated. The dotted line represents tumor cell counts in the absence of bispecific antibodies. BsAb were incubated at the indicated concentrations with PBMC of healthy donors together with LNCaP- expressing high and low amounts of PSMA, respectively. After three days, cytokine release were determined by flow cytometry as described in the methods section. Mean values and standard deviations of triplicate measurements are indicated. BsAb were incubated at the indicated concentrations with PBMC of healthy donors together with 22Rv1low-cells expressing high and low amounts of PSMA, respectively. After three days, CD4+ and CD8+ T cell activation were determined by flow cytometry as described in the methods section. Mean values and standard deviations of triplicate measurements are indicated. The dotted line represents the baseline T cell activation in the absence of bispecific antibodies. BsAb were incubated at the indicated concentrations with PBMC of healthy donors together with 22Rv1low-cells expressing high and low amounts of PSMA, respectively. After three days, target cell depletion were determined by flow cytometry as described in the methods section. Mean values and standard deviations of triplicate measurements are indicated. The dotted line represents the tumor cell counts in the absence of bispecific antibodies. BsAb were incubated at the indicated concentrations with PBMC of healthy donors together with 22Rv1low-cells expressing high and low amounts of PSMA, respectively. After three days, cytokine release were determined by flow cytometry as described in the methods section. Mean values and standard deviations of triplicate measurements are indicated.
| Vaccination with a VSR-deficient mutant virus induces protection against secondary infection in neonatal mice.Figure 1A, At 2 days post vaccination with NoVΔB2, UV- inactivated NoVΔB2 (UV- ΔB2) or culture medium DMEM, BALB/c mice were challenged with WT NoV. The survival data were analyzed by the log rank test, **** indicates p<0.0001, n=7 per group. NoV levels in the vaccinated mice hindlimb muscle tissue at 2, 3 or 4 days post infection (dpi) with WT NoV were determined by Western blotting to detect the viral coat protein (CP) and B2 Staining of β-actin was used as a loading control (B). , NoV levels in the vaccinated mice hindlimb muscle tissue at 2, 3 or 4 days post infection (dpi) with WT NoV were determined by quantitative RT-qPCR to detect the viral genomic RNA1, n=7 per group (C). The viral RNA1 level was normalized by β-actin mRNA levels (C). D, E, Time course analysis of the accumulation of NoVΔB2 RNA1 determined by RT-qPCR (D) and 21-to 23-nt vsiRNAs determined by deep sequencing (E, two independent sets) from BALB/c suckling mice infected with NoVΔB2. Mice, n=3, in each independent analysis. Data are shown as mean±SD. Normalization was done by β-actin mRNA levels for NoVΔB2 RNA1 (D) or by mature miRNAs for vsiRNAs (E). Accumulation levels of NoVΔB2 RNA1 or vsiRNAs at 3 dpi are set as 100%, respectively. F, Properties of viral siRNAs (per million mature miRNAs) cloned and sequenced from the time course series of NoVΔB2-infected BALB/c suckling mice. Size distribution, 5' terminal nucleotide and duplexes by 22-nt vsiRNAs are indicated. The percentage of 1U vsiRNAs (21- to 23-nt) in each library is shown in parentheses.
a,b) Two representative images of fluorescent microscopy of LC3 and LINE-1 fused to six MS2-binding sites detected with MS2-GFP.f1(a,b) Two representative images of fluorescent microscopy of LC3 and LINE-1 fused to six MS2-binding sites detected with MS2-GFP. (c) Fluorescent microscopy of LC3 and Alu fused to six MS2-binding sites detected with MS2-GFP. Li's correlation coefficient of co-localization (0.0232, s.e.m. 0.048). (d) Relative number of foci of LINE-1-MS2, or number of foci of LINE-1-MS2 co-localized with LC3 per cell in randomly selected fields of cells treated with BAF1 (20 h, 400 nM). n=25 cells, total number of LINE-1-MS2 foci: control=104, BAF=175, total number of LINE-1-MS2 foci co-localized with LC3: control =68, BAF 167. *Total P=0.015, Co-localized with LC3 P=5 × 10−8, t-test). Li's correlation coefficient of co-localization (without BAF 0.19 s.e.m. 0.027; with BAF 0.213, s.e.m. 0.02). (e,f) Two representative images of fluorescent microscopy of FISH probes recognizing endogenous LINE-1 RNA and LC3. (g) Three-dimensional reconstruction of confocal microscopy z-stacks of endogenous LINE-1 RNA and LC3 prepared in Imaris. (h) Western blot of fractions enriched in autophagosomes compared with total cell extracts and ER/mitochondria-enriched fractions. RT-qPCR analysis of mRNA levels of two transmembrane proteins (GLUT4 and APP) in total cell RNA and autophagosome-enriched fractions. n=3, P>0.05. (i) RT-qPCR analysis of LINE-1 ORF1 and AluYa5 in RNAse-treated autophagosome-enriched fractions or total cell RNA. n=3, *P=0.043 LINE-1, P-0.01 AluYa5, t-test (j) Model of system to measure degradation of GFP-tagged substrates by autophagy. (k) Western blot analysis of GFP and alpha-tubulin (control) in cells transfected with MS2-GFP or LINE-1-MS2 and MS2-GFP and treated with control or Bafilomycin (2 h, 400 nM). All error bars represent s.e. of the mean. All experiments were replicated a minimum of three times.
Figure 7(A) The bacterial burden was evaluated 20 h after treatment of CLP mice with rRv2626c-CA or its mutants (n=10 mice per group). (B) The percentage of F4/80+ macrophages cells and rRv2626c-CA-His or its mutants were found in the spleen or lung using Fluorescence-activated cell sorting analysis in the background of CLP-induced sepsis. (C) CD86, iNOS, CD163 and Arg1 mRNA levels were determined by real-time PCR. (D) Expression of SR-A, FcR, TLR2, TLR4, NRP1 or CXCR2 were measured by flow cytometry analysis.
Figure 8(A) IHC scores of TFAM in 429 paired tissues from HCC patients. The relative expression ratio of tumor to non-tumor was log2-transformed. T, tumor tissues; NT, non-tumor liver tissues. (B) mRNA level of TFAM in 50 HCC patients' paired tissues from TCGA database. The relative expression ratio of tumor to non-tumor was log2-transformed. (C) Representative images of IHC staining of TFAM (top panel) and scores (bottom panel) in HCC tumor tissues. One-way ANOVA. *P < 0.05; **P < 0.01. MT, metastatic tumor tissues; NMT, non-metastatic tumor tissues. Scale bars, 25μm. Boxes: first quartile to third quartile; Whiskers: minimum value to maximum value; Central band: median. (D Kaplan-Meier curves for overall survival of HCC patients stratified by TFAM expression. (F Kaplan-Meier curves for overall survival of HCC patients stratified by nuclear F-actin positive rates. E) the recurrence of HCC patients stratified by TFAM expression. G) the recurrence of HCC patients stratified by nuclear F-actin positive rates. (H) Representative images of IHC staining of TFAM, FN1 and ITGB3 from two HCC patients. Scale bars, 25μm. (I) Spearman correlation analysis between the levels of TFAM and FN1, IL-6, TGFB1 or ITGB3.
Figure 2(A) Competitive binding experiments were performed to determine relative affinity of Gal4 to UASwt (green) and UASmut (blue) motifs. Gal4 occupancy on UASwt Cy3/Cy5 DNA was determined by measuring protein induced fluorescence enhancement (PIFE) on 51 bp oligos containing either Gal4 UASwt or UASmut site 1 bp away from Cy3 fluorophore). (B) Titrating unlabeled UASwt or UASmut competitor DNA reduces UASwt Cy3/Cy5 DNA occupancy. IC50UASwt = 4.0 ± 0.6 nM, IC50UASmut = 17.3 ± 3.5 nM. n = 3. Error bars indicate SD. (C) Comparison between relative affinities of Gal4 UASwt versus UASmut in naked or nucleosomal DNA shows 4.3 ± 1.1x difference in KD from naked DNA and 6.8 ± 1.7x from nucleosomal DNA. n = 3. Error bars indicate SD. (D) Experimental setup for smFRET experiments to measure Gal4 binding at nucleosomal DNA. Gal4 binds to site 8 bp into nucleosome. A FRET pair in the entry/exit region provides readout on binding events (one fluorophore on DNA, one on histone). In the absence of Gal4, nucleosomes are in the high FRET state. A Gal4 binding event traps nucleosome in low FRET state. (E) Gal4 affinity to nucleosomes containing UASwt or UASmut sequences gives S1/2 of 7.2 ± 0.8 nM and 48.9 ± 10.8 nM, respectively. n = 3. Error bars indicate SD. (F) Example smFRET traces showing Gal4 binding to UASwt in nucleosomal DNA at two different Gal4 concentrations. States are determined using HMM fit. (G) Same as F but for UASmut. (H) Binding-rates of Gal4 are concentration dependent, but are similar for UASwt and UASmut: kon UASwt = 0.011 ± 0.002 s-1 nM-1, kon UASmut = 0.009 ± 0.001 s-1 nM-1. n = 2 for all, except n = 4 for UASwt 10 nM Gal4 and n = 3 for UASwt 20 nM Gal4. Error bars indicate SD. (I) The dissociation-rate of Gal4 at UASwt is ~5-fold slower compared to the UASmut: koff UASwt = 0.20 ± 0.01 s-1, koff UASmut = 0.92 ± 0.05 s-1. n = 2 for all, except n = 4 for UASwt 10 nM Gal4 and n = 3 for UASwt 20 nM Gal4. Error bars indicate SD. (J) Histogram of Gal4 dwell time to nucleosomal DNA containing UASwt and UASmut sequences. n = 11 for UASwt, n = 8 for UASmut. Error bars indicate SE. (K) Scatter plot showing the average number of nascent RNA at TS (from smFISH) vs affinity of Gal4 to different UAS sequences in nucleosomal DNA (from in vitro measurements). In vivo transcription levels correlate with in vitro affinity of Gal4, but transcription saturates above wildtype sequence. UAS sequences are shown in the box. For mean number of nascent RNA from smFISH, n = 2 for UASconsensus, UAS-2C, UAS-8T and n = 8 for UASwt, UASmut and errors indicate SEM. For in vitro affinity measurements, n = 3 and errors indicate SD.
Figure 5A Gene sets enriched in astrocytes 2 days after microglial ablation, demonstrated using g:Profiler with the KEGG database. B Expression level of phagocytic receptors and their extracellular adaptor molecules (n = 3 independent experiments). Values show the mean ± SEM. N.S.: no significance; ***q < 0.001. The probability values for differential expression analysis were computed with edgeR followed by a correction of multiple testing with q-value.
Figure 8A and B. Cytoplasmic (A) or mitochondrial (B) Ca2+ flux was measured in N2a-IP3R3 cells treated with PRE-084 (5 µM) or NE-100 (5 µM) for 1 h prior to fluorescent imaging. Cytoplasmic and mitochondrialCa2+ flux were detected by fluo-4 and Case12-mito, respectively. The fluorescent intensity was normalized by the resting state at 0 s, and plotted as mean ± SD. n = 10 each. **: p < 0.01, *: p < 0.05. Two-way ANOVA with subsequent post hoc Tukey's test. C and D. Calpain activity were measured in N2a-IP3R3 cells treated with PRE-084 (5 µM) or NE-100 (5 µM) for 24 h. Data are plotted as mean ± SEM from three independent experiments. *: p < 0.05 vs. mock control. A one-way ANOVA with subsequent post hoc Tukey's test. C and D. intracellular ATP levels were measured in N2a-IP3R3 cells treated with PRE-084 (5 µM) or NE-100 (5 µM) for 24 h. Data are plotted as mean ± SEM from three independent experiments. *: p < 0.05 vs. mock control. A one-way ANOVA with subsequent post hoc Tukey's test. E and F. SOD1G93Amice were intraperitoneally administered with saline or PRE-084 (0.25 mg/kg, 3 times per week) from postnatal day 35 to 95. Lumbar spinal cord sections were stained by using anti-Sig1R (white), IP3R3 (green), and βIII-tubulin (red) at 95 days old. Arrowheads indicate the neurons with normal distribution of Sig1R and IP3R3. Scale bars: 50 µm.
PGC specification is sinhcaf-dependent in zebrafish.Figure 2(A and B) Whole-mount in situ hybridization was performed in WT, MZsinhcaf -/-, Tg(CMV:EGFP;CMV:sinhcaf) and MZsinhcaf -/-;Tg(CMV:EGFP;CMV:sinhcaf) zebrafish embryos with PGCs markers nanos3 (A) and vasa (B). Scale bars, 100 μm. (C) Quantitative analysis of PGCs in bud stage WT, MZsinhcaf -/-, Tg(CMV:EGFP;CMV:sinhcaf) and MZsinhcaf -/-;Tg(CMV:EGFP;CMV:sinhcaf) zebrafish embryos. Data shown are mean ± SEM (n = 10, the number of embryos analyzed). Statistical analysis was performed using one-way ANOVA followed by Tukey's post-hoc test. The different letters (a, b, c) indicate a significant difference between the means, p<0.05. (D) Maximal intensity projection of a confocal z-stack of 24hpf Tg(piwil1:EGFP) and MZsinhcaf -/-; Tg(piwil1:EGFP) zebrafish embryos, dorsal views. Arrowheads point to PGCs. Scale bars, 100 μm. (E) Quantitative analysis of PGCs in 24hpf Tg(piwil1:EGFP) and MZsinhcaf -/-; Tg(piwil1:EGFP)zebrafish embryos. Data shown are mean ± SEM (n = 13, the number of embryos analyzed). Statistical analysis was performed using unpaired two-tailed Student's t-test. (F) Relative mRNA level of vasa, dnd and nanos3 in 24 hpf WT and MZsinhcaf -/- mutant embryos, respectively, as measured by RT-qPCR. Data shown are mean ± SEM (n = 3, biological replicates). Statistical analysis was performed using 2-way ANOVA followed by Holm-Sidak post-hoc test.
USP26 variants might be responsible for the origin of Klinefelter syndrome.Figure 1A The karyotype of KS patients and controls. B The cohorts used in this study. The first cohort that contained 108 unrelated KS patients with the classical 47, XXY karyotype was selected to perform WES, the second cohort that contained 354 unrelated KS patients was selected to perform Sanger sequencing in the coding regions of USP26, and the third cohort that contained 558 unrelated KS patients was selected to perform Sanger sequencing in the mutated haplotype (c.370-371insACA/494T>C/1423C>T) of USP26. C WES data analysis pipeline. A total of 179,579 sequence variants in 20,800 genes were identified in 108 unrelated KS patients, and among of them nonsense, missense, frameshift, and essential splice-site variants were further investigated. These variants were divided into four subtypes (high, likely high, medium, low), and most of the medium to high variants were localized on the X chromosome. The genes on the X chromosome that contained more than two types of medium to high variants were selected. As the extra paternal-origin X chromosome in KS might be caused by impaired recombination between sex chromosomes during meiosis I, only the testis-specific genes ESX1, LOC101059915, and USP26 were further investigated. The variant frequencies of these three genes in the KS patients were compared to the variant frequencies of these three genes in the 1000 Genomes Project. The frequency of USP26 variants had the highest fold increase. D The genomic localization of sequence variants in 108 KS patients. E Genes with more than two medium- to high-level variants on the X chromosome. The testis-specific genes are labeled in red. F The frequency difference between KS patients and the 1000 Genomes Project.
Figure 5a, Schematic depiction of ISX and deletion constructs used. b GFP-tagged truncated ISX mutant proteins or HA-tagged BRD4 were detected in anti-HA or GFP immunoprecipitates by Western blotting analysis. Data information: Each experiment was repeated at least three times. A putative interaction surface of the ISX/BRD4 complex was presented through structural modeling. A putative interaction surface of the ISX/BRD4 complex was presented through structural modeling. The electrostatic surfaces are drawn either blue for positive or red for negative charge (e and f).
TMZ activation of the UPR pathway and subsequent enhancement of glioblastoma cell killing in vitro by the RGD4C/AAVP-Grp78-HSVtk and GCV.Figure 2TMZ induction of phospho-eIF2α and ATF6-p90 expression. Human glioblastoma cells transduced with RGD4C/AAVP-Grp78 were analyzed by Western blot following treatment with TMZ (100 μM for LN229 and SNB19, 60 μM for U87). GAPDH was used as a control. RT-PCR analysis of constitutive expression and splicing of XBP1 in glioblastoma cells transduced with RGD4C/AAVP-Grp78, in the presence of TMZ. Sizes of the PCR products were 289 bp for unspliced XBP1 and 263 bp for spliced XBP1, the lower size band is not specific. Time points were selected among those tested in the Western blot in (A), subsequently the image containing 0, 1 and 2 hours was juxtaposed to images of 6, 12 and 24 hours. A white line has been added between the gel pieces that have been juxtaposed. Tumor cell killing in vitro by the HSVtk/GCV approach. Glioblastoma cells stably transduced with RGD4C/AAVP-Grp78-HSVtk or RGD4C/AAVP-CMV-HSVtk were treated with either GCV (10 μM) or TMZ (100 μM for LN229 and SNB19, 60 μM for U87) or combination of both GCV and TMZ. Cells were stained with MitoSOX and analyzed by FACS at day 4 post-treatment. Data shown are representative of three experiments, n=3. Two-way ANOVA with Tukey's multiple comparison test (GraphPad Prism 6) was used for data analysis.
Figure 6A, B CUT&RUN coverage normalised by spike-ins from 1kb upstream to 1kb downstream for all ribosomal protein genes in L1236 HL cells (A) and L428 HL cells (B). CUT&RUN data analyses were performed on two biological replicates. C, D Mean difference between H2A.B and IgG CUT&RUN coverage normalized by spike-ins aligned between -1 and +1 kb from the TSS ranked according to their level of expression in L1236 HL cells (C) and L428 HL cells (D). Below is the position of H2A.B nucleosomes relative to the TSS; the colour reflects nucleosome abundance (red reflects the highest abundant H2A.B nucleosome located at the TSS). E Genome browser screen shots of gene examples (RPs RPLP1 and RPL23A, and HDAC5 and TAF10) that contain H2A.B at their promoters identified by the CUT&RUN peak calling and whose expression decreased significantly following H2A.B knockdown in the RNA-seq analysis. Green and purple indicates L1236 and L428cells, respectively.
ALX1165F/165F NCC show a migration defect and a difference in BMP secretion.Figure 5A. Mutant ALX1165F/165F NCC (blue) exhibited a migration defect in timed coverage of the central clearing of the wound assay when compared with control ALX1165L/165L NCC (black). Data is presented as percent area recovery of the central circular clear area of the wound assay by migrating NCC at the end of a 24-hour period. For fluorescent pictures, cells were stained in serum free media containing 3.6µM CellTracker Green CMFDA (Life Technologies) for 30 min at 37°C and allowed to recover for 30 min before starting the experiment. Images were acquired every 6 hours using a Keyence BZ-X800 microscope. Surface area analyses and percentages of coverage were measured using ImageJ software (NIH). The NCC were monitored over 24 hours. The data are represented as the average of the percentage of closure ± SEM. Scale bar = 200µm. Data obtained of each clone from three independent experiments were pooled and the mathematical mean was calculated. SEM was used to determine the standard error. To test statistical significance, an ANOVA test was performed. A p-value <0.05 was considered to be statistically significant.*: Significantly different from control NCC (p<0.0001). B. Multiplex analysis of BMP2 and BMP9 in the supernatant of cultured NCC showed that ALX1165F/165F NCC (blue) secrete less BMP2 and more BMP9 compared to control ALX1165L/165L NCC (green). Data are represented as pooled mean ± SEM of three clones from each genotype. Statistical significance was determined with an ANOVA test. A p-value <0.05 was considered significant. *: Significantly different from control NCC (p =0.0424 for BMP2 and p=0.0192 for BMP9). C. Addition of soluble BMP2 or CV2, a BMP9 antagonist, to the culture medium could partially rescue the migration defect of ALX1165F/165F NCC. At the beginning of the assay, 10, 50, or 100 ng/ml of soluble BMP2, CV2, or a combination of the two at 100 ng/ml each were added to the culture medium, and the cells were monitored over the next 24 hours. The data are represented as the average of the percentage of closure ± SEM. Scale bar: 400µm.
Figure 2High-resolution imaging of mitochondria in live cells using the Airyscan module of Zeiss LSM880 confocal microscope. Mitochondria from HeLa cell, co-stained with NAO and TMRE. Area from dashed white box, zoomed to right, shows the red and green intensities of TMRE and NAO colocalized. Scale bars = 500 nm. N = 3 independent experiments. Mitochondria in L6 myoblast, co-stained with NAO and TMRE. Area from dashed white box, zoomed to right, shows colocalizing NAO and TMRE at the cristae membrane (arrowheads). Scale bars = 500 nm. N = 3 independent experiments. High-resolution imaging of mitochondria in live cells using the Airyscan module of Zeiss LSM880 confocal microscope. Mitochondria in HeLa cells, stained with NAO alone (top row) vs. NAO + TMRE (bottom row), and simultaneously excited with 488- and 561-nm lasers. Note that mitochondria stained with NAO alone do not emit noticeable fluorescence in the red channel; only after adding TMRE does strong signal appear in the red channel, showing negligible bleed-through. Scale bars = 500 nm. N = 2 independent experiments.
Figure 1A - Schematic overview of part of the biofilm regulatory network, highlighting the roles of OmrA and OmrB. Grey arrows: transcriptional activation, black lines with bars: post-transcriptional negative regulation, dotted lines: direct protein-protein or protein-c-di-GMP interactions. B - Congo red plates with colonies of E. coli strain MC4100 relA+ and its target mutation csgD:M4-ompR:mut2 derivative, harboring sRNA-producing plasmids pOmrA, pOmrB, or an empty vector (pControl). Incubation at 28°C for 48 h. C - Schematic showing published interaction sites between OmrA or OmrB and targets csgD or ompR Mutations introduced to disrupt base-pairing are in grey boxes.
A-BRD impairs the transition of PCa cells into a dedifferentiated, cancer stem-like state.Figure 5A. Heatmaps showing the relative BAZ2A-BRD binding intensity at modified histones peptides upon incubation with BAZ2A-BRDi BAZ2-ICR or GSK2801 displayed in Fig. EV4A. Binding intensity was calculated as average of fold change from peptides signal over background controls of 2 different arrays. B. Cell proliferation curves of PC3 cells treated with siRNA-BAZ2A. Error bars represent the standard deviation from three independent experiments. C. Cell proliferation curves of PC3 cells treated with 5μM BAZ2-ICR and GSK2801. Error bars represent the standard deviation from three independent experiments. D. Representative images of tumorspheres generated from PC3 cells treated with BAZ2-ICR (1μM) or GSK2801 (1μM). E. Half-maximal effective concentration (EC50) of BAZ2-ICR. Measurements were performed 5 days after treatment of PC3 cells and initiation of tumorspheres using the indicated BAZ2-ICR concentration. The amount of tumorspheres was assessed by measurement of cell viability. Error bars represent SD. Values are from three independent experiments. F. Representative images showing tumorspheres derived from the metastatic PCa cell line DU-145 treated with DMSO or BAZ2-ICR (1μM) inhibitor for 5 days.
Figure 7A, B Relative expression of NPFs in M. truncatula roots grown in FP and exposed to different NaCl treatments. Data information: *p<0.05, Student's t-test; biological replicates: n=3 (3 pooled root organs in each replicate) ±SEM C Relative expression of NPFs in M. truncatula roots grown in FP and exposed to different concentration of KCl for 48h. Data information: *p<0.05, Student's t-test; biological replicates: n=3 (3 pooled root organs in each replicate) ±SEM D Relative expression of NPFs in M. truncatula roots grown in FP and exposed to 100mM KCl treatments for different times. Data information: *p<0.05, Student's t-test; biological replicates: n=3 (3 pooled root organs in each replicate) ±SEM