EMBO/sd-character-level-ner huggingface dataset:
Lamplight can be used for switchable and tuneable control Go activity in Hek293T cellsA) Hek293T cells expressing Lamplight-1D4 (anti-1D4 = green, DAPI = blue, Top) and schematic of BRET assay, measuring interaction of liberated Gβγ-Venus with GRK3 fragment-Nanoluciferase (Bottom).Figure 1A) Hek293T cells expressing Lamplight-1D4 (anti-1D4 = green, DAPI = blue, Top) and schematic of BRET assay, measuring interaction of liberated Gβγ-Venus with GRK3 fragment-Nanoluciferase (Bottom). Scale bar = 20µm.F) Schematic of Lamplight photoequilibrium. In dark, Lamplight is mostly in inactive (R) state. 405nm light shifts equilibrium (blue arrow) so opsin is mostly in active (M) state, while 525nm light shifts (green arrow) Lamplight into R state. Shifts caused by concurrent 525nm and 405nm partially cancel out leading to a small increase of opsin in M state. G) Response to 405nm alone or with 525nm light   H-I) Representative time courses of Lamplight-driven responses to 8s mixed stimuli (grey bar) with different 405nm: 525nm ratios from 1 (100% 405nm) to 0 (100% 525nm) from (H) dark or (I) after 8s 405nm light (blue bar) J) Sustained Lamplight-driven activity (mean BRET ratio 20-80s after stimulus) after exposure to mixed stimuli from dark (ON) or 405nm light-adapted state (OFF). Lamplight data fit with linear trendline. Best fitting parameters for ON, slope = 0.311, Y-intercept = 0.04, R2 = 0.85. For OFF, slope = 0.30, Y-intercept = 0.07, R2 = 0.69).
TFG depletion causes the accumulation of cargo‐laden COPII transport carriers throughout cellsHuman RPE‐1 cells stably expressing low levels of mannosidase II‐mApple were transfected with a TFG siRNA for 60 h.Figure 7A HumanRPE‐1 cells stably expressing low levels of mannosidase II‐mApple were transfected with a TFG siRNA for 60 h. Cells were subsequently treated with brefeldin A (BFA) for 1 h, followed by a wash into fresh media and further incubation for 2 h in the absence of BFA, prior to fixation. Images shown are projections of 3D data sets (4 μm in z). Merged images with Sec31A in green and ManII in red are shown (representative of at least 15 cells analyzed). Scale bar, 5 μm. B HumanRPE‐1 cells stably expressing low levels of mannosidase II‐mApple were transfected with a TFG siRNA for 60 h. Cells were subsequently treated with BFA for 1 h, followed by a wash into fresh media and further incubation for 1 h in the absence of BFA, prior to fixation and staining using α‐Sec16A and α‐Sec31A antibodies. Images shown are projections of 3D data sets (4 μm in z). Merged images with Sec31A (green), ManII (red), and Sec16A (blue) are shown (representative of at least 15 cells analyzed). Scale bar, 5 μm.C Higher magnification views of the indicated regions in (B, boxed) are shown. Arrows highlight COPII‐positive transport carriers that contain the cargo ManII, which are not juxtaposed to Sec16A‐labeled sites on the ER. Additionally, arrowheads point out distinct foci in which COPII continues to associate with Sec16A‐labeled sites, indicating that COPII vesicle formation continues in the absence of TFG. Scale bar, 1 μm.
expression in human breast cancer and patient survival(A) Representative IHC images of CXCL12 intensity and staining pattern in the TMA.Figure 8(A) Representative IHC images of CXCL12 intensity and staining pattern in the TMA. N = 219 tumors. Upper panel, scale bar, 400 μm; lower panel, scale bar, 100 μm. Arrows point at punctate staining in the stroma. (B, C) Histological score of CXCL12 staining in TNC+ and TNC- tumors in combination with CD8+ (above median) and CD8- (below the median). * p = 0.0206, ** p = 0,0040, Unpaired t-test. The central band represents the median, the ends of the box the first and the third quartiles, and the whiskers reach from each quartile to the minimum or maximum. (D) Kaplan Meier survival analysis of breast cancer patients upon stratification into tumors with a CXCL12 Hscore below or above the median, p = 0.002. Mann-Whitney test. (E) Survival analysis of breast cancer patients in the GSE 19783-GPL6580 cohort upon stratification into tumors with high or low expression of TNC and CXCL12. Log-rank test. HR and p values are indicated,
GBP1, its GTPase activity and prenylation are required for IFNγ-enhanced cell death upon Toxoplasma infection (A) IFNγ enhances macrophage host cell death after type I (RH) and type II (Pru) Toxoplasma gondii (Tg) infection.Figure 1IFNγ enhances macrophage host cell death after type I (RH) and type II (Pru) Toxoplasma gondii (Tg) infection. Graphs show cell death as measured by LDH release assay (left) and viability measured by XTT assay (right) in untreated or IFNγ-primed THP-1 cells infected with type I or type II Tg for 24 h. LDH release assays from THP-1 cells left untreated or primed with IFNγ, transfected with siRNA against indicated GBPs or non-targeting control (CTRL) and infected with indicated strains of Tg for 24 h. LDH release assay from primary monocyte-derived macrophages (MDM) left untreated or treated with IFNγ, transfected with siRNA against GBP1 or non-targeting control (CTRL) and infected with indicated strain of Tg for 24 hours. Mean ± SEM of n = 4 donors shown. LDH release assay from indicated wildtype, GBP1 knockout (∆GBP1) or GBP1 reconstituted (∆GBP1+Tet-GBP1) cells infected with type I or type II Tg for 24 hours. Cells were untreated or treated with IFNγ or additionally treated with doxycycline (Dox) as indicated. Realtime propidium iodide (PI) uptake assay from indicated THP-1 cells infected with type I or type II Tg, and untreated or treated with IFNγ and/or Dox as labelled. AU, arbitrary units. LDH release assay from wildtype THP-1 or ∆GBP1 cells stably reconstituted with Dox-inducible expression plasmids of the indicated mutants of GBP1. Cells were pre-treated with IFNγ and Dox and infected with either type I or type II Tg for 24 h.
Figure 3A. Depleting TRAPPC9, but not TRAPPC8, increased lipid droplet size in HEK293T cells. The efficiency of siRNA depletion of TRAPPC8 and TRAPPC9 in Hela cells is shown by immunoblotting in the top left panel. A. Depleting TRAPPC9, but not TRAPPC8, increased lipid droplet size in HEK293T cells. Representative fluorescence images show the status of lipid droplets in these siRNA treated cells after oleic acid incubation. Bar =10 μm. B. After loading with oleic acid, human skin fibroblasts containing a R475* mutation in TRAPPC9 accumulated lipid droplets of greater sizes than skin fibroblasts similarly isolated from a TRAPPC9 wildtype human individual. Bar = 10 μm.
Figure 5(A) Summary and representative immunoblots of total PKCα and p-PKCα in dPMNs that were pretreated without or with PKC inhibitor Go6976 for 1 h, and then treated without or with PMA (A) for 3 h. Panels A is summary analyses of immunoblots that were calculated based on the arbitrary unit (A) (B) Immunoblot (IB) detection of the phospho-lamin B and total lamin B with lamin B protein purified by immunoprecipitation (IP) with anti-lamin B from human dPMNs that were pretreated without or with PKC inhibitor Go6976 for 1 h, and then treated by PMA (B) for 3 h. (C) Confocal microscopy images of human dPMNs that were pretreated without or with PKCα Go6976 for 1 h, and then treated without or with PMA for 3 h, followed by staining of lamin B (primary anti-lamin B, and FITC-labeled secondary antibody), and phosphorylated PKCα (primary anti-human p-PKCαS657, and PE-labeled secondary antibody). The green/white arrows indicate the site of nuclear envelope rupture. Scale bars, 10 μm (C). (D) Schematic representation of lamin B domain structure and several mitotic serine phosphorylation sites (indicated by blue font) that exhibit PKC consensus motifs ([30]) and overview of lamin B reporters containing serine (S) to alanine (A) mutations at PKC consensus phosphorylation sites. (E) The endpoint analysis of % cells with NET formation was detected with dPMNs that were transfected with plasmids of either wildtype lamin B (WT control), or mutants with single or multiple defined point mutations at PKCα-consensus-phosphorylation sites (S395A, S405A, S408A) of lamin B, and treated for 3 h by 10 μM PAF in medium containing cell-permeable dye SYTO Red (500 nM) for the total cell count, while the cells with NET formation were stained by cell impermeable dye SYTOX Blue (5 μM). Then the images were taken with Olympus confocal microscopy, followed by automated quantification of NETs using ImageJ for quantification of % cells with NET formation. percentage of cells with NET formation by immunofluorescent imaging quantification using ImageJ (E)
Figure 5Cross and longitudinal sections of ablated (control) and implanted (graft) TA were marked with LacZ and immunoassayed for MHC (red), LAM (green). Cross- and longitudinal sections were immunostained against MHC and LAM and overlaid to LacZ labeled images. Dashed area indicates the enlarged view displaying sarcomeres (asterisks) in combination with LacZ positive nuclei (arrowheads).
Figure 3A Western blot analysis of various structural subunits of complex I in fibroblasts from Subjects 1 and 3 (S1 and S3 respectively) with three age-matched controls (C1, C2, and C3). B BN-PAGE analysis of OXPHOS complex assembly in enriched mitochondria from subject and control fibroblasts solubilised using DDM. Immunoblotting was performed using antibodies to structural subunits from each OXPHOS complex (CI [NDUFB8], CII [SDHA], CIII [UQCRC2], CIV [MT-CO1], and CV [ATP5A]). C Western blot analysis of whole cell lysates from controls (C1 and C2) and subject cell lines (S1 and S3) that were either untransduced (-) or transduced with the lentiviral vector (pLVX) containing wild-type NDUFC2 (+). Transduced cell lines were either uninduced or induced with 100 or 200 ng/ml doxycycline (for 48 and 96 hours respectively) where indicated. D BN-PAGE analysis of OXPHOS complex assembly in enriched mitochondria isolated from controls (C1 and C2) and subject cell lines that were either untransduced (-) or transduced with the lentiviral vector (pLVX) containing wild-type NDUFC2 (+). Transduced cell lines were either uninduced or induced with 200 ng/ml doxycycline for 72 hours.
Fig.Figure 4Immunoblotting of His-GID1 and His-SLR1 expressed in E. coli. Lipid-protein blotting assay of PA, PC, PE, PG, and PS with GID1 and SLR1. Lipids (0.5 μg) were spotted on nitrocellulose strips. PA, PC, PE, and PG were from egg yolk, and PS was from porcine brain. Purified proteins (GID1 and SLR1, 0.5 mg/ml) were used, followed by immunoblotting with anti-His-tag antibodies and color development. Liposomes were made from di18:1-PC only or di18:1-PA/PC (PA:PC = 1:3 mole ratio). 1× and 10× refer to the concentration of PC or PA/PC liposomes used. NL, no liposome was added to the binding mixture. Schematic diagram showing serial deletions of GID1. GID1 fragments were expressed in E. coli and used for defining the PA binding region. CD1/2 denote conserved domain HGG and GXSXG. Catalytic triad including three conserved amino acids of GID1, S, D, and H (vertical red bars). Immunoblotting of His-GID1 proteins using constructs shown in (D). Proteins were separated by SDS-PAGE, followed by immunoblotting with anti-His-tag antibodies. The PA binding activity of different truncation mutations were analyzed by fat-immunoblotting. The red arrowheads indicate truncated proteins with different molecular weights. Sequence alignment of the PA-binding fragment of GID1 with that of the PA binding motifs in chicken Raf1, abscisic acid insensitive 1 (ABI1), constitutive triple response1 (CTR1) and werewolf (WER) from Arabidopsis. Residues in bold are basic, potentially involved in PA binding and were mutated to Ala in GID1. Immunoblotting of His-GID1 mutants and lipid-immunoblotting of PA binding by GID1 mutant proteins on a filter. Liposomal binding of GID1 proteins to PA. Liposomes were made from di18:1-PA/PC (PA:PC = 1:3 mole ratio). Liposomal associated proteins were subjected to SDS-PAGE and immunoblotting using anti-His antibodies. The band intensity was analyzed by Image J and the intensity of input was set as 100%.
Structures or domain schematics are shown for Class 1 depolarization and PARKIN-dependent diGly sites (PDB codes, Supplementary Table 5).f4Structures or domain schematics are shown for Class 1 depolarization and PARKIN-dependent diGly sites (PDB codes, Supplementary Table 5). a, Legend for colour-coded circles that indicate the conservation of Lys at homologous positions in M. musculus, D. rerio and D. melanogaster (Supplementary Table 5). For structures, regulated sites are shown in red space-filled models. b, Cytoplasmic proteins. RPN10, red circle. c, MOM proteins with structures. d, MOM proteins without structures.
3D recordings of apoptotic mitochondria suggest the formation of a Bax-ring induced pore in the mitochondrial outer membrane.Figure 6Shown are seven optical sections through a mitochondrion of an apoptotic U2OS cell. Top and middle row: Tom22 and Bax (recorded in STED) channel, respectively. Bottom row: overlay (Tom22: red; Bax: green). Note the absence of Tom22 signal in the area enclosed by the Bax-ring. Displayed are raw data. Scale bar: 0.5 µm.
Lon deficiency stimulates PINK1 activity in vivo.Figure 5A) The eye severity score of flies co-overexpressing PINK1 with Control-R (n = 74), Lon-R1 (n = 163), or Lon-R2 (n = 90) using the ey-GAL4 driver. (B) Comparison of the frequency of thoracic indentations in flies expressing the PINK1-RNAi construct (PINK1-R) in an otherwise wild-type background to the frequency in animals that also bear heterozygous mutations in Lon: PINK1-RNAi +/+ (n = 306), PINK1-RNAi LonEP/+ (n = 74), and PINK1-RNAi LonDf/+ (n = 83). LonEP refers to the LonG3998 P-element allele of Lon, and LonDf refers to the Df(3L) Exel9011 deletion that removes the Lon gene along with several other genes. The Dmef2-GAL4 driver was used to drive expression of the PINK1-R construct in experiments described in panel B. Error bars represent s.e.m. *p<0.05, **p<0.005 by Student t test.
Figure 6A) Immunofluorescence-based antibody feeding assay performed using HUAECs seeded on laminin 411, 511 or 111-coated coverslips, reveals more VE-cadherin at junctions and less in vesicles in cells plated on laminin 511 (arrow heads mark VE-cadherin positive vesicles). Scale bar is 10 µm. B) Quantification of VE-cadherin positive vesicles/ cell in HUAECs plated on laminin 411, 511 or 111. Values are means ± s.e.m from 300 cells from 3 independent experiments, unpaired t-test. C) Western blot of biotinylated (surface) VE-cadherin in HUAECs plated on different laminins versus total VE-cadherin (input) confirms more junctionally located VE-cadherin in cells bound to laminin 511. D) Quantification of Western blots expressed as relative signal proportions (surface VE-cadherin/ total VE-cadherin). Values shown are means ± s.e.m of 6 experiments, unpaired t-test. E) Western blot of p120 catenincoimmunoprecipitated with VE-cadherin (input) from HUAECs plated on different laminins Asterisks are isotope controls. Values shown are means ± s.e.m of 4 experiments, Mann-Whitney test. E) Western blot of p120 catenin coimmunoprecipitated with VE-cadherin (input) from HUAECs plated on different laminins, and (F) corresponding quantification of the p120 signal intensity relative to the total VE-cadherin signal. Asterisks are isotope controls. Values shown are means ± s.e.m of 4 experiments, Mann-Whitney test.
Fig.Figure 5ApoE-/- (-/-) and ApoEYF (YF) mice were subjected to partial ligation of the left carotid artery (LCA) and given a cholesterol-rich diet. The right, non-ligated artery (RCA) was used as a control. Proximity ligation assays showing S-nitrosated PKM2 (yellow) in the RCA and LCA 2 days after ligation. Phalloidin staining (blue) was included to highlight the vessel wall; n=4-5 mice per group (2-way ANOVA and Bonferroni). Scale bars: 50 µm. ApoE-/- (-/-) and ApoEYF (YF) mice were subjected to partial ligation of the left carotid artery (LCA) and given a cholesterol-rich diet. The right, non-ligated artery (RCA) was used as a control. GSH (green) levels in the RCA and LCA 2 days after ligation. CD31 (red) was included to label endothelial cells. n=4-5 mice per group (2-way ANOVA and Bonferroni). Scale bars: 50 µm. ApoE-/- (-/-) and ApoEYF (YF) mice were subjected to partial ligation of the left carotid artery (LCA) and given a cholesterol-rich diet. The right, non-ligated artery (RCA) was used as a control. 3-nitrotyrosine (3NT, green) levels in the RCA and LCA 2 days after ligation. CD31 (red) was included to label endothelial cells. n=5 mice per group (2-way ANOVA and Bonferroni). Scale bars: 50 µm. ApoE-/- (-/-) and ApoEYF (YF) mice were subjected to partial ligation of the left carotid artery (LCA) and given a cholesterol-rich diet. The right, non-ligated artery (RCA) was used as a control. Acetylcholine (ACh)-induced relaxation of the phenylephrine contracted LCA 7 days after ligation, n=7-8 mice per group (2-way ANOVA and Bonferroni). Acetylcholine (ACh)-induced relaxation of the phenylephrine contracted aorta 7 days after ligation, n=7-8 mice per group (2-way ANOVA and Bonferroni).
AGO4 and AGO6 mediation of canonical and non‐canonical RdDM pathwaysVenn diagram showing the overlapping patterns among DNA hypomethylation loci identified in ago4‐6 ago6‐2, nrpd1‐3, and nrpe1‐11.Figure 3A Venn diagram showing the overlapping patterns among DNA hypomethylation loci identified in ago4‐6 ago6‐2, nrpd1‐3, and nrpe1‐11. B DNA methylation levels in different mutants. The 2,174 loci where AGO4 and AGO6 are mutually dependent are numerically clustered (y‐axis). Cytosines were examined as CG, CHG, and CHH. C DNA methylation levels at TAS1a, TAS1c, and TAS3a loci. Snapshots from whole‐genome bisulfite sequencing results are shown. D RT-qPCR measurements of transposon RNA levels. Double: ago4‐6 ago6‐2 double mutant. Actin 2 was used as an internal control. RNA levels in the mutants are relative to those in the wild‐type (Col‐0). Means ± SD are shown, n = 3.
Previously unknown nucleolar proteins.Figure 2A. KRI1 localized to the nucleoli in MCF-7 cells (HPA043574). Data information: Protein of interest is shown in green, nuclear marker DAPI in blue and the reference marker of microtubules in red. Scale bar 10 µm. B. GPATCH4 localized to the nucleoli in U-2 OS cells (HPA054319). Data information: Protein of interest is shown in green, nuclear marker DAPI in blue and the reference marker of microtubules in red. Scale bar 10 µm. C. FOXL2NB localized to the fibrillar center in SiHa cells (HPA061017). Data information: Protein of interest is shown in green, nuclear marker DAPI in blue and the reference marker of microtubules in red. Scale bar 10 µm. D. FAM71E1 localized to the fibrillar center in U-2 OS cells (HPA048111). Data information: Protein of interest is shown in green, nuclear marker DAPI in blue and the reference marker of microtubules in red. Scale bar 10 µm. E. LRRC23 localized to the nucleoli in U-2 OS cells (HPA057533). Data information: Protein of interest is shown in green, nuclear marker DAPI in blue and the reference marker of microtubules in red. Scale bar 10 µm. F. ZNF853 localized to the nucleoli in U-2 OS cells (HPA067690). Data information: Protein of interest is shown in green, nuclear marker DAPI in blue and the reference marker of microtubules in red. Scale bar 10 µm. G. METTL5 localized to the nucleoli in U-2 OS cells (HPA038223). Data information: Protein of interest is shown in green, nuclear marker DAPI in blue and the reference marker of microtubules in red. Scale bar 10 µm.
Figure 1A NPF6 clade from M. truncatula, O. sativa, and A. thaliana, including two Z. mays NPFs. All NPFs from M. truncatula, O. sativa, and A. thaliana were used for tree construction and only NFP6s are presented. Sequences were aligned using ClustalOmega, the phylogenetic tree was constructed using PhyML. B The AtNPF6.3 sub-clade in rosids can be grouped into three haplotypes with residues corresponding to AtNPF6.3 T101, H356, T360 and F511, designated as Types A, B and C. All legumes have Types A and B, while a subset also have Type C, which is found only in legumes of the Hologalegena clade. All NPF6s from M. truncatula, and A. thaliana were used for tree construction and only the AtNPF6.3 sub-clade is presented. Rosid orders represented: Cucurbitales: Cucumis sativa; Rosales: Prunus persica, Trema orientalis, Parasponia andersonii; Fagales: Morella rubra; Malpighiales: Populus alba; Fabales: Trifolium pratense, Lotus japonicus, Phaseolus vulgaris, and Glycine max. Sequences were aligned using MUSCLE, the phylogenetic tree was constructed using PhyML. Branches with maximum likelihood support >0.80 are indicated by filled circles.
Ex vivo stimulation of human breast tissue microstructures with different PR agonists.Figure 2A Experimental scheme showing the preparation of human breast tissue microstructures from reduction mammoplasty specimens, the ex vivo stimulation with R5020 and progesterone and epithelial purification by FACS-sorting based on EpCAM expression. B PCA plot showing global gene expression profiles of EpCAM+ cells isolated from ethanol-, P4- and R5020-treated human breast tissue microstructures, n=3. C Venn diagram showing differentially expressed genes (adjusted P-value < 0.05) in response to either P4 or R5020 relative to CTRL samples. D Boxplot showing log-scaled variant stabilized gene expression values for RANKL and WNT4 in human tissue microstructures, n=3. Shown are the non-corrected P-values estimated by DESEQ2. ** P < 0.01. Vertical lines outside the box end at maximum and minimum values, upper and lower borders of the box represent lower and upper quartiles, and line inside the box identifies the median. E Scatter plot showing a linear relationship between the log2FC of shared differentially expressed genes between R5020 and P4 relative to CTRL samples shown in (B). Coefficients, P-value on the estimated coefficient and adjusted R squares were calculated by linear regression model (lm function in R). The dashed red line shows the diagonal. F GSEA of differentially expressed genes between P4 (red) and R5020 (yellow) relative to CTRL samples. The red dashed lines indicate NESs. G Scatter plot showing the genes which are commonly altered upon R5020 stimulation (adj. P-value < 0.05) in both mouse and human organoids relative to their CTRL samples. Color scale as a function of the averaged human and mouse logFC values. The red dashed lines represent an adj. P-value threshold of 0.001. H Bar graph showing GSEA of genes altered in both mouse and human EpCAM+ cells upon R5020 stimulation of tissue microstructures. Bars define the averaged NES between ex vivo R5020-treated human and mouse tissue, ticks indicate averaged P-values given by GSEA.