EMBO/sd-character-level-ner huggingface dataset:
Figure 3A Percentages of MDSCs (CD11b+Gr1+) gated from the CD45+ cell population in peripheral blood and spleen [n=11, tumor-free (-) WT and Tet2-/-（KO） mice; n=12, tumor-bearing (+) WT and KO mice, peripheral blood; n=10, tumor-free (-) WT and KO mice; n=13, tumor-bearing (+) WT and KO mice, spleen]. B Percentages of G-MDSCs (CD11b+Ly6G+) gated from the CD45+ cell population in peripheral blood and spleen [n=14, tumor-free (-) WT and KO mice; n=15, tumor-bearing (+) WT and KO mice, peripheral blood; n=15, tumor-free (-) WT and KO mice; n=15, tumor-bearing (+) WT and KO mice, spleen]. C Percentages of M-MDSCs (CD11b+Ly6C+) gated from the CD45+ cell population in peripheral blood and spleen [n=5, tumor-free (-) WT and KO mice; n=6, tumor-bearing (+) WT and KO mice, peripheral blood; n=6, tumor-free (-) WT and KO mice; n=6, tumor-bearing (+) WT and KO mice, spleen]. D Representative images of Ly6g and Ly6c immunohistochemistry in Hepa1-6 cell tumors from WT and KO mice. Corresponding bar charts display quantification of positive staining using Image Pro Plus 6.0 software (n=3). Pictures were captured at 400X magnification. Scale bar = 50 μm. E Percentages of CD8+ T cells gated from the CD45+ cell population in peripheral blood and spleen [n=11, tumor-free (-) WT and KO mice; n=5, tumor-bearing (+) WT and Tet2-/- KO mice, peripheral blood; n=16, tumor-free (-) WT and KO mice; n=12, tumor-bearing (+) WT and KO mice, spleen]. F Percentages of CD4+ T cells gated from the CD45+ cell population in peripheral blood and spleen [n=8, tumor-free (-) WT and KO mice; n=5, tumor-bearing (+) WT and KO mice, peripheral blood; n=16, tumor-free (-) WT and KO mice; n=11, tumor-bearing (+) WT and KO mice, spleen].
Figure 3Inhibitory effect of the indicated doses of BO-110 (in µg/ml) or vehicle (V) on MDK mRNA expression determined by qRT-PCR in SK-Mel-147 (16 h after treatment). Data correspond to average mRNA levels in three experiments with technical replicates normalized to vehicle control ± SD. Statistical significance was determined by ANOVA.   qRT-PCR analysis of relative mRNA levels of MDA5 16 h after treatment of SK-Mel-147 with the indicated doses of BO-110 (in µg/ml) (BO). Data correspond to the mean ± SD of three experiments with three technical replicates. Statistical significance was determined by ANOVA.   Analysis of apoptotic cells at the indicated time points. SK-Mel-147 cells were treated with vehicle (V) or 0.5 µg/ml BO-110 (BO) for the indicated time points. Cells were collected and apoptosis was analyzed by flow cytometry as indicated in methods. Data correspond to the mean ± SD of 3 experiments. Statistical significance was determined by t-test. Luciferase signal driven by MDK-promoter transduced into SK-Mel-147 cells treated with vehicle (v) or BO-110 (BO) as indicated in Materials and Methods. Results were normalized to vehicle control. Data correspond to the mean ± SD of four biological replicates. Statistical significance was determined by ANOVA. MDK secretion by ELISA in SK-Mel-147 melanoma cells treated with vehicle (V) or 0.5 µg/ml BO-110 (BO). Data correspond to the mean ± SD of 3 biological replicates. Statistical significance was determined by Two-Way ANOVA. Immunohistochemical analysis of MDK repression (pink staining) in SK-Mel-147 xenografts and PDX lesions after treatment with BO-110 (BO, 0.8 mg/kg, 2 doses/week). Histological staining in tumors extracted from animals treated with vehicle control (V) are included as a reference. Nuclei were counterstained in with hematoxylin.
Figure 7A mRNA decay measurement using Roadblock-qPCR RNA was isolated from HEK-293 cells at indicated timepoints following transfection with indicated siRNAs and treatment with 400 µM 4sU and 50 µg/mL puromycin. mRNA half-lives were estimated by fitting the data to a single-phase exponential decay model. Puromycin treatment was compared to the vehicle control and siUPF1LL was compared to the siNT in the absence and presence of puromycin treatment using the extra-sum-of-squares F test. Error bars indicate mean±SD (n = 4 biological replicates). B RT-qPCR analysis of indicated transcripts following transfection of HEK-293 cells with indicated siRNAs and treatment with 50 µg/mL puromycin for 4hr. 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 C Density plot of changes in relative mRNA abundance as determined from RNA-seq following treatment of HEK-293 cells with 50 µg/mL of puromycin for 4hr. mRNAs were subdivided by PTBP1 and/or hnRNP L motif density within the first 400 nt of 3'UTR. Statistical significance was determined by K-W test, with Dunn's correction for multiple comparisons. D Density plot as in (C), following UPF1LL-specific knockdown.
UnaG-based hypoxia sensor kinetics can be tuned by fusion to different protein destabilization domains.Figure 2a) Schematic representation of the different variants of UnaG used in the four indicated hypoxia-reporter constructs. dUnaG is identical to Fig. 1a, oUnaG is destabilized by in frame fusion with the oxygen-dependent degradation domain sequence (ODDD) from HIF1a (aa338-608), doUnaG is destabilized by both degradation motifs. b) Microscopic assessment of the averaged fluorescence intensity (AFI) of CHO cell bulk cultures stably expressing the indicated UnaG variants or destabilized EGFP. Expression under hypoxia was induced by incubation in 1% oxygen for 16 hrs and then culture was continued for 24 hrs under normoxia. Fluorescence hysteresis after switching to normoxia was markedly reduced in the singly-destabilized UnaG-variants, while the doubly destabilized doUnaG displayed the most rapid switching behavior, but the least brightness. To reduce phototoxicity, illumination intensity was kept minimal. Data points represent one viewfield of 425 µm2 ± SEM. c) Flow cytometry analysis of the induction and decay kinetics of the fluorescence intensity in CHO cells stably expressing either dUnaG or oUnaG. Open bars represent control cultures grown under normoxia. Solid bars indicate culture for 4, 8, 12, 16, 20 or 24 hrs under hypoxia (1% oxygen). To assess deactivation, cultures previously kept under hypoxia (1% oxygen) for 24hrs were shifted to normoxia (21% oxygen) for the indicated times. Error bars represent standard errors. d) Representative maximum intensity projections (MIPs) from the life cell cultures depicted in b. Cells were grown for the indicated times at hypoxic (1% oxygen) conditions and subsequently switched for the indicated times to normoxia (21% oxygen)(red arrows in b) to illustrate the dynamics of the different UnaG-based hypoxia sensors. Increasing destabilization is associated with reduced brightness but, also reduced background and improved switching behavior. Scale bars = 50 µm.
Model of Ykt6 phosphoregulation during autophagy.Figure 5Atg1 kinase phosphorylates the SNARE protein Ykt6 on autophagosomes, which prevents interaction of Ykt6 with the vacuolar SNARE proteins. Upon dephosphorylation by an unknown phosphatase, Ykt6 becomes competent for formation of a SNARE bundle with the vacuolar SNAREs Vam3, Vam7 and Vti1, allowing subsequent fusion of the autophagosome with the vacuole.
a) Immunoblot analysis of Irgm1 in lysates of flow cytometry–sorted naive wild-type CD4+ lymphocytes activated for 48 h with immobilized agonistic mAb to CD3 (10 μg/ml) alone or with mAb to CD28 (2 μg/ml) in the presence or absence of IFN-γ-neutralizing mAb.figf2(a) Immunoblot analysis of Irgm1 in lysates of flow cytometry-sorted naive wild-type CD4+ lymphocytes activated for 48 h with immobilized agonistic mAb to CD3 (10 μg/ml) alone or with mAb to CD28 (2 μg/ml) in the presence or absence of IFN-γ-neutralizing mAb. Actin, loading control. (b) Enzyme-linked immunosorbent assay of IFN-γ produced by OT-II and Irgm1−/− OT-II CD4+ T cells stimulated with OVA peptide in the presence of splenic DC in polarized conditions (TH1 or TH2) or nonpolarized conditions (N), assessed in 72-hour cultures. ND, not detected. (c) Accumulation of polarized and nonpolarized OT-II (open symbols) or Irgm1−/− OT-II (filled symbols) CD4+ T cells after peptide stimulation, quantified by counting of viable cells. (d) Viable OT-II and Irgm1−/− OT-II CD4+ T cells 72 h after restimulation with OVA peptide in the presence of T cell-depleted APC samples. (e) Flow cytometry of the viability and division of OT-II and Irgm1−/− OT-II CD4+ T cells stimulated with peptide, allowed to 'rest' for 7 d in IL-2, then labeled with CFSE and restimulated with OVA peptide in the presence of T cell-depleted APC samples and assessed 72 h later. (f) Flow cytometric analysis of the population expansion of CFSE-labeled OT-II and Irgm1−/− OT-II CD4+ T cells activated with OVA peptide in the presence or absence of IFN-γ-neutralizing mAb. Results in e,f obtained after gating on CD4+ T cells; numbers in outlined areas (e) and in top left corners (f) indicate percent CD4+ cells. PI, propidium iodide. (g) [3H]thymidine incorporation by wild-type, Irgm1−/−, Ifng−/− and Irgm1−/−Ifng−/− CD4+ T cells stimulated for 72 h with soluble agonistic mAb to CD3 and irradiated syngenic Ifng−/− splenocytes. Data are representative of two independent experiments with similar results (mean and s.d. of triplicate cultures, b-d,g).
Figure 2A, Teratoma volume (mm3) 20-25 days after subcutaneous injection of wild-type iPSCs or miiPSCs expressing GFP. Data are represented as mean ± s.e.m. (n=8 tumors per genotype; 292,8 ± 66,67 versus 1216 ±140,1; difference between means: 923,5 ± 155,2; 95% confidence interval: 1256 to -590,7); ***P<0.001 (Student´s t-test). B, Incidence and representative images of specific highly differentiated tissues in teratomas. The number of tumors included in the analysis is indicated in the panel. ***P<0.001 (Student´s t-test). Scale bars, 50 μm. C, Table showing the frequency of nude mice with embryo-like structures (E-Ls) in their abdominal cavity 20-30 days after intraperitoneal (i.p.) injection of 400.000-500.000 in vivo (iv)-generated wild-type iPSCs, un-induced iPSCs, or miiPSCs. The number of independent clones tested per condition is indicated in the panel (each animal was inoculated with a different clone). D, Representative example of E-Ls generated after i.p. injection of GFP-expressing miiPSCs. H/E, hematoxylin and eosin. The following antigens were detected by immunohistochemistry: Sox2 (ectoderm), Cd34 (mesoderm), Gata4 (endoderm), AFP and CK8 (visceral endoderm of the yolk sac) and Ter119 (nucleated erythroid cells). Scale bars, 500 μm and 100 μm for higher magnifications. E, Embryo tetraploid complementation assays with un-induced iPSCs, miiPSCs or wild-type ESCs (n=3 clones per condition). Pictures on the right show a representative example of a viable "all-miiPSC" mouse (black) generated from miiPSCs and litters obtained from "all-miiPSC" adult mice, which efficiently contributed to germline transmission.
| Pten activates IgD expression and development of Fo.Figure 4A | Schematic overview of the transduction procedure: Purified Ptenf/f x mb1-cre x MD4tg-derived mature splenic B cells were cultured in presence of LPS 2.5 μg/ml for 1.5 days and subjected to retroviral transduction with expression vectors encoding the constitutively active FoxO1 (FoxO1-A3) or EV, respectively. B | 3 days post transduction the surface expression of IgM/IgD (dot plots, top), IgD and CD23 (histograms, bottom) was measured by flow cytometry and compared between EV- and FoxO1-A3-transduced cells. Fluorescence minus one (FMO) stainings, lacking α-IgD or α-CD23 antibodies respectively, served as controls (ctrl). Bar diagrams below flow cytometry data display the percentages of IgD+ cells (n = 12; left) and MFI of CD23 (as relative units, RU), normalized to the MFI measured in the respective EV transduced cells; n = 12; middle). Fcer2 transcript levels were measured by qRT-PCR (right, n = 4). Mean ±SD, symbols indicate data from individual mice. Statistical significance of IgD expression was determined by applying the Wilcoxon matched-pairs signed rank test for CD23 and by applying the one sample two-tailed t-test for Fcer2 expression. C | Cstf64 (n = 8), Ell2 (n = 8) and Zfp318 (n = 11) expression levels were measured at 3 days post transduction by qRT-PCR. FoxO1-A3- and EV-transduced splenic B cells were FACS-purified according to the gating strategy Mean ±SD, symbols indicate the expression in individual mice. Statistical significance was calculated by using the one-sample two-tailed t-test. D | Schematic overview of transduction procedure: Purified FoxO1f/f-derived mature splenic B cells were cultured in presence of 2.5 μg/ml LPS for 1.5 days and subjected to retroviral transduction with expression vectors encoding either cre or EV, respectively. E: IgD (n = 6), CD23 (n = 4) and Fcer2 (n = 4). F: Cstf64 (n = 4), Ell2 (n = 4) and Zfp318 (n = 4).
Figure 5(A) Multiple sequence alignment of AsiA homologs from different phage genomes at residue positions 50-61. Highlighted red residues indicate positions that are mutated in AsiA-m2.1. (B) CRISPRi in S. enterica and K. oxytoca using CasTA2.1. (C) CRISPRa in S. enterica and K. oxytoca using CasTA1.0 (dCas9-AsiA_wt), CasTA2.1 (dCas9-AsiA_m2.1) or ancestral strain with basal promoter expression (none).
Corticosteroid treatment affects H.Figure 6Corticosteroid treatment affects H. influenzae clearance by antibiotic treatment in the mouse airwayA, B Mice infected intranasally with 1 × 108 CFU H. influenzae and treated by inhaling PBS with or without 50 μM beclomethasone. Azithromycin was administered at a concentration of 100 mg/kg/24 h, daily for 3 days after infection. On day 3-4 after infection, the mice were harvested, and bacterial loads were determined in lung (A) and spleen (B) homogenates. Values represent the mean ± standard deviation (SD). The data are pooled from three independent experiments. Statistical significance by two-tailed Student's t-test is indicated: *P < 0.05, **P < 0.01.
Schistosomal-miRNAs are found in lymph node-derived Th cells.Figure 6Image of a schistosome-infected mouse. The lymph nodes, from which the Th cells were isolated, are indicated. Th cells were isolated from the indicated lymph nodes and spleen. In the case of uninfected mice, the Th cells from different lymph nodes and the spleen were mixed. qRT-PCR was performed with specific Taqman primers to either schistosomal-miR-10 or -Bantam. The presence of the miRNAs is presented as arbitrary units calculated as 2∆∆CT of the miRNA divided by snRNA U6. The mean +/- SEM was calculated from 4 independent experiments.
Figure 3A. Schematic of the quantitative BIR assay. Modified chr. VIIL (red) and chr. IIR (blue) share a 6,272 bp homology (grey shadow) used to repair an HO-induced DSB by BIR. Black boxes indicate hybridization probes used in Southern blotting experiments to monitor re-synthesis of resected DNA (RS-probes, red) and BIR (BIR-probes, blue) respectively. Numbers next to the one-ended arrows indicate distances (in bp) from the homology to the distal restriction sites of the DNA fragments analysed by Southern blotting using the corresponding probes. Numbers between the restriction sites indicate the sizes of restriction fragments detected by the corresponding hybridisation probes. B. Southern blot analysis of re-synthesis of resected DNA during BIR in SRS2 and srs2Δ corresponding to the data quantifications in C. DNA was digested with EcoRI and BamHI, resolved on 0.7% agarose gels, transferred onto charged Nylon membrane and hybridised to the mixture of 4 probes (three RS probes and a reference probe, REF, hybridizing to an ARS522-containing fragment on chr. V which is not involved in the repair). A representative image of one of the three repeats is shown.C. Re-synthesis of resected DNA on chr.VIIL in SRS2 and srs2Δ cells (solid and dashed lines, respectively) at the distance of 15.2 (RS15.2), 6.8 (RS6.8) and 2.6 (RS2.6) kb away from the homology region. Average ± SD (n=3) is shown for each time point. D. Southern blot analysis of BIR-dependent DNA synthesis in SRS2 and srs2Δ corresponding to the data quantifications in D. DNA was digested with EcoRI and BamHI, resolved on 0.7% agarose gels, transferred onto charged Nylon membrane and hybridised to the mixture of 4 probes (three BIR probes and a reference probe, REF, hybridizing to an ARS522-containing fragment on chr. V which is not involved in the repair). A representative image of one of the three repeats is shown. C - control strain NK3980.E. BIR-dependent DNA synthesis in SRS2 and srs2Δ cells (solid and dashed lines, respectively) at the distance of 6 (BIR6), 36 (BIR36) and 77 (BIR77) kb away from the homology region. Average ± SD (n=3) is shown for each time point.
Figure 4Photosynthetic CO2 assimilation (A) in dark-adapted WT and stp1stp4 plants in response to a step increase in CO2 concentrations from 0 to 1000 ppm. Data shown are means ± SEM; n=3 per genotype. Quantification of leaf glucose (Glc). Data for two independent experiments are shown; means ± SEM; n ≥ 17 per genotype and time point. Quantification of leaf fructose (Fru). Data for two independent experiments are shown; means ± SEM; n ≥ 17 per genotype and time point.   Quantification of leaf sucrose (Suc). Data for two independent experiments are shown; means ± SEM; n ≥ 17 per genotype and time point.   Quantification of leaf starch. Data for two independent experiments are shown; means ± SEM; n ≥ 17 per genotype and time point.   Representative Red Green Blue (RGB) images of 3-(day 0) and 4-week-old (day 6) WT and stp1stp4 plants grown under 600 ppm CO2. Projected rosette area over the phenotyping period. Data shown are from two independent experiments; means ± SEM; n ≥ 24 per genotype and time point. Representative confocal laser microscopy images of propidium iodide-stained guard cell starch granules of intact leaves of WT and stp1stp4 plants grown under 600 ppm CO2. Scale bar, 10 µm. Starch dynamics in guard cells of intact leaves of WT and stp1stp4 plants grown under 600 ppm CO2 at the end of the night (EoN) and after one and three hours of illumination with 150 µmol m-2 s-1 of white light. Data for two independent experiments are shown; means ± SEM; n = 80 individual guard cells per genotype and time point.
Anti-MAPK therapy re-routes developmental trajectories in CRC organoids.Figure 6A Activities of gene expression signatures, as indicated, in single cell transcriptomes from control (DMSO) condition CRC organoids, ordered along latent time. Correlation between cell state distributions and latent time was calculated using Pearson´s r. For correlations and significances, see Table EV6. Color code red: high activity; blue: low activity. B UMAPs of organoid single cell transcriptomes, color coded by treatment conditions, as indicated. C Dendrograms of transcriptome similarities across treatment conditions, per organoid line. Height of dendrogram is obtained by hierarchical clustering on the overlap of KNN neighborhoods across conditions. Preferred treatment conditions are marked by asterisk. D Heatmap of signature gene expression, from scRNA seq data. Average activities of gene signatures are given as log fold-change for preferred treatment condition, after normalization to DMSO control condition. Range of color scale was adjusted for each signature. E RNA velocity analysis of organoids under DMSO and preferred treatment conditions. Latent time is given as color code in the control (DMSO) condition UMAP, dynamical velocities are displayed as arrows. F Scaled signature expression and single gene expression moments per cell, under control (grey) and preferred treatment (color) conditions, as indicated. 10% of cells populating the end of latent time are displayed in bold. It is of note that latent time is linear and thus cannot capture multiple populations located at developmental end points. Densities at the side lines display expression in all cells.
Figure 3A, B, Representative images from scaf1+/+ and scaf1-/- (A) female and (B) male adult zebrafish. c-f, Size of scaf1Δ1/ Δ1 and scaf1Δ2/ Δ2 (scaf1-/-) fish in comparison with their respective scaf1+/+ lines, (C) length and (E) weight of females (Δ1 +/+ n=10, Δ1 -/- n=12, Δ2 +/+ n=24, Δ2 -/- n=18); (D) length and (F) weight of males (Δ1 +/+ n=16, Δ1 -/- n=13, Δ2 +/+ n=13, Δ2 -/- n=23). G-K, Adipose tissue measurements on hematoxylin-eosin (H&E)-stained adult zebrafish sagittal sections. (G, I) Adipose tissue area per total section area (average of 3 sections/biological replicate) and (H, J) adipocyte size (average of 20-30 adipocytes of ventral adipose tissue per biological replicate) of females (G, H) (Δ1 n=8, Δ2 n=8, same number of animals for homozygous mutants and controls) and males (I, J)(Δ1 n=8, Δ2 n=7, same number of animals for homozygous mutants and controls). K, Representative images of ventral fat deposits in females (dotted lines). L-N, Effect of Scaf1 loss of function on female fertility. (L) Number of eggs per clutch (Δ1 +/+ n=12, Δ1 -/- n=13, Δ2 +/+ n=13, Δ2 -/- n=10). (M) Quantification of mature ovary follicles per ovary section (average of three sections/biological replicate) (Δ1 n=5, Δ2 n=8; same number of animals for homozygous scaf1+/+ and scaf1-/-). (N) Representative images of H&E-stained ovaries. Dotted lines delineate adipose tissue.
Mitochondrial proteins represent a significant fraction of locally synthesized proteins in synaptoneurosomes (A) Workflow of the experiment presented in panel (C), depicting synaptoneurosomes (SN) isolation and in vitro stimulation in the presence of radioactive 35S-labelled methionine/cysteine mix.Figure 1(A) Workflow of the experiment presented in panel (C), depicting synaptoneurosomes (SN) isolation and in vitro stimulation in the presence of radioactive 35S-labelled methionine/cysteine mix. B) Western blot on fractions obtained during SN preparation revealed the enrichment of both pre- and postsynaptic markers, but not glia marker (GFAP) in the SN fraction. Cytosolic markers were depleted, while nuclear markers were barely detected in the SN (n=4 biological replicates, *** p<0.001, ** p<0.01, * p<0.05; one sample t-test; error bars indicate SEM). C) Synaptoneurosomes were NMDAR-stimulated and incubated with radioactive 35S-methionine/cysteine mix for 15-120 min. After labeling, proteins were separated on SDS-PAGE and newly synthesized proteins in SN labeled with [35S] were visualized by autoradiography (left panel). To determine the contribution of mitochondrial and cytosolic translation the labeling was preceded by treatment with chloramphenicol (CAP, 50 μg/ml) or with puromycin (PURO, 3mM) respectively (right panel). After 1 hour after the stimulation, a significant increase in 35S-labelled proteins was observed (n=6 biological replicates, *** p<0.001; repeated measures one-way ANOVA, post-hoc Sidak's multiple comparisons test) and the CAP treatment did not affect the overall levels of de novo synthetized proteins (n=6 biological replicates, ns; p=0.220). In contrast, PURO treatment significantly inhibited de novo protein synthesis in synaptoneurosomes (n=6 biological replicates, ** p=0.0091, repeated measures one-way ANOVA, post-hoc Sidak's multiple comparisons test). Error bars indicate SEM. (D-F) Results of mass spectrometry (label free quantification - LFQ) analysis identifying proteins with increased abundance in NMDAR-stimulated (20 min) synaptoneurosomes. (D) Volcano plot (Whitney-Mann test, n=4 per group) showing abundance of identified proteins in stimulated synaptoneurosomes as compared to unstimulated. Mitochondrial proteins are depicted in orange. The vertical line defines the p-value statistical significance cut-off. (E) Biological functions of identified proteins annotated using DAVID. Third top functional category identified as mitochondrial ones. (F) Pie-chart showing percentage share of mitochondrial proteins among all identified upregulated proteins.