EMBO/sd-character-level-ner huggingface dataset:
Figure 4A, B The expression level of OsHKT2;1 and OsHKT1;5 in osprr73 mutant and the wild control Dongjin (DJ) plants with or without salinity stress in multiple time points. DJ Data represent means ± SD. n = 3, technical replicates. The representative data is from at least two individual biological replicates. The asterisks represent significant difference among means by student's t-test with (*) P ≤0.05, (**) P ≤ 0.01 and (***) P ≤ 0.001. C Transient expression assay in the infiltrated leaves of N. benthamiana showing OsPRR73 can repress OsHKT2;1 transcription. Left panel showing the bioluminescence signal of OsHKT2;1 and OsPRR73. Bioluminescence of co-transformation of either GFP or 35S: GFP-OsPRR73 (GFP-OsPRR73) with OsHKT2;1pro: LUC in N. benthamiana. D Quantiﬁcation of bioluminescence intensity of OsHKT2;1pro: LUC shown in (C). Data represent means ± SD. n = 10. The asterisks indicate the significant difference by student's t-test with (***) P ≤ 0.001. At least two biological replicates were conducted with similar results. E ChIP-qPCR assay showing the enriched DNA fragments including the S3 and S4 regions of OsHKT2;1 promoter by OsPRR73, compared to wild type DJ controls. The amplicon of Ubiquitin was taken as a negative control. Two-week-old seedlings were harvested at ZT12. Data represent means ± SD (n =3, technical repeats). The experiments were performed at least two biological replicates with similar result. Top scheme indicates the locations of amplicons for ChIP analysis. The asterisks represent significant difference among means by student's t-test with (**) P ≤ 0.01. F EMSA assay with the OsPRR73 incubated with the probes designed for the S4 region of OsHKT2;1 promoter. Unlabelled probes were used as competitor with indicated folds. MBP alone was used as negative control. Arrowhead marks the shifted positive bands.
Figure 3A Electron micrographs of VPS34IN1- and DMSO-treated synapses following four consecutive trains of 200 AP (40 Hz, 5 s; 90 s gap between each train) stimulation. The postsynaptic compartment and the postsynaptic density are highlighted in green and purple, respectively. Note the reduced SV numbers and plasma membrane-derived endocytic intermediates in VPS34IN1-treated synapses. Scale bars, 500 nm (left) and 200 nm (right). B High magnification views of endocytic intermediates accumulated in stimulated hippocampal neurons treated with VPS34IN1. Scale bar, 200 nm. C 3D-reconstruction of a synaptic terminal treated with VPS34IN1 and stimulated as in A. The plasma membrane is colored in turquoise. Synaptic vesicles (yellow), plasma membrane-derived invaginations (blue), endosome-like vacuoles (green) and clathrin-coated vesicles (red) are highlighted. Scale bar, 500 nm (top) and 200 nm (bottom). Data information: Data presented as mean ± SEM n.s. not significant; ***p < 0.001; ****p < 0.0001 D-H Morphometric quantification of data shown in A. Synapses of VPS34IN1-treated stimulated neurons display reduced numbers of SVs/ bouton area in (D) and an accumulation of endocytic membrane invaginations (E), endosome like vacuoles (ELV) (F), clathrin-coated pits (CCP) (G), and clathrin-coated vesicles (CCV) (H). n = 100 (SV) and n = 200 (invagination, ELV, CCP, CCV) profiles per condition; Two-way ANOVA; Tukey's Multiple Comparison Test. Data information: Data presented as mean ± SEM n.s. not significant; ***p < 0.001; ****p < 0.0001
Figure 6(A) Ifnar1-/- MEFs were transfected with the indicated Cy5-labeled dsRNA and the next day were infected with SFV-Rluc at the indicated multiplicities of infection (MOI). Renilla luciferase activity was measured 24 hours later. Each bar represents mean + SD of biological duplicates and numbers above each pair of bars depict the fold-reduction in viral activity achieved with dsRNA-RL vs. dsRNA-GFP treatment. (B) As in (a) but SFV-Rluc virus titre was measured by plaque assay and expressed as plaque forming units (PFU) per ml of supernatant at 24 h post-infection (MOI=0.1). (C) As in (a) but with Ifnar1-/- or Mavs-/- MEFs and a single dose of SFV-Rluc (MOI=0.1). (D) As in (a) but with two Mavs-/- Ago2-/- clones (clone 5.2 and 5.3) individually complemented to express wild-type (HA-mAgo2 WT) or a catalytic mutant (HA-mAgo2 D597A) version of mAgo2 or transduced with a control vector (as described in Fig 5). SFV-Rluc was used at an MOI=0.1.
a, Maximal treadmill running distance for mice of indicated genotype.f3a, Maximal treadmill running distance for mice of indicated genotype. Data represent mean ± s.e.m. of 5 mice per group. b, Representative haematoxylin and eosin (H E) and periodic acid-Schiff (PAS) staining in tibialis anterior muscle sections from mice of indicated genotype. Scale bar, 20 µm. c, d, Plasma glucose (c) and insulin (d) levels in mice of indicated genotype at rest, after 80 min exercise (∼900 m), or maximal exercise. Data represent combined mean ± s.e.m. for 9-11 mice per group from three independent cohorts; similar results were observed in each cohort. e, Representative images of GLUT4immunofluorescence microscopy staining in vastus lateralis muscle of mice of indicated genotype before and after maximal exercise. Scale bar, 20 µm. For b and e, similar results were observed in 3 mice per group. f, Western blot analysis of AMPK phosphorylation (p-AMPK (Thr172)) in vastus lateralis muscle lysates from mice of indicated genotype at indicated time after exercise. g, Maximal treadmill running distance for mice of indicated genotype. Data represent mean ± s.e.m. of 4-6 male mice per group. h, Soleus muscle14C-deoxyglucose uptake during treadmill exercise in mice of indicated genotype. Data represent mean ± s.e.m. of 3 mice per group. *P<0.05, **P<0.01, ***P<0.001, one-way ANOVA for comparison between groups; †P<0.05, ††P<0.01, two-way ANOVA for comparison of magnitude of changes between different groups in mice of different genotypes. NS, not significant.
Figure 7(A) HIV-1-infected primary CD4 T cells (strain CH058) were incubated with sera of HIV negative (HIV-; n=9) or ART-treated HIV-1-infected (HIV+; n=9) individuals (serum dilution 1:1000). The levels of IgG binding on infected (Gag+) cells were analyzed by flow cytometry. Results are expressed as the MFI on infected (Gag+) cells. Error bars indicate SEM. ****P<0,0001; Mann-Whitney test. (B) CH058-infected primary CD4 T cells were cultured for 24h with sera of healthy (HIV-; n=9) or ART-treated HIV-1-infected (HIV+; n=9) individuals (serum dilution 1:100) and 50% NHS from a healthy donor. C3 surface levels on infected (Gag+) cells were analyzed by flow cytometry. Results are expressed as the MFI on infected (Gag+) cells. Error bars indicate SEM. ****P<0,0001; Mann-Whitney test. (C) CD4 T cells not infected (NI) or infected with WT, ∆Nef, ∆Vpu or ∆Nef∆Vpu HIV-1 (strain CH058) were incubated with the serum of HIV-1-infected ART-treated individuals (sKB104 or sKB105, dilution 1:1000). The levels of IgG binding on infected (Gag+) cells were analyzed by flow cytometry. One representative experiment is shown on the left. On the right, results are expressed as MFI of staining. Each dot represents a single donor of CD4 T cells. n=6 donors of CD4 T cells. Error bars indicate SEM. *P<0.05, Wilcoxon test. Only significant comparisons are depicted. (D) Primary CD4 T cells either not infected (NI) or infected with WT, ∆Nef, ∆Vpu or ∆Nef∆Vpu HIV-1 (strain CH058) were incubated with 50% NHS from a healthy donor and serum of HIV-1-infected ART-treated individuals (sKB104 or sKB105, dilution 1:100). The levels of C3 deposition on infected (Gag+) cells were analyzed by flow cytometry. One representative experiment is shown (left panel). Results are expressed as MFI of staining (right panel). Each dot represents a single donor of CD4 T cells. n=6 donors of CD4 T cells. (E) Correlation between patients' sera (sK104 and sKB105) antibody binding and C3 deposition on CD4 T cells either mock-infected or infected with WT, ∆Nef, ∆Vpu or ∆Nef∆Vpu HIV-1 (strain CH058). Each dot is the mean of 6 donors of CD4 T cells. Correlation was analyzed by Spearman correlation. Correlation coefficient (r) and p-value are indicated.
Figure 3A. HeLa cells transfected with siCtrl or siBclaf1-1 were treated with TNF (10 ng/ml) for the indicated time periods followed by western blotting analysis with the indicated antibodies. B. MEFs treated with TNF (10 ng/ml) for 4 hours were subject to FADD immunoprecipitation, and then analyzed by western blotting. MEFs cells pretreated with CHX (1 μg/ml) for 30 min and then stimulated with TNF (10 ng/ml) in the presence of CHX for indicated times were subject to FADD immunoprecipitation, and then analyzed by western blotting. D. HeLa (D) cells pretreated with CHX (1 μg/ml) for 30 min and then stimulated with TNF (10 ng/ml) in the presence of CHX for indicated times were subject to FADD immunoprecipitation, and then analyzed by western blotting. E, F. Bclaf1 WT, KO-1 and -2 HeLa cells (E) or MEFs transfected with siCtrl or siBclaf1-1 and -2 (F) were pretreated with CHX (1 μg/ml) for 30 min and then stimulated with TNF (10 ng/ml) in the presence of CHX for 12 hours followed by western blotting analysis. G, H. HeLa cells transfected with siRNAs against Bclaf1 or c-FLIP were pretreated with CHX (1 μg/ml) for 30 min and then stimulated with TNF (10 ng/ml) in the presence of CHX for 12 hours followed by Annexin V/7AAD staining and flow cytometry analysis (G), or protein extractions followed by western blotting analysis (H). Data information: Data are shown as mean ± SD. n=3 biological replicates. **p<0.01; ****p<0.0001. One-way ANOVA test. I. After transfection with siBclaf1-1 for 12 hours, the HeLa cells were transfected with Flag-FLIP for 24 hours, and then treated as described in H followed by western blotting analysis.
Figure 1A Schematic representation of the UbG76V-GFP chimeric repoter and its associated internal control (mRFP or mCherry, in red). The amino acid sequences for ubiquitin are in yellow, whereas the sequences for GFP are in green. The UbG76V-GFP chimera, which contains a mutation in the terminal residue of ubiquitin, cannot be cleaved. The resulting protein is a substrate for poly-ubiquitination (indicated by the circles labeled with "u") and degradation by the proteasome. High levels of reporter poly-ubiquitination and/or proteasome activity result in little or no GFP fluorescence. B Quantified fluorescence of UbG76V-GFP normalized to mRFP in the hypodermis from animals of the indicated timepoint (in hours) after the L4 stage. Animals have been exposed to the indicated knockdown RNAi bacterial strains or a strain that only contains the empty RNAi vector. *P<0.001, Student t-test corrected for multiple comparisons at each time point using the Holm-Sidek method. N=20 animals per genotype and timepoint. Error bars indicate SEM. C,D Co-expression via the col-19 promoter of UbG76V-GFP (green) and mRFP (red) in C. elegans hypodermis from a single integrated transgene at L4+48 hours. Wild-type animals are shown. Bar, 50 microns. UbG76V-GFP fluorescence is barely detectable.E,F Co-expression via the sur-5 promoter of UbG76V-GFP (green) and mCherry (red) in C. elegans from two separate integrated transgenes (with focus on the intestine) at L4+48 hours. Wild-type animals are shown. Bar, 50 microns. UbG76V-GFP fluorescence is barely detectable.G,H Co-expression of UbG76V-GFP (green) and mRFP (red) in C. elegans hypodermis at L4+48 hours in dop-1(vs100) mutants. Bar, 50 microns. Abundant, stable UbG76V-GFP fluorescence is observed.I,J Co-expression of UbG76V-GFP (green) and mCherry (red) in C. elegans from the sur-5 promoter at L4+48 hours in dop-1(vs100) mutants. Bar, 50 microns. Abundant, stable UbG76V-GFP fluorescence is observed, particularly in the intestine. K Quantified fluorescence of UbG76V-GFP normalized to mRFP in the hypodermis from animals of the indicated timepoint (in hours) after the L4 stage and of the indicated genotype. *P<0.001, Student t-test corrected for multiple comparisons at each time point using the Holm-Sidek method. N=20 animals per genotype and timepoint. Error bars indicate SEM. L Quantified fluorescence of UbG76V-GFP normalized to mRFP in the hypodermis of animals at L4+48 hours and of then indicated genotypes. ***P<0.001, ANOVA with Dunnetts posthoc comparison to the wild-type control. N=20 animals per genotype and timepoint. Error bars indicate SEM. M Quantified fluorescence of UbG76V-GFP normalized to mCherry (both expressed by the sur-5 promoter) in the intestine of animals at L4+48 hours and of then indicated genotypes. ***P<0.001, **P<0.01, *P<0.05, ANOVA with Dunnetts posthoc comparison to the wild-type control. N=20 animals per genotype and timepoint. Error bars indicate SEM.
Detection of disease-associated myeloid cell clusters.Figure 7a) Schematic diagram depicting the experimental setup. b) Isolation of single myeloid cells from the retina under healthy conditions (d0) and three (d3) or 7 days (7d) after laser pulse for unbiased sampling and single-cell RNA-seq (scRNA-seq). Increased cellular heterogeneity in lesioned mice compared to healthy controls (d0), shown by May-Grünwald-Giemsa stained cytospins. Scale bars represents 10 µm. c c) t-SNE representation of individual cells from all conditions analyzed (control represents homeostatic microglial cells) (122 cells), CNV d3, CNV d7 with scRNA-seq. Each dot represents an individual cell. Color code indicates the different experimental conditions. d) t-SNE plot depicting different cell types. e) Unbiased cluster analysis of hematopoietic cell subpopulations found at different conditions. f) Marimekko chart showing the cluster-wise distribution of 511 cells color-coded for the experimental conditions (control 122 cells, CNV d3 209 cells, CNV d7 180 cells). Hypergeometric testing revealed significantly enriched clusters for the different conditions (control: cluster 4 and 5; CNV d3: cluster 1, 6 and 7; CNV d7: cluster 11, 12 and 14), indicated by asterisks (*p < 0.05, **p < 0.01, ***p < 0.001). g) Marimekko chart presenting the proportion of cell types among all assigned cells (see fig. 5d) during different experimental conditions demonstrating almost pure microglia populations present at d0, whereas clusters of dendritic cells (DC), lymphocytes, monocytes, granulocytes and macrophages are present at d3 and d7 of CNV. h h) Heatmap of the 30 most regulated genes per cluster (adjusted p-value < 0.05 based on the negative binomial distribution). Asterisks highlight genes presented in t-SNE plots shown in i).The scale bar represents the color-coded z-scores. i) Selection of t-SNE plots representative for different cell subsets using Hexb, C1qa and Sparc to show differential expression of these genes between microglia subpopulations; Grp34, Csf1r and Gapdh to depict cluster 6 as the highest activated microglia cluster; Cd74, H2-Aa and Clec10a to present dendritic cell cluster; Ly6c2, Plac8 and Ms4a4b to illustrate blood-derived cell cluster.
Characterization of IKur and IK,ACh in hESC-ventricular and hESC-atrial CMs.Figure 6A Expression of KCNA5 (left) and KCNJ3 (right) in GFP+ pools of VM and AM CMs at day 31, as well as in ventricles and atria of human heart. B, C Typical examples (left) and current-voltage relationships (right) of (B) IKur and (C) IK,ACh in VM and AMCMs.Data information: Data are presented as mean ± SEM. In (A), *P < 0.05 by unpaired t-test. In (B, C), *P < 0.05 by two-way repeated measures ANOVA followed by pairwise comparison using the Student-Newman-Keuls test for (B) and Mann-Whitney rank-sum test for (C). In (B), P = 0.778, 0.350, 0.03, 0.02, 0.002, 0.001, < 0.001 and < 0.001, respectively, for comparison between VM and AM within membrane potentials of −20, −10, 0, 10, 20, 30, 40 and 50 mV. In (C), P = 0.01, 0.01, 0.01, 0.01, 0.03, 0.397, 0.397, 0.397, 0.671, 0.207, 0.207, 0.09, 0.01, 0.039 and 0.015, respectively, for comparison between VM and AM within membrane potentials of −120, −110, −100, −90, −80, −70, −60, −50, −40, −30, −20, −10, 0, 10 and 20 mV. CMs = cardiomyocytes; hESC-atrial (AM) and hESC-ventricular (VM) CMs; IK,ACh = acetylcholine-activated potassium current; IKur = potassium ultra-rapid delayed rectifier current. 4-AP = 4-aminopyridine; CCh = carbachol. D, E Representative APs of VM and AM at 1 Hz in response to (D) IKur block by 4-AP and (E) IK,ACh activation by CCh. AP parameters are shown in Supplementary Table S5.
FBXO2 is required for GAS xenophagy.Figure 5A Wester blotting of FBXO2 in human cell lines. B Bacterial invasion rates into host cells (CFU recovered at 2 h post-infection/CFU at 1 h post infection) which were normalized to that in wild-type cells. C Intracellular bacterial survival rates (CFU recovered at 6 h post-infection/CFU at 2 h post-infection) which were normalized to that in wild-type cells. D HeLa WT or FBXO2-KO cells transfected with mCherry-Gal3 (magenta) were infected with WT GAS for 2 or 4 h, and percentages of Gal3-positive GAS-containing cells were quantified. E - J Localization of ubiquitin, LC3 and p62 in FBXO2 KO cells. HeLa WT or FBXO2-KO cells were infected with WT GAS for 4 h, fixed, and immunostained for ubiquitin (FK2: magenta) or LC3 (magenta) or p62 (magenta). Representative confocal images (E, G, and I) and percentages of cells with ubiquitin, LC3, or p62 -positive GAS (F, H, and J). K, L Accumulation of LC3-II during GAS infection. (K) Representative western blot of LC3 during infection. (L) LC3-II intensities normalized to non-infection condition of wild-type cells. M, N GlcNAc recognition of FBXO2 required for GAS xenophagy. Non-transfected HeLa WT cells and HeLa FBXO2-KO cells transfected GFP, GFP-FBXO2, GFP-FBXO2 YW/AA were infected with GAS WT for 4 h, fixed, and immunostained for ubiquitin. Percentages of cells with LC3 (M) or ubiquitin (N) -positive GAS were quantified.
Figure 5(A) Role of E10 in the RING of TRIM25 (blue) in stabilizing the closed E2~Ub conformation by contacting K11 from the proximal ubiquitin (salmon) and N71 of the opposite RING (cyan) which in turn contacts K33 of the same ubiquitin. (B) E2~Ub discharge and K63 poly-ubiquitination assays to test the role of E9 and E10 in TRIM25 activity. Substitution of Glu9 with Arg has no significant effect on activity, whereas the E10R mutation almost completely abolishes catalytic activity. (C) Discharge and K63 poly-ubiquitination assays to test the role of the equivalent residue E16 in TRIM32 and the role of I85R. Mutation of E16R abolishes catalytic activity indicating that the role of the glutamate is conserved. (D and E) Quantification of UBE2D1~UbAtto discharge assays. The loss of E2~Ub is plotted as the average of experimental duplicates (± s.d.).
AAV-split-dCas9 Activator System (AAV-dCAS) based reprogramming of astrocytes.Figure 4A, Representative photomicrographs taken from the dorsal striatum 13 weeks after AAV-injection. In the GFP control condition, virtually all GFP positive cells depict an astrocytic morphology, many GFP positive cells in ALNe-218 and ALN-treated animals show a neuron-like morphology. Data information: Scale bars indicate 20 µm. B, Immunohistochemical analysis showing GFP+/GFAP+ double positive cells 13 wpi. Arrows indicate double positive GFP+/GFAP+ cells, arrowheads indicate GFP+/GFAP- cells. Quantification of GFAP+/GFP+ cells shows a significant decrease upon ALNe-218 and ALN-treatment (GFP vs. ALNe-218 P=0.0083, GFP vs. ALN P<0.0001 and ALN vs. ALNe-218 P=0.0006, multiple comparison ANOVA F(2,6)=79.76). C, Photomicrographs showing GFP+/NeuN+ neurons 13 wpi. Arrowheads indicating GFP+/NeuN+ induced neurons. Quantification demonstrate a significant increase in NeuN+/GFP+ cells upon ALNe-218 and ALN-induction (GFP vs. ALN P=0.0008, ALN vs. ALNe-218 P=0.0092, multiple comparison ANOVA F(2,7)=21.74). Data information: Scale bars indicate 20 µm. Error bars represent mean ± SD. n = 3-4 mice (b,c) per condition. Tukey's multiple comparisons test * P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. D, In ALN-induced neurons, confocal analysis is demonstrating a co-localization of GFP and glutamic acid decarboxylase (Gad65/67), a marker specific for GABAergic neurons. E, Quantification of GAD6567+/GFP+ cells with neuronal morphology shows that the majority of ALN-induced neurons are colocalizing with this GABAergic marker both in the AAV-dCAS as well as in the dCAM setting. Data information: Scale bars indicate 20 µm. Error bars represent mean ± SD. n= 6-8 mice (e) per condition. Tukey's multiple comparisons test * P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.
Figure 7Comparison of NUP58 mRNA abundance between late (P19) and early (P1) passage in wild-type cells (WT, top) and clone C5 (bottom). mRNA was quantified using qPCR and five independent probe sets amplifying different regions of NUP58 mRNA. Annealing of the probe sets is indicated at the bottom of the NUP58 structure. Relative mRNA abundance between P19 and P1 is color-coded for each exon analysed (according to the color bar shown below). Red triangles highlight the position of the identified mutations in C5. Diagram at the top represents G-banding pattern of chromosome 13 and highlights chromosome locations of tested genes (NUP58, GRK1, ADPRHL1). Graph at the bottom reports qPCR results for indicated genes preformed on the gDNA of indicated samples at the indicated passage number. Representative images of NUP58 gene-specific FISH hybridization for control HAP1 cell lines (WT P3-WT P19) and mutant C5 at P19. Each dot represents a NUP58 locus of cluster thereof. Scale bar, 31.3 μm
Figure 3A-C, Zoom-in analysis of fibroblasts identified in Fig 2. A, tSNE plot colored by FB subpopulations. B, Dot plot showing expression of ECM genes in FB subpopulations. C, Bar plot showing relative proportion of FB subpopulations in FB at each time point. Expression of fn1a, col12a1a (D) in FB at each time point. Expression of tagln (E) in FB at each time point. F, In situ hybridization showing temporal spatial expression patterns of postnb. The white boxed region is shown in zoom-in images at the bottom. Scale bar = 25 μm. White dashed lines outline the heart and the yellow dashed lines indicate approximate resection plane. A stands for arium, V stand for ventricle and OFT stands for outflow tract. G, Expression of tnfrsf1a and tgfbr2a in FB at each time point. H, Fluorescent in situ hybridization for postnb, tnfα, and tnfrsf1a in the injury area at 7 dpf, respectively. The white boxed regions are shown in their respective zoom-in images to the right. White dashed lines outline cardiac apex and the yellow dashed lines indicate approximate resection plane. Scale bar = 25 μm. I, tSNE plot colored by eEC subpopulations. Bar plot showing relative proportion of eEC subpopulations in eEC at each time point. J, Violin plots showing expression of cxcl18b, sele, atf3, and fosl1a in each eEC subpopulation. K, Fluorescent in situ hybridization for fosl1a at 7 dpi. The white boxed region is shown in a higher magnification image to the right for the injury area. cdh5 was used to label all EC cells and the red boxed region is shown in a higher magnification image to highlight a pan-EC cdh5 expression in green. White dashed lines outline the heart and the yellow dashed lines indicate approximate resection plane. Scale bar = 25 μm. A stands for arium, V stand for ventricle and OFT stands for outflow tract.