EMBO/sd-character-level-ner huggingface dataset:
Figure 6A - HeLa cells were infected or not (NI) with C. trachomatis (MOI = 1), then lysed 24 or 48 hpi. After separation with SDS-PAGE, proteins were transferred to a membrane, probed with anti-O-GlcNAcylation antibody followed with HRP-conjugated secondary antibodies. After extensive washes the membrane was blotted again with anti-GFPT and anti-actin antibodies before revelation with HRP-conjugated secondary antibodies. B - HeLa cells were infected or not with C. trachomatis (MOI = 1). Twenty-four hours later, 8 µM ionomycin (or DMSO alone) and 0.5 mM BP were added. After 6 h of treatment, cells were lysed and proteins revealed as in (A). C - HeLa cells treated for 72 h with siRNA targeting GFPT1 or GFPT2 were lysed. After separation with SDS-PAGE, proteins were transferred to a membrane, probed with anti-GFPT and anti-actin antibody before revelation with HRP-conjugated secondary antibodies. D - HeLa cells were transfected with control siRNA or siRNAs against GFPT or TG2. Two days later the cells were infected for the indicated times (MOI=0.3) before fixation, rupture of the cells and measurement of bacterial diameter. The mean diameter ± SD and p-values of Student's paired t-test on 4 independent experiments are shown. E - HeLa cells treated for 48 h with siRNA targeting GFPT1 or not (siCTRL) were infected with C. trachomatis (MOI = 0.15). Thirty hours later cells were fixed and analyzed by flow cytometry. The percentage of infected cells (top) and the mean fluorescence of the infected population (middle) ) ± SD are shown for at least four independent experiments. Duplicate wells were lysed and used to re-infect fresh HeLa cells to determine the bacterial titer (bottom). P-values of Student's ratio-paired t-test are indicated. F - Schematic view of the outcome of TG2 activation in infection. The increase in TG2 expression and activity in cells infected with C. trachomatis results in the up-regulation of the expression of glucose transporters. Increasing quantities of glucose are thus imported in the host cytoplasm and redirected to the vacuole, where they fuel bacterial growth. Parallel to this transcriptional outcome, the transamidating activity of TG2 targets the host enzyme GFPT, thereby boosting the hexosamine biosynthesis pathway. The bacteria consume the resulting UDP-GlcNAc, or an intermediate along this pathway, in particular to sustain bacterial division.
Figure 4(A) Locomotory behavior of a representative rd1 and ReaChR-treated mouse during illumination with light at 590 nm in an open field arena (irradiance measured at the level of the mice's eyes was ~1015 photons cm-2 s-1, cumulative activity trajectories are shown as red traces after 2 min of illumination), see also EV Movie 1 and EV Movie 2. (B) Mean velocities of the ReaChR-treated and rd1mice analyzed over a duration of 2 min at light onset. The mean velocity of ReaChR-treated mice were significantly reduced than rd1mice (P = 0.006, one-tailed t-test) upon illumination. Mean ± SEM are shown for each group. ReaChR-treated rd1 (n = 11), rd1 (n = 12). (C) Light/dark box test. Percentage of time mice spent in the light compartment as a function of light intensity (WT, n = 9; rd1, n = 9; ReaChR-treated n = 7). Mean ± SEM are shown for each group. (D) Percentage of time rd1 (n = 9), WT (n = 9) and ReaChR-treated (n = 7) mice spent in the light compartment for an illumination intensity of 2.11 × 1015 photons cm-2 s-1. Both WT as well as ReaChR-treated mice spent significantly less time in the light compartment than the rd1mice (P < 0.0001, ordinary one-way ANOVA). *** and **** indicates significance levels determined by Tukey's multiple comparisons test for rd1 vs. WT and rd1 vs. ReaChR-treated. See also EV Movie 3 for the response of a representative ReaChR-treated rd1 mouse plotted in (D).
Figure 5(A) Survival of WT mice pre-treated with vehicle, LicoB (20 mg/kg), LicoB (40 mg/kg), MCC950 (40 mg/kg), or LicoB (40 mg/kg) + MCC950 (40 mg/kg), followed by i.p. injection with LPS (20 mg/kg); mice survival was monitored for 72 h (n=10 mice per group). Mice were pre-treated with LicoB or MCC950 for 1 h, then i.p. injected with LPS (20 mg/kg) and treated for 3 h. The levels of IL-1β in the serum and peritoneal lavage fluid were measured using ELISA. Mice were pre-treated with LicoB or MCC950 for 1 h, then i.p. injected with LPS (20 mg/kg) and treated for 3 h. The levels of IL-1β and TNF-α (D, E) in the serum and peritoneal lavage fluid were measured using ELISA. Mice were pre-treated with LicoB or MCC950 for 1 h, then i.p. injected with LPS (20 mg/kg) and treated for 3 h. Quantification of peritoneal exudate cells (PECs) (F) and macrophages (F4/80+ cells) (G) using flow cytometry (n=8 mice per group). C57BL/6 mice received vehicle or LicoB (40 mg/kg) for 17 consecutive doses, once every two days for 34 days. Following that treatment, the serum levels of ALT (H) (n=8 mice per group). C57BL/6 mice received vehicle or LicoB (40 mg/kg) for 17 consecutive doses, once every two days for 34 days. Following that treatment, the serum levels of AST (I), and CRE (J) were measured, (n=8 mice per group). C57BL/6 mice received vehicle or LicoB (40 mg/kg) for 17 consecutive doses, once every two days for 34 days. Following that treatment, the serum levels were measured, along with the body weight (K) (n=8 mice per group).
Figure 2A) Strategies to establish HP1 conditional triple-KO mESC lines named HP1-cTKO. After the disruption of HP1β/γ by CRISPR/Cas, an expression construct of loxP-flanked Myc-hHP1β was introduced into an mESC line, TT2. Next, we disrupted the HP1α allele by CRISPR/Cas and subsequently introduced an expression construct for mER and Cre fusion protein (MerCreMer). When HP1-cTKO cells are cultured in the presence of 4OHT, Myc-hHP1β transgene can be removed by Cre-loxP recombination, thereby generating mESCs that lack HP1 completely. B) Protein expression levels of H3K9 MTs and histone modification levels in HP1-cTKO cells were monitored by immunoblotting. The intensities were quantified using ImageJ software and summarized on the right. The thin gray line and bold colored line show individual data and means of the experimental replicates (n ≥ 3, biological replicates), respectively. C) Comparison of H3K9me3 levels between HP1-deficient cells and Suv39h/Setdb1-deficient cells. H3K9me levels were quantified using ImageJ software and are summarized at the bottom; the horizontal line represents the mean intensity of each cell. More than 75 nuclei were counted for each staining. **p < 0.01 (Tukey's honestly significant difference test). D) Electron microscopy images of HP1-cTKO cells in the absence of 4OHT (left) and of cells treated with 4OHT for 4 days (right). Boxes represent enlarged images.
2019-nCoV S binds human ACE2 with high affinity.Figure 3.SPR sensorgram showing the binding kinetics for human ACE2 and immobilized 2019-nCoV S. Data are shown as black lines and the best fit of the data to a 1:1 binding model is shown in red. Negative-stain EM 2D class averages of 2019-nCoV S bound by ACE2. Averages have been rotated so that ACE2 is positioned above the 2019-nCoV S protein with respect to the viral membrane. A cartoon depicting the ACE2-bound 2019-nCoV S protein is shown (right) with ACE2 in blue and S protein monomers colored tan, pink and green.
Figure 1(A) O-GalNAc type O-glycosylation pathway. Biosynthesis of Core 1 type structures is shown. (B) Strategy for generation and characterization of GALNT isoform knock outs in HaCaT keratinocytes. (C) Expression of GALNT isoforms in primary keratinocytes and HaCaT cell line. The scatter plot depicts individual RPKM values of 2 biological replicates. (D) Expression of GalNAc-T1, -T2, and -T3 in human skin (upper panel) and HaCaT keratinocyte organoids (lower panel). Frozen human skin or HaCaT keratinocyte organotypic skin models were stained using antibodies for the GalNAc-T isoforms. Scale bar - 25 μm. (E) Phenotypic characterization of organotypic models made with HaCaT WT or GALNT1/2/3 KO keratinocytes. IHC of tissue sections stained for differentiation marker keratin 10 (upper panel) or proliferation marker Ki67 (lower panel). Scale bar - 50 μm. Red arrows - flattened cells; red asterisks - K10-negative region in suprabasal/granular layers; purple asterisks - pyknotic nuclei; green asterisks - increase in Ki67 positive cells. (F) Quantification of epidermal thickness of skin organotypic models. Epidermal thickness was measured in 5 distinct images (4 positions/image) of 4 clones of GALNT isoform KO or WT (4 different tissues) and is presented as averages +SD. Due to high ZFN KO phenotypic inter-clonal variation and to exclude off target effects, 5 clones of ZFN and 3 clones of CRISPR KO (each targeted by a different gRNA) were used for GALNT3 KO. ANOVA followed by Dunnet's multiple comparison test was used to compare mean epidermal thicknesses of different KOs to WT. * p ≤ 0.05; **** p ≤ 0.0001. (G) Quantification of keratin 10-negative area in skin organotypic tissue sections. Keratin 10-negative area was measured in 5 distinct images of 4 clones of GALNT isoform KO or WT (4 different tissues) and is presented as averages +SD. Due to high ZFN KO phenotypic inter-clonal variation and to exclude off target effects, 5 clones of ZFN and 3 clones of CRISPR KO (each targeted by a different gRNA) were used for GALNT3 KO. ANOVA followed by Dunnet's multiple comparison test was used to compare mean areas of different KOs to WT. **** p ≤ 0.0001. (H) RNA Seq expression data of select differentiation markers, expressed in spinous and granular layers of the epidermis (cartoon). Expression data is presented as log2(average KO/average WT). Significantly changed transcripts, identified in R analysis, are marked with asterisks.
Figure 3A-C Confocal images of primary hepatocytes after ribopuromycylation experiment combined with mRNA in situ hybridization for (A) Hadha, (B) Pcx and (C) Hmgcs2. Scale bar, 4 µm. D-F Fluorescence profile of 100-pixel line from the merged channel shown in (A-C). The cells from which the enlarged areas (400 μm2) have been magnified are shown in Appendix Figure S2 for each individual channel. Cells analyzed were isolated from 6 different mice with similar results.
Figure 3(A) Morphological phenotypes of WT, summ3-17, mekk1-1 and summ3-17 mekk1-1 plants grown on soil for three weeks. (B-C) Expression levels of PR1 (B) and PR2 (C) in the indicated genotypes as determined by quantitative RT-PCR. Values were normalized to the expression levels of ACTIN1. The data are shown as mean ± SD (n=3) with one-way ANOVA analysis and Tukey test (P < 0.01). Different letters indicate significant differences. The experiments were repeated three times with similar results. Two-week-old seedlings grown on half MS plates were used for assays in (B)-(C). (D) Morphological phenotypes of WT, mpk4-3, summ3-1 mpk4-3 and summ3-2 mpk4-3 plants. Photos were taken on three-week-old soil grown plants. (E-F) Expression levels of PR1 (E) and PR2 (F) in the indicated genotypes as determined by quantitative RT-PCR. Values were normalized to the expression levels of ACTIN1. The data are shown as mean ± SD (n=3) with one-way ANOVA analysis and Tukey test (P < 0.01). Different letters indicate significant differences. The experiments were repeated three times with similar results. Two-week-old seedlings grown on half MS plates were used for assays in (E)-(F).
Figure 8RNase J1 is depicted as a dimer. RNase J1, after the nascent transcript has been endonucleolytically cleaved, degrades RNA in the 5'-to-3' direction. Upon encounter with RNAP, conformational changes are induced, likely resulting in dissociation of the complex, and liberating RNAP from DNA thereby removing obstacles potentially causing transcription-replication clashes.
Defective autophagy underlies major phenotypic signatures of CCM disease.Figure 3Cd44, PAI1 (also known as Serpine1), and Id1 mRNA expression levels in KRIT1 wt and KRIT1-KO endothelial cells were assessed by quantitative real-time PCR. Where indicated, KRIT1 wt and KRIT1-KO endothelial cells were treated with 100 nM Torin1 or 500 nM rapamycin for 16h. The data are expressed as the mean ± s.e.m. Cd44: *P = 0.02848 (KO ctrl vs. KO Rapa); *P = 0.02605 (KO ctrl vs. KO Tor1). PAI1: *P = 0.04446 (KO ctrl vs. KO Rapa); *P = 0.03996 (KO ctrl vs. KO Tor1). Id1: *P = 0.00266 (KO ctrl vs. KO Rapa); *P = 0.01554 (KO ctrl vs. KO Tor1). n = 3 independent experiments. Immunoblot analysis of CD31/Pecam-1, vascular endothelial cadherin (VE-cadherin), and actin in KRIT1-KO endothelial cells that were treated with 100 nM Torin1 or 500 nM rapamycin for 24 h. The results are representative of three independent experiments. Immunoblot analysis of CD31/Pecam-1, vascular endothelial cadherin (VE-cadherin), N-cadherin, alpha-smooth muscle actin (alpha-SMA), and actin in HUVECs transfected with control siRNA or ATG7 siRNA. Formation of capillary-like structures measured by tube formation assays. HUVECs were transfected with control siRNA or ATG7 siRNA for 72 h. Representative phase-contrast microscopy (Scale bar, 100 μm) and calcein-fluorescent (Scale bar, 50 μm) images were reported. All data are presented as percentage ± s.e.m from three different experiments performed in duplicate. *P = 1.29e−11. Immunoblot analysis of p62, LC3I/II, and rapamycin in CCM3 wt and CCM3-KO rapamycin treated with 100 nM Torin1 or 500 nM rapamycin for 4 h. The results are representative of three independent experiments. Representative immunostaining of retina sections from wt and a model of inducible and endothelial-specific CCM3-KO (CCM3-ECKO) mice at postnatal day 14. Endothelium was stained with isolectin B4 (ISOB4) (blue). A, artery; V, vein. p62 aggregates can be observed in endothelial cells forming retinal lesions in CCM3-ECKO animals (scale bar: 200 μm). Scale bar of magnifications: 100 μm. Representative immunostaining of brain sections from wt and a model of inducible and endothelial-specific CCM3-knockout mice (CCM3-ECKO) at postnatal day 9. p62 aggregates can be observed in the proximity of CCM lesions (arrows). Cell nuclei (DAPI) are in blue. Scale bar, 30 μm. Smaller panel shows the magnifications of blood vessels (green). Scale bar, 10 μm.
Figure 7(A) Primary root length of the different genotypes was measured using the ImageJ program 3 days after 5 mM KNO3 or KCl treatments. Tukey box plot show results from 3 independent biological replicates per experimental condition (n = 8-10 roots each replicate). The box plot shows the data within the interquartile range (25th and 75th percentiles) and a solid black line represents the median. Whiskers show maximum and minimum values no further than 1.5x IQR (interquartile range). Outlier data are plotted individually. Asterisk indicate statistically significant difference between means analyzed by unpaired, two-tailed and assuming equal variance t-test (*p < 0.05, **p < 0.01). (B) Number of initiating and emerging lateral roots for all genotypes were counted using light microscopy 3 days after 5 mM KNO3 or KCl treatments. Tukey box plot show results from 3 independent biological replicates per experimental condition (n = 8-10 roots each replicate). The box plot shows the data within the interquartile range (25th and 75th percentiles) and a solid black line represents the median. Whiskers show maximum and minimum values no further than 1.5x IQR (interquartile range). Outlier data are plotted individually. Asterisk indicate statistically significant difference between means analyzed by unpaired, two-tailed and assuming equal variance t-test (*p < 0.05, **p < 0.01).
Figure 1Cortical malformations observed in six novel BBSOAS patients Patient (P) 1 shows abnormally elongated occipital convolutions (arrowheads in A), whereas abnormal gyrification of supramarginal and angular gyri is present in five out of six individuals (P2-P6 B', C' and C'' highlight the abnormally convoluted regions boxed in B and C. Cortical malformations observed in six novel BBSOAS patients Patient (P) 1 shows abnormal gyrification of supramarginal and angular gyri is present in five out of six individuals (P2-P6; (G) Schematic representation of the human NR2F1 protein sequence, showing novel variants of a new BBSOAS patient cohort. Key aminoacids for the functioning of the DNA-binding domain (DBD) or the ligand-binding domain (LBD) are listed in boxes. (H,H') TBR2 (green in H), SOX2 (red in H) and NR2F1 (yellow in H') immunofluorescence (IF) of a GW11 section of human neocortex Data information: Nuclei (blue) were stained with DAPI. Scale bars: 50µm. CP: cortical plate; IZ: intermediate zone; oSVZ: outer subventricular zone; SVZ: subventricular zone; VZ: ventricular zone. (I) NR2F1 expression (quantified by pixel intensity) at different L-M levels and in different regions along the A-P extent of the cortex, as indicated. n ≥ 3 sections from n = 1 fetal brain. Data information the number of positive cells was quantified in 100µm-width boxes, randomly placed across the cortex. In graphs, data are represented as means ± SEM. 2-way ANOVA (I) (*P<0.05, ***P<0.001). (J-L') NR2F1 (red) IF of human GW14 neocortex, showing expression levels in the CP (K,L) and VZ (K',L') around primary convolutions in the posterior-most cortex High NR2F1 is detected in the neurogenic area of a gyrus (L'). Data information: Nuclei (blue) were stained with DAPI. In Scale bars: 50µm. CP: cortical plate; IZ: intermediate zone; oSVZ: outer subventricular zone; SVZ: subventricular zone; VZ: ventricular zone. (M,M') NR2F1 level as measured on single cells in the VZ (M) and CP (M') of sulci and gyri n ≥ 4 sections from n = 1 fetal brain. (N-O) NR2F1 (red) and TBR2 (green) immunostaining NR2F1 level as measured in single TBR2+ IPs by pixel intensity is shown in graph (O). n ≥ 4 convolutions from n = 1 fetal brain (n = 2 sections). Data information In graphs, data are represented as means ± SEM. Student t-test (M,M',O (*P<0.05, ***P<0.001). (P-R) NR2F1 (red) and HOPX (green) immunostaining Virtually all HOPX+ bRGs are also NR2F1+ (magnification in P'') and their number is greatly increased in gyri (P'') compared to sulci (P'), as quantified in (R). At a single cell level, NR2F1 pixel intensity is higher in oSVZ HOPX+ cells in gyri compared to sulci (Q). n ≥ 4 sections from n = 1 fetal brain. Data information: Nuclei (blue) were stained with DAPI. the number of positive cells was quantified in 100µm-width boxes, randomly placed across the cortex. In graphs, data are represented as means ± SEM. Student t-test Q,R) (*P<0.05, ***P<0.001). oSVZ: outer subventricular zone
AMPK regulated longitudinal microtubule dynamics through the phosphorylation of CLIP-170 in cardiomyocytes.Figure 3(A) GFP time-lapse images of neonatal rat cardiomyocytes expressing EGFP-CLIP-170 WT 0 min and 15 min after treatment with 20 μM Compound C (left side panel) and expressing EGFP-CLIP-170 S311A (right side panel). Higher magniﬁcation of white square (upper row) showing CLIP-170 migrated longitudinally toward the cell-cell junctions. (B) Beeswarm plots of comet length of EGFP-CLIP-170. The comet length from multiple cells in different fields was analyzed. Number of comets analyzed, Pre: n=32, Cpd. C: n=36, CLIP S311A: n=68. Data means ±S.D. Differences among multiple groups were compared by one-way ANOVA, followed by a post hoc comparison using the Tukey method. **, P<0.01 versus Pre. (C) GFP time-lapse images of neonatal rat cardiomyocytes expressing EGFP-CLIP-170 WT treated with control siRNA (siCL, left) or siRNA targeting both AMPKα1 and α2 (siAMPKα1α2, right). White dotted lines in the images showed the connected cardiomyocyte not expressing EGFP-CLIP-170 WT. (D) Beeswarm plots of comet length of EGFP-CLIP-170. The comet length from multiple cells from different fields was analyzed. Number of comets analyzed, siCL: n=178, siAMPKα1α2: n=184. Data means ±S.D. Two-tailed Student's t-test was used to analyze differences between two groups. **, P<0.01 versus siCL.
Figure 4(A) SCOT-deficient murine cardiac endothelial cells were generated using the CRISPR/Cas9 technology by targeting the SCOT-encoding gene Oxct1. A non-targeting control construct was used to generate the non-targeting control cells (nt ctrl). Immunoblot of SCOT and β-actin in non-targeting control cells (nt ctrl) and Oxct1 knockout cells (Oxct1 ko). (B, Angiogenic capacity of Oxct1 knockout cells in response to ketone body treatment was analyzed using a spheroid-based sprouting assay. Spheroids were treated with media containing 30 mM D-β-hydroxybutyrate (βOHB), 30 mM acetoacetate (AcAc) or the respective controls (final concentration of reagents is diluted to approximately 10 %) for 48 hours. The average number of sprouts per spheroid was quantified. Data are presented as mean ± SD. n=4; *, p<0.05; **, p<0.01; unpaired Student's t-test. C) Angiogenic capacity of Oxct1 knockout cells in response to ketone body treatment was analyzed using a spheroid-based sprouting assay. Spheroids were treated with media containing 30 mM D-β-hydroxybutyrate (βOHB), 30 mM acetoacetate (AcAc) or the respective controls (final concentration of reagents is diluted to approximately 10 %) for 48 hours. the accumulated sprout length was quantified. Data are presented as mean ± SD. n=4; *, p<0.05; **, p<0.01; unpaired Student's t-test.
Dissection of genetic interactions between AGO4 and AGO6 based on comparative methylome analysesPie chart showing proportions of different groups of hypomethylation loci in the ago4‐6 ago6‐2 double mutant.Figure 2A Pie chart showing proportions of different groups of hypomethylation loci in the ago4‐6 ago6‐2 double mutant. Group I are loci where DNA hypomethylation is observed in the double mutant but not in the single mutants, indicating that DNA methylation at these loci is redundantly regulated by AGO4 and AGO6; Group II are loci where DNA hypomethylation occurs in one of the two single mutants but not in the other, and DNA methylation is not further decreased in the double mutant, indicating that DNA methylation at these loci requires AGO4 or AGO6 specifically; Group III are loci where DNA methylation is similarly reduced in the two single mutants, and the double mutant shows no additive effects on DNA hypomethylation, indicating that AGO4 and AGO6 are each required to mediate RdDM at these loci; and Group IV are loci where DNA hypomethylation is observed in the single mutants while the double mutant shows more severe DNA hypomethylation, indicating more complex genetic interactions between the two AGO proteins. B Box plots showing methylation patterns of different groups of loci as categorized in (A). Gene regions targeted by AGO4 and AGO6 are enriched in Group III loci. Y‐axis shows the numbers of DNA hypomethylation loci in ago4‐6 ago6‐2. TEs: transposons; Gene: genic regions; IGR: intergenic regions that do not overlap with TEs.