EMBO/sd-character-level-ner huggingface dataset:
Figure 3scRNAseq was performed on sorted CD45+, Annexin V-, BM cells from a pool of two control mice and two IFNγ-treated mice at day 12, and on a pool of two control and two pools of two IFNγ-treated mice on day 17 (blue and yellow dots in Fig. 1B indicate the individual mice from the CTRL and IFNγ groups, respectively). (A) Uniform manifold approximation and projection (UMAP) visualization of the complete dataset. The left panel shows cells coloured according to unsupervised clustering (Louvain - resolution 0.6), the middle panel shows the distribution of cells according to experimental conditions, whereas the right panel displays cell-type classification according to singleR immgen annotation, as described in supplementary methods. ( scRNAseq was performed on sorted CD45+, Annexin V-, BM cells from a pool of two control mice and two IFNγ-treated mice at day 12, and on a pool of two control and two pools of two IFNγ-treated mice on day 17 (blue and yellow dots in Fig. 1B indicate the individual mice from the CTRL and IFNγ groups, respectively). (B) UMAP visualisation of myeloid compartment. From left to right panel cells are coloured according to: unsupervised clustering (Louvain - resolution 1.2), experimental condition and markers-based, custom cell-type classification. scRNAseq was performed on sorted CD45+, Annexin V-, BM cells from a pool of two control mice and two IFNγ-treated mice at day 12, and on a pool of two control and two pools of two IFNγ-treated mice on day 17 (blue and yellow dots in Fig. 1B indicate the individual mice from the CTRL and IFNγ groups, respectively). (C) Heatmap showing top marker genes (obtained from unsupervised clustering) used for custom classification of the myeloid compartment. Interferon-stimulated, inflammatory & proliferating monocytes belong to the group of classical monocytes. scRNAseq was performed on sorted CD45+, Annexin V-, BM cells from a pool of two control mice and two IFNγ-treated mice at day 12, and on a pool of two control and two pools of two IFNγ-treated mice on day 17 (blue and yellow dots in Fig. 1B indicate the individual mice from the CTRL and IFNγ groups, respectively). (D) UMAP visualisation of the lymphoid compartment. From left to right panels, cells are coloured according to: unsupervised clustering (Louvain - resolution 1.2), experimental condition and custom marker-based cell-type classification. scRNAseq was performed on sorted CD45+, Annexin V-, BM cells from a pool of two control mice and two IFNγ-treated mice at day 12, and on a pool of two control and two pools of two IFNγ-treated mice on day 17 (blue and yellow dots in Fig. 1B indicate the individual mice from the CTRL and IFNγ groups, respectively). (E) Heatmap showing top marker genes (obtained from unsupervised clustering) used for custom classification of cells that belong to the lymphoid compartment.
H2O2 directly inhibits the activity of HsAtgA.Figure 5(A) Cleavage activity was tested by incubation of recombinant His6‐HsAtg4A (0.1 μg) and His6‐GATE‐16‐HA (0.3 μg) in 50 KT reaction buffer at 30°C for 45 min in the presence of indicated concentrations of DTT followed by Western blot analysis, using anti‐His monoclonal antibodies. The resulting bands from three separate experiments were quantified with a densitometer using the Bio‐Rad Multi‐Analyst program and are presented as the average percentage of cleaved form out of the total GATE‐16. (*) indicates non‐cleaved His6‐GATE‐16‐HA and (**) indicates cleaved His6‐GATE‐16. (B) His6‐HsAtg4A was incubated in the presence of 200 μM DTT at 4°C for 10 min. Reduced His6‐HsAtg4A (0.1 μg) was then incubated in 50 KT (to obtain 15 μM DTT) with the indicated concentrations of H2O2 at 25°C for 5 min, after which recombinant His6‐GATE‐16‐HA (0.3 μg) was added and incubation proceeded at 30°C for 45 min. Reaction mixtures from three separate experiments were analyzed and are presented as explained in (A). (C) His6‐HsAtg4A (0.1 μg) was incubated with recombinant His6‐GATE‐16‐HA (0.3 μg) after the following procedures: no treatment (lane 1); pretreatment with 200 μM DTT at 25°C for 5 min (lane 2); treatment with 200 μM DTT followed by treatment with 1 mM H2O2 at 25°C for 5 min (lane 3); and treatment with 200 μM DTT followed by treatment with 1 mM H2O2 for 5 min and then 2 mM DTT (lane 4). Reaction mixtures were analyzed by Western blot using anti‐His monoclonal antibodies.
a, Survival ratio after TAC (n = 7–14 per group).f1a, Survival ratio after TAC (n = 7-14 per group). b-g, Ten days after TAC. b, Gross appearance of hearts. Scale bar, 2 mm. b-g, Ten days after TAC. c, Echocardiography. Scale bars, 0.2 s and 5 mm. Echocardiographic parameters (d) (n = 7-13 per group). LVIDd and LVIDs, end-diastolic and end-systolic left ventricular internal dimension, respectively; LVFS, left venticular fractional shortening; b-g, (e) Ten days after TAC.physiological parameters (n = 7-13 per group). Haematoxylin and eosin-stained (f) and azocarmine and aniline blue (AZAN)-Mallory-stained (g) heart sections. Scale bar, 100 µm. Data are mean ± s.e.m. *P  0.05 versus all other groups, **P  0.05 versus sham-operated controls.
Figure 7A. Data are mean 525em ratio from 488/445 nm excitation intensities of internalized FDx particles vs. pH of clamping buffer fit to a standard curve from two independent experiments. B. Representative images of PBS- and DR-EV-treated MDCK-SIAT1 cells. Scale bars for full size images = 50 μm. Scale bars for cropped images = 10 μm. FDx particles are shown green and DR-EV particles are shown purple. White arrows are FDx endosomes containing DR-EVs. C. Measured pH of FDx endosomes in PBS- (left bar) and DR-EV-treated (right bar) MDCK-SIAT1 cells. The pH of FDx endosomes containing DR-EVs in DR-EV-treated cells (middle bar) (corresponding to white arrows in Fig 7B). Data are the means of individual data points representing the average calculated pH of each endosome population within 1 hpf in each of 5 independent experiments.
a,b) Flow cytometry of CD8+ T cell obtained from chimeras generated (as in Supplementary Fig.figf6(a,b) Flow cytometry of CD8+ T cell obtained from chimeras generated (as in Supplementary Fig. 5a) by reconstitution of wild-type mice with a mixture of bone marrow cells from Atg7fl/fl C57BL/6 and wild-type C57BL/6C57BL/6 (Atg7fl/fl vs C57BL/6) or from Atg7fl/flGzmb-Cremice and wild-type C57BL/6mice and (Atg7fl/flGzmb-Cre vs C57BL/6), gated (far left) as populations specific for H-2Db-gp33 (a) or H-2Db-NP396 (b) and then assessed, on days 8, 15 and 30 after infection of recipients with LCMV Armstrong infection, as CD45.2+ (Atg7fl/fl or Atg7fl/flGzmb-Cre) donor cells or CD45.1+ (C57BL/6) donor cells (middle). Far right, appearance of tetramer-positive T cells (key, donor source) in the peripheral blood of chimeras from day 8 to day 30 after infection, presented relative to that at day 8 after infection, set as 100%. (c) Frequency of CD45.2+ antigen-specific CD8+ T cells in the spleen, liver and lungs on day 30 after infectionwith LCMV Armstrongas in a,b. Data are representative of two independent experiments with three to four mice per group (error bars, s.e.m.).
Figure 3Expression patterns of Hoxa5 and Hoxc8 mRNAs, as revealed by in situ hybridization, aligned with corresponding sections showing Hoxa5 and Hoxc8 immunostainings in MNs (demarcated with pink/white dashed lines) along the cervical spinal cord (cervical C5 to C7) in mouse embryos at E12.5. Pink scale bar, 50 μm. White and pink dash lines demarcate the postmitotic MN region. Schematic illustration of the strategy to apply single-cell RNA sequencing (scRNAseq) on FACS-collected homogenous Hb9:GFPon MNs. Visualization of E12.5 cervical MNs (right panel) and the subset of LMCs (left panel) using Uniform Manifold Approximation and Projection (UMAP), a 2D non-linear transformation of high-dimensional data that assigns proximal x-y coordinates to cells (points) with similar expression profiles. Individual cells are colored according to their cluster assignments. Details are provided in the Materials & Methods. Dot-plots showing expression of genes (rows) that distinguish MN subtypes (columns). The height of each colored shape is proportional to the percentage of cells expressing the marker (≥1 UMI), and its width reflects the average transcript count within expressing cells. scRNAseq results reflect that Hoxa5/Hoxc8 mRNAs largely co-exist along the RC axis of E12.5 spinal cord, particularly at the middle boundary region. Schematic illustration of Hoxc8 lineage tracing. Hoxc8-expressing cells and all of their progeny are indelibly marked by tdTomato (RFP) expression in Hoxc8:Cre;ROSA26-loxp-STOP-loxp-tdTomato (lsl-tdTomato) embryos. Analysis of the Hoxc8 lineage (RFPon cells) in Hoxc8:Cre;ROSA26-lsl-tdTomato E12.5 spinal cord sections (cervical segments 5-6). RFP is expressed in a broad domain that includes Hoxc8on cells and a subset of Hoxa5on cells (Hoxa5on plus RFPon, indicated by arrowheads). Pink scale bar, 50 μm. White dash lines demarcate the postmitotic MN region. Hoxa5on and Hoxc8on cells adopt an "all or none" segregated cell fate despite coexistence of Hoxa5/Hoxc8 mRNAs, indicating a potential post-transcriptional regulatory mechanism for cell fate segregation.
Expression levels, efficient deletion of Dot1L in B cells and its effect on cellularity of B lineage subsets in the bone marrow.Figure 1A. Expression of Dot1L in different B-cell populations (Shi et al., 2015). B1: B1 cells, MZB: marginal zone B, FOB: follicular B, GCB: germinal center B, SPLPB: spleen plasma blast, SPLPC: spleen plasma cells, and BMPC: Bone marrow plasma cells. Expression is shown as transcript per million (TPM). Results represent the data from two biological replicates except SPLPB and BMPC which lack replicates (WT; n=2)..Bars indicate mean values. B. Intracellular flow-cytometry staining for H3K79me2 in bone marrow B-cell subsets as well as splenic B and T cells from MB1Cre; Dot1Lwt/wt (WT) and MB1Cre; Dot1Lfl fl (KO) mice. Results represent the data from two independent experiments. C. Statistical analysis of absolute number of total nucleated cells from bone marrow B-cell subsets. Results represent the data pooled from three independent experiments and numbers represent biological replicates for each group (WT; n = 5, KO; n = 7). Bars and error bars indicate mean ± SD.
Figure 66-week-old male CD36 wild-type (WT) or knockout mice (CD36KO) were administered with AAV-GFP (Ctrl) or AAV-KLF10 (KLF10OE) through tail vein injection, and then kept on WD/CCl4 for 12 weeks. n=5 per group. (A) Protein levels of KLF10 and CD36 in the liver (left), and their normalization to β-actin (right). Data information: * P <0.05, ** P <0.01, *** P <0.001. Results are shown as mean ± SD. 1-way ANOVA (A 6-week-old male CD36 wild-type (WT) or knockout mice (CD36KO) were administered with AAV-GFP (Ctrl) or AAV-KLF10 (KLF10OE) through tail vein injection, and then kept on WD/CCl4 for 12 weeks. n=5 per group. (B) Representative immunofluorescence staining of KLF10 and CD36 (left), and the quantitative results (right). Data information: * P <0.05, ** P <0.01, *** P <0.001. Results are shown as mean ± SD. 1-way ANOVA 6-week-old male CD36 wild-type (WT) or knockout mice (CD36KO) were administered with AAV-GFP (Ctrl) or AAV-KLF10 (KLF10OE) through tail vein injection, and then kept on WD/CCl4 for 12 weeks. n=5 per group. (C) H&E and Sirius red staining of liver sections. (D) Quantitative results of Sirius red staining. Data information: * P <0.05, ** P <0.01, *** P <0.001. Results are shown as mean ± SD. 1-way ANOVA 6-week-old male CD36 wild-type (WT) or knockout mice (CD36KO) were administered with AAV-GFP (Ctrl) or AAV-KLF10 (KLF10OE) through tail vein injection, and then kept on WD/CCl4 for 12 weeks. n=5 per group. (E) Body weights and liver/body weight ratio. Data information: * P <0.05, ** P <0.01, *** P <0.001. Results are shown as mean ± SD. 1-way ANOVA 6-week-old male CD36 wild-type (WT) or knockout mice (CD36KO) were administered with AAV-GFP (Ctrl) or AAV-KLF10 (KLF10OE) through tail vein injection, and then kept on WD/CCl4 for 12 weeks. n=5 per group. (F) Hepatic TG contents. Data information: * P <0.05, ** P <0.01, *** P <0.001. Results are shown as mean ± SD. 1-way ANOVA 6-week-old male CD36 wild-type (WT) or knockout mice (CD36KO) were administered with AAV-GFP (Ctrl) or AAV-KLF10 (KLF10OE) through tail vein injection, and then kept on WD/CCl4 for 12 weeks. n=5 per group. (G) Serum AST and ALT levels. Data information: * P <0.05, ** P <0.01, *** P <0.001. Results are shown as mean ± SD. 1-way ANOVA
Figure 4(A) NSC-34 and SH-SY5Y cells were reverse-transfected with siPRPH or siNTC for 48 hrs prior to transfection with EV-A71 RNA. The culture supernatants were harvested for viral titer determination at 24h.p.t. (B) Schematic drawing of LucEV-A71 replicon. (C) NSC-34 and SH-SY5Y cells were reverse-transfected with siPRPH or siNTC for 48 hrs prior to transfection with LucEV-A71 replicon. Luminescence signal was read at 24h.p.t.
CircSLC8A1 is reduced under neuroprotection (A) qPCR measurements of circSLC8A1 and SLC8A1 mRNA after SH-SY treatment of statins or the PF LRRK2 inhibitor, T-test p**=0.Figure 5(A) qPCR measurements of circSLC8A1 and SLC8A1 mRNA after SH-SY treatment of statins or the PF LRRK2 inhibitor, T-test p**=0.007 and p**=0.01 for PF-06447475 and p*=0.047 and p=*0.0136 for Statins for cirSLC8A1 and SLC8A1 mRNA respectively (B,C) Protein gel and quantification of SLC8A1 and Tubulin as a loading control in Statin and PF treated neuronal cell cultures, T-test p*=0.027 for Statins and p*=0.011 for PF-06447475, n=3 biological replicas for each condition. Data presented as mean ± SD. (D) SLC8A1 immunostaining of statin-treated SH-SY cells.
a) A C.figf6(a) A C. elegans embryo expressing mCherry::RAB-5 and GFP::LGG-1. The arrows point to two apoptotic phagosomes. See Supplementary Movie S11. Scale bar, 10 μm. (b) Cropped time-lapse images showing RAB-5 and LGG-1 recruitment to an apoptotic phagosome (from a, left arrow). (c) Representative images of apoptotic phagosomes in embryos from control-RNAi- or bec-1-RNAi-fed worms. The insets show higher magnifications of the areas outlined in the main panels. (d) Quantification of GFP::LGG-1- and mCherry::RAB-5-positive phagosomes in control- or bec-1-RNAi embryos determined by time-lapse imaging. Control, 21 embryos, 52 phagosomes; bec-1, 9 embryos, 25 phagosomes; ***P0.0001, chi-squared test. (e) Representative DIC images from control- and bec-1-RNAi embryos at different stages of development (minutes after first division). The yellow arrowheads mark apoptotic corpses. Scale bar, 10 μm. (f) Quantification of apoptotic corpses in control- and bec-1-RNAi embryos; data show ± s.d.; ***P0.0001. (g) Representative images of CED-1::GFP embryos with control or bec-1 RNAi. The yellow arrowheads mark engulfed corpses surrounded by CED-1::GFP. Scale bar, 5 μm.
TSLP secreted by mutantGFP epidermal cells induces hyper-proliferation of non-mutantTom keratinocytes A.Figure 6Experimental design for induction of mutantGFP KCs in vitro and neutralization of secreted TSLP by anti-TSLP. Primary keratinocytes were cultured from the skin of Junblox/lox; c-Junlox/lox; RosaKi/+; K5cre-ERT+/+ mice. 50% of mutantGFP KCs were induced by infection with Cre Adenovirus, the non-infected cells were non-mutantTom KCs. Adeno-empty was used as control. EdU was added for 5 hours to label proliferating tomato+ cells in 6-day co-culture. Quantification was performed by Confocal imaging analysis. TSLP production in conditioned media quantified by ELISA three days after infection with Ad-empty or Ad-cre. n=3 independent experiments. Percentage of EdU+ Tomato+ keratinocytes after blocking with anti-TSLP. Anti-TSLP neutralizes the hyper-proliferation of non-mutantTom KCs in vitro after infection with Ad-cre. Gene expression of Tslpr after TSLP neutralization is reduced. Gene expression of IL-7ra after TSLP neutralization is increased. Gene expression of VEGFα after TSLP neutralization is reduced. Experimental design for TSLP neutralization in vivo. TSLP neutralization was performed by triple intra-dermal injections of 20 μg of anti-TSLP or IgG to the right ears of DKO* and DKO*15 mice five days after of psoriasis-like induction. EdU was added into mice by IP injection 2 hour before euthanasia. Representative images of treated ears after 9 days of anti-TSLP or IgG treatment. Longitudinal analysis of ear thickness in DKO* mice at different time points during psoriasis-like disease progression Trans-epidermal water loss (TEWL) (J) in DKO* mice at different time points during psoriasis-like disease progression Percentage of EdU+ epidermal cells (CD45-CD49f+) after blocking with anti-TSLP in DKO* and DKO*15 mice Representative immune-fluorescence images of ear sections from control or DKO* mice treated with IgG or anti-TSLP) stained for Ki67. Ki67 expression was reduced in mice treated with anti-TSLP (n =3).
Figure 9Overall proposed model for CEP41-mediated angiogenesis via endothelial cilia. (1) Potential interactions of CEP41 with MAP and/or polyglutamylase and their transportation from the centrosome to the cilium, (2) tubulin glutamylation in endothelial cilia by fluid flow-driven shear stress or hypoxia, (3) activation of AURKA dependent on CEP41 under shear stress or hypoxia, (4) induction of cilia disassembly via AURKA activation, and (5) upregulation of pro-angiogenic regulators, such as VEGFA and VEGFR2.
Figure 5a) LEFT. E3 auto-ubiquitination assays were performed using various Ub mutants (I44A, R42A, Q49A, Q49E, and V70A) and the RBR E3s HHARIRBR (left) and ParkinRBR (right). Products were visualized by western blotting against HA-Ub. Samples were analyzed 30 minutes after ATP addition. b) The hydrophobic patch of Ub (PDB 1ubq) is colored yellow on a surface representation and positions of each mutation are noted. c) UbcH7~Ub conjugates were preformed with either with WT-Ub or I44A-Ub. After the addition of apyrase to quench the charging reaction, UbcH7~UbWT (left) or UbcH7~UbI44A (right) was incubated with ParkinRBR. The disappearance of each UbcH7~Ub species and appearance of auto-ubiquitinated E3 was visualized under non-reducing conditions by western blotting for HA-Ub. Time was recorded post addition of ParkinRBR. E2~Ub conjugated with I44A-Ub does not disappear over the time course of the reaction. d) UbcH7 conjugated with WT-Ub, I44A-Ub, or V70A Ub as indicated was incubated with either H887A-HOIPRBR-LDD (left) or H359A-HHARIRBR (right) mutants that allow trapping of the E3~Ub thioester with WT-Ub (Stieglitz et al, 2013;[29]). While a HOIP~Ub thioester species is observed when UbcH7 was charged with WT-Ub, no detectable transfer occurs with the I44A-Ub conjugate (left). Similarly, V70A-Ub shows reduced formation of the HHARI~Ub thioester (right). In addition to blotting for HA-Ub, the blot on the right was also blotted for GST-HHARI.
The cyclin E/Cdk2 kinase‐dependent phase of Mcm2‐7 loading requires the canonical pre‐RC assembly pathwaySchematic of the experiment.Figure 3A Schematic of the experiment. B FACS profiles of DNA content for each cell population assayed. C Western blot analysis for Orc2 and Mcm2‐7 in the nuclear chromatin fraction and Orc2, Mcm2‐7, Dup/Cdt1, and cyclin E in the cytoplasmic fraction for each condition assayed.
Pathogen infection induces DNA-damage and a DNA-damage-response through the mitochondrial apoptosis pathway HeLa cell derivatives were infected with viral, bacterial or protozoan pathogens.Figure 2A, The caspase-3-reporter cell line (labelled DEVD) and the control cell line (DEVG; see methods) were infected and analysed at various time points post-infection by flow cytometry (Chlamydia trachomatis (Ctr; MOI=1, 30 h); Salmonella Typhimurium (STy; MOI=50, 24h); Toxoplasma gondii (MOI=5, 16 h); MVA (MOI=10, 16h); Influenza A virus (IAV; MOI=1, 16h); Herpes simplex virus 1 (HSV-1; MOI=1, 16h). Staurosporine treated cells (1 μM, 3h) were used as a positive control. Percentages of cells positive for caspase-3-activation (FRET-loss) were determined. Data are means/SEM of three independent experiments. (n=3) B, C Cells were infected with modified vaccinia virus Ankara (MVA) (MOI=10). The caspase-inhibitor zVAD-fmk was used at 100µM; cells were analysed 16 h post-infection. D, E Cells were infected with influenza A virus (IAV) (MOI=1; analysis was 16 h post-infection). D, detection of viral M2 protein was used as infection control. F, G Cells were infected with Herpes simplex virus 1 (HSV-1) (MOI=1; analysis was 16 h post-infection). H Cells were infected with Chlamydia trachomatis (Ctr) (MOI=1; analysis was 30 h post-infection). I Cells were infected with Salmonella Typhimurium (Sty) (MOI=50; analysis was 24 h post-infection). J Cells were infected with the protozoan parasite Toxoplasma gondii (MOI=5; analysis was 16 h post-infection).
Figure 3mTORC2 in adipose tissue is not required for cold-induced lipid droplet mobilization. (A) Representative H&amp;amp;E staining of sWAT sections from AdRiKO and control mice (n=5/group). (B) Non-esterified fatty acids (NEFAs) in plasma of AdRiKO and control mice (n=6/group). (C) Glycerol in plasma of AdRiKO and control mice (n=6/group). (D) Representative H&amp;amp;E staining of BAT sections from AdRiKO and control mice (n=5/group). (E) Triglycerides (TGs) in BAT of AdRiKO and control mice housed at 22°C or 4°C for 8h (n=6/group). (F) NEFAs in BAT of AdRiKO and control mice (n=6/group). Data represent mean ± SEM. Statistically significant differences between AdRiKO and control mice were determined with unpaired Student's t-test and are indicated with asterisks (*=p<0.05; **=p<0.01, ***=p<0.001). Statistically significant differences between temperatures were determined with unpaired Student's t-test and are indicated with a number sign (#=p<0.05; ##=p<0.01; ###=p<0.001). The exact p-value for each significant difference can be found in Appendix Table S2.
Kinesin‐mediated intracellular trafficking functions together with autophagy to facilitate necrotic cell death.Figure 7(A) Kinesin 1 heavy chain (UNC‐116) deficiency. (B) Monomeric kinesin UNC‐104 deficiency. In both cases, no significant synthetic protection of neurons is observed in LGG‐1/LC3‐depleted animals with impaired autophagy. Error bars denote s.e.m. values (n>250 for all populations examined; P>0.5, compared with single mutant control animals, unpaired t‐test).