EMBO/sd-character-level-ner huggingface dataset:
Xeno-GVHD induction with MitoTpos-PBMCs is associated with improved survival and reduced tissue inflammation in a mouse model of disease.Figure 6A In vivo experimental design of human xenogenic model of GVHD in NSG immunodeficient mice, with MSC-mitocepted human PBMC transplant. B Representative FACS plots of mitoception (% of MitoTracker+) in CD4+ or CD8+T cells from mitocepted PBMC compared to control PBMC, before intravenous injection. C Percent of mouse weight after GVHD induction with IV injection of mitocepted PBMC or control PBMC cells (n=3, with a total of 25 mice per group). Control group in grey, were untreated mice (n=6 mice). * p<0.05 by Mann Whitney t-test. D Kaplan Meier plot showing percent survival of GVHD mice after transplantation of an equal amount (12x106 cells) of mitocepted PBMC or control PBMC cells (n=3, with a total of 25 mice per group) (p=0.0218 by log-rank test). E FACS analysis of human Treg (CD4+CD25+FoxP3+) cell engraftment in mouse spleen following transplantation of PBMC-MitoT or control PBMC cells (n=3, with a total of 25 and 27 mice per group, respectively). F FACS analysis of human pro-inflammatory T helper1 (CD4+IFNγ+, left panel) and T cytotoxic (CD8+IFNγ+, right panel) cell engraftment in mouse spleen following transplantation of mitocepted or control PBMC cells (n=3, with a total of 25 and 27 mice per group, respectively). G Representative histopathologic images of GVHD in liver and small intestine sections obtained from PBMC-MitoT, PBMC and non-transplanted control samples, stained with Masson's trichrome staining. Asterisk showed increased periportal collagen deposits in the liver and increased piknotic cells in the small intestine crypts. Arrowheads represents a reduced number of Paneth cells. H-I Semiquantitative scoring system for pathologic damage in the liver and small intestine of mitocepted PBMC (n=8 independent mice) or control PBMC transplanted mice (n=5 independent mice). J Quantification of Paneth cells per crypt, from at least 50 crypts per PBMC-MitoT (n=8 mice), PBMC transplanted (n=5 mice) or non-transplanted control (n=3) mouse. Representative histopathologic images of GVHD in lung sections obtained from PBMC-MitoT, PBMC and non-transplanted control, stained with Masson's trichrome staining. L Semiquantitative scoring system for pathologic damage in lung of mitocepted PBMC (n=8 independent mice) or control PBMC transplanted mice (n=5 independent mice). M FACS analysis of total human CD4+ cell engraftment in mouse lungs following transplantation of mitocepted PBMC or control PBMC cells (n=3, with a total of 25 and 27 mice per group, respectively).
Figure 4Sleep phenotype of OctβR mutants. Total sleep was shown in (A-C). For A, from left to right mean±SEM: 377.8±7.389, n=95; 479.3±7.039, n=134; 492.6±6.590, n=95; 582.8±8.937; n=134; For B, from left to right mean±SEM: 392.7±6.385, n=126; 484.2±8.537, n=121; 452.0±11.69, n=77; 516.4±10.58; n=55; 496.7±5.618, n=126; 575.6±8.820, n=121; 546.1±11.37, n=77; 657.9±3.756, n=55; For C, from left to right mean±SEM: 372.3±6.483, n=120; 560.8±6.881, n=123; 494.4±5.862, n=120; 544.8±7.190; n=123; The sleep amount after OA treatment in OctβR mutants comparing with w1118 control. (D)The sleep amount after OA treatment in Octβ1R mutants (From left to right mean±SEM: 916.0±13.51, n=74; 765.1±27.27, n=60; 1067±18.23, n=70; 1007±18.61; n=71). (E) The sleep amount after OA treatment in Octβ2R mutants (From left to right mean±SEM: 916.0±13.51, n=74; 765.1±27.27, n=60; 1066±19.12, n=70; 956.2±22.36; n=60; 987.7±27.66, n=32; 803.3±51.34, n=23). (F) The sleep amount after OA treatment in Octβ3R mutants (From left to right mean±SEM: 916.0±13.51, n=74; 765.1±27.27, n=60; 1124±18.18, n=72; 1047±27.28; n=58). Sleep phenotype of OctβR double and triple mutants. (From left to right mean±SEM: 392.0±12.91, n=60; 395.1±8.56, n=79; 433.7±13.03, n=58; 422.8±10.34; n=62; 474.4±8.36, n=73; 517.4±6.30, n=119; 513.2±7.10, n=79; 536.6±9.05 n=78; 499.1±11.95, n=60; 546.0±8.51, n=79; 519.6±10.31, n=58; 518.9±13.13; n=62; 584.3±8.59, n=73; 579.2±8.35, n=119; 560.9±6.80, n=79; 606.4±6.38, n=78). The sleep amount after OA treatment in OctβR double and triple mutants comparing with w1118 control. (From left to right mean±SEM: 932.6±15.23, n=38; 792.2±28.87, n=40; 978.6±19.51, n=48; 890.5±25.17; n=38; 1030±25.66, n=36; 931.6±38.73, n=32; 1019±18.33, n=35; 876.2±31.15, n=38; 1109±16.16, n=32; 1003±26.65, n=44; 1067±13.31, n=44; 963.6±19.95; n=57; 1110±16.15, n=36; 1055±13.47, n=24; 1136±14.21, n=64; 1117±16.77, n=46). OA treated OctβR mutants and control flies have different percentage of sleep recovery after sleep deprivation. (For I, from left to right mean±SEM: 13.31±2.760, n=51; 1.394±4.107, n=25; 30.35±3.473, n=44; 7.198±8.511; n=53. For J, from left to right mean±SEM: 13.31±2.760, n=51; 1.394±4.107, n=25; 21.37±2.244, n=54; 31.06±2.545; n=55. For K, from left to right mean±SEM: 13.31±2.760, n=51; 1.394±4.107, n=25; 23.48±3.409, n=70; 21.46±2.629; n=67). Sleep phenotype of Octβ2R mutants by the Video-Based Method. (From left to right mean±SEM: 630.5±27.96, n=52; 738.0±36.62, n=20; 867.6±42.99, n=24; 926.5±17.25, n=52).
3D architecture of membrane rearrangements induced 36 h after HCV infection.Figure 7(A) Left: slice of a dual axis tomogram taken from Huh7.5 cells 36 h after infection with 100 TCID50/cell of Jc1. Right: 3D reconstruction of the tomogram. Note the extensive membrane reorganization and the appearance of MMVs predominating at late time points after infection. Black arrows show invaginations of DMVs. The white star indicates a large MMV; due to its complexity only its middle part could be rendered. (B) Left: slices through the same tomogram highlighting a DMT enwrapping a DMV and presumably leading to the formation of a MMV. Right: 3D surface rendering of this structure. (C) Left: slices through the same tomogram highlighting a 'self-invagination' event of a DMV, also leading to the formation of a MMV. Right: 3D surface rendering of this late structure, revealing an opening of this MMV towards the cytosol as a result of the self-invagination. Panels B and C are part of the tomogram displayed in panel A and their positions are highlighted by yellow dashed squares in panel A. Scale bars represent 100 nm. For further details see legend to Figure 6. This tomogram is shown in movie S2.
TMEM59 induces LC3 activation.Figure 1(A) Clone P15 induces HA-LC3 lipidation. 293T cells were transfected with P15 plus plasmids expressing HA-LC3A and/or the apoptotic inhibitor p35 (as shown), and lysed for western blotting against the indicated molecules (anti‐HA for HA-LC3A). The figure shows that expression of P15 induces HA-LC3A conversion to a lower molecular weight form indicative of protein lipidation. This activity remains unchanged by p35. (B) P15 induces GFP-LC3 translocation to a vesiculated pattern. 293T cells were transfected with the indicated plasmids mixed with vectors expressing GFP-LC3A and p35. The known autophagic inducer bNIP3L constituted a positive control. Representative confocal microscopy pictures are shown. (C) TMEM59 induces HA-LC3 lipidation in different cell lines lacking the SV40 large T−antigen plasmid amplification system. Cells were transfected with the shown plasmids, vectors expressing HA-LC3A (left) or HA-LC3B (right) and GST (as transfection control), and lysed for western blotting against the indicated molecules. (D) TMEM59 induces GFP-LC3 activation. 293 cells were transfected with the indicated plasmids and GFP-LC3A (top) or GFP-LC3B (bottom). Representative confocal microscopy pictures are shown. (E) Quantification of the phenotype in D. The number of GFP-LC3‐positive vesicles per transfected cell was scored for at least fifty cells. Data are expressed as means ±s.d. of one representative experiment of three repetitions.Source data for this figure is available on the online supplementary information page.
Figure 2A) Pearson correlation matrix for the H3K27Ac genomic profiles in different PDAC cell lines. The matrix was hierarchically clustered. Source data were deposited in GEO. B) Histogram showing common and grade-specific genomic regions based on their histone acetylation. The number of genomic regions in each category is indicated in the pie-charts in panel C. Detailed computational and statistical procedures for the identification of different groups of genomic regions are provided in the methods section. C) Genomic distribution of common and grade-specific H3K27Ac peaks.D) Distance of grade-specific enhancers identified by H3K27Ac from genes expressed in PDAC cells of different grade (*p ≤ 1E-50; ** p ≤ 1E-100, two tailed Welch's t-test). Central values represent the median. E-F) Heatmaps showing grade specific, TSS-distal (E) or TSS-proximal (F) acetylated peaks and their relation to other histone marks. The box-plots indicate the signal intensity for each of the modifications (normalized values were transformed into a 0-1 range; central values represent the median). G) A representative snapshot of a genomic region with several enhancers selectively marked by H3K27Ac and H3K4me1 in Lo-G (CAPAN-1 and CFPAC-1) but not Hi-G (MiaPaca2 and PANC1) PDAC cell lines. The NFKBIZ gene in this region is selectively expressed in Lo-G PDACs.
CRY-independent rhythms are regulated post-transcriptionally(A) Mean number of PER2::LUC molecules per cell at the estimated peak of PER2 expression for each cell line (mean of three experiments, n=3 each).Figure 3(A) Mean number of PER2::LUC molecules per cell at the estimated peak of PER2 expression for each cell line (mean of three experiments, n=3 each). (B) PER2::LUC binding to BMAL1 in WT and CKO cells. Cells were harvested at the peak of PER2 expression, BMAL1 was immunoprecipitated, and PER2::LUC binding was measured by bioluminescence measurements (n=3, mean ±SD). (C) Per2 mRNA levels in WT (left) and CKO (right) cells were determined by qPCR over one circadian cycle (bottom), while PER2::LUC bioluminescence (top, min-max normalised) was recorded from parallel cultures. Per2 mRNA reported relative to Rns18s (bottom), n=3, ±SEM; PER2::LUC (top) presented as mean (solid) ±SEM (dashed), n=3. The WT mRNA trace was preferentially fit by a circadian damped sine wave compared with straight line (p=0.0412, extra sum-of-square F-test) whereas CKO data was not (ns). (D) Detrended Per2 and Nr1d1 promoter activity in WT, CKO and quadruple Cry1/2-Per1/2 knockout (CPKO) mouse embryonic fibroblasts (MEFs) recorded at 37°C, n=3, mean (solid) ±SEM (dashed). Nr1d1 data were preferentially fit with a circadian damped sine wave over straight line (p<0.0001, extra sum-of-squares F-test) (right hand graphs, solid lines; error bars, SEM).
An UnaG-based hypoxia sensor efficiently indicates hypoxic growth conditions in CHO cells.Figure 1a) Schematic representation of sensor constructs for the hypoxia-inducible expression of fluorescent proteins. Five HRE-sequence motifs derived from the enhancer region of the Vegfa gene confer hypoxia-specific activity upon the CMV minimal (mCMV) promoter. Fluorescent proteins are destabilized by in frame fusion of a PEST sequence from mouse ornithine carboxylase gene. The EGFP-encoding construct was obtained from Addgene (#46926). b - d) Comparative characterization of CHO cell bulk transfection cultures, selected for stable expression of the UnaG- or EGFP-encoding reporter constructs.b) Flow cytometry revealed efficient induction of green fluorescence, after treatment of both UnaG and EGFP-expressing CHO bulk cultures with CoCl2, while growth under hypoxia (1% oxygen) for 12 hours, selectively induced green fluorescence only in dUnaG expressing cells. Under normoxia (21% oxygen) only background fluorescence was observed.c) Assessment of the mean fluorescence intensity (MFI) to determine the activity of the 5xHRE-CMV promoter after 12 and 24hrs. Fluorescence intensity increased between 12 and 24hrs. d) Representative images of dUnaG- or dEGFP-expressing CHO cells grown either under normoxia (21% oxygen), CoCl2-treatment or hypoxia (1% oxygen) for 12 hours. Scale bars= 50µm e) Fluorescence intensity in dUnaG- or dEGFP-expressing CHO cell bulks grown under increasingly hypoxic conditions of 10%, 5% and 1% oxygen for 24 hrs indicated efficient induction of the dUnaG sensor at oxygen concentrations below 5%. f) Maximal fluorescence of the UnaG-based hypoxia reporter in CHO cells grown at 1% oxygen for 24 hrs hours was only observed at serum concentrations of 5% FCS or higher, reflecting the dependence of UnaG on its serum cofactor bilirubin.
Figure 2(A) A549 cells were infected with WT P. aeruginosa for various time points and cell lysates were evaluated by immunoblotting. (B) A549 cells infected with WT or ΔpscD P. aeruginosa for 4 hours or left uninfected (NI) in the presence of lysosomal inhibitor CQ (50 nM). The cell lysates were evaluated by immunoblotting. (C) A549 cells stably expressing GFP-LC3 were left non-infected or infected, for 4 hours, with WT or ΔpscD P. aeruginosa, and evaluated by fluorescence microscopy. Cell nuclei were counterstained with DAPI (blue) and autophagosome were visualized as green puncta. Graph represents quantitative analysis of GFP-LC3 positive autophagosome per cell, scale bars: 10 µm. Each bar represents mean value of three independent experiments, error bars represent standard deviation. Statistical significance between assays was determined using One-way ANOVA followed by Dunn's Multiple Comparison Test. **p ≤ 0.01. (D-E) A549 cells were treated for 16 hours with rapamycin 0.8 μg/ml or DMSO, then infected with either WT (D) or ΔpscD P. aeruginosa and followed by 1 hour of gentamycin treatment (E). The circles and squares represent the individual test (three technical replicates per individual test) with DMSO and rapamycin treatment. The bars represent the means of CFU from three independent experiments, error bars represent standard deviation. The significance of differences between different drug treatment was determined using two-tailed student's t test with Welch's correction. NS: not significant; *p ≤ 0.05; **p ≤ 0.01.
Membrane-tethered Smaducin-6 inhibits TLR4 signaling.Figure 3A Pre-treatment of Smaducin-6 reduces LPS-induced interleukin-6 (Il6) gene expression in a dose-dependent manner in RAW264.7 cells. Il6 gene expression was analyzed by qRT-PCR. B, C Pre-treatment of 100 nM Smaducin-6 or scrambled peptide (Pal-Scram #1) for 30 min inhibits (B) NF-κB-mediated luciferase gene expression when RAW264.7 cells are treated with LPS for 2 h. Luciferase activity in (B) was normalized to β-galactosidase activity. B, C Pre-treatment of 100 nM Smaducin-6 or scrambled peptide (Pal-Scram #1) for 30 min inhibits (C) IκBα degradation and IKKα/β phosphorylation when RAW264.7 cells are treated with LPS for 2 h. D Peli1 knockdown or wild-type humanTHP1 cells were pre-treated with 100 nM Pal-Scram peptide and Smaducin-6 for 30 min and subsequently treated with LPS for 2 h. Il6 and Peli1 gene expression were analyzed by qRT-PCR. Data show the mean ± SD of three independent experiments. E RAW264.7 cells were treated with 100 nM FITC-conjugated Smaducin-6 and TAT-S6(422-441), and localization was observed by confocal microscopy. Scale bar, 10 μm (magnification, 200×). Nuclei were stained with DAPI. F After pre-treating RAW264.7 cells with biotin-conjugated Smaducin-6 (100 nM) for 30 min, cells were treated with LPS for 2 h. Subsequent precipitation by streptavidin-agarose showed that endogenous Pellino-1 binds to the Smaducin-6 peptide. A biotin-conjugated scrambled peptide was used as a negative control. G-I Immunoprecipitation assays in primary peritoneal macrophages show that Smaducin-6 inhibits the formation of endogenous (G) IRAK1-, (H) RIP1-, or (I) IKKε-mediated signaling complexes induced by 2 h LPS treatment, compared to a scrambled peptide. IB, immunoblot; TCL, total cell lysates. Data shown are representative of three independent experiments.
a, Starvation (3 h)-induced autophagy results in a downregulation of H4K16ac in histone extracts of MEF cells.f1a, Starvation (3 h)-induced autophagy results in a downregulation of H4K16ac in histone extracts of MEF cells. b, Upon rapamycin treatment (300 nM) LC3 conversion and downregulation of H4K16ac are observed in WT MEF cells but not in the autophagy-deficient atg5−/− and atg7−/− MEF cells. c, Rapamycin treatment increased the LC3-II/LC3-I ratio and promoted H4K16ac decrease in sirt1−/− and WT MEF cells. d, Rapamycin-induced autophagy led to downregulation of H4K16ac at 48 h in histone extracts of HeLa and U20S cells, and after 6 h in U1810 cells. e, Quantification of H4K16 acetylation by immunoblotting is depicted for rapamycin-treated cells. Data are expressed as mean ± s.e.m. (n = 3-5); *P 0.05; **P 0.01.
hst-1 suppresses movement of endogenous miR395 and miR160, and alters xylem pole specification by mobile miR165/166(A) miR395 northern analysis in scions (S) and hyl1-2 rootstocks (R), in the indicated genotypes' combinations, under SO4-sufficient (+SO4) or SO4-starved (-SO4) conditions, in two biological replicates (rep.Figure 7miR395 northern analysis in scions (S) and hyl1-2 rootstocks (R), in the indicated genotypes' combinations, under SO4-sufficient (+SO4) or SO4-starved (-SO4) conditions, in two biological replicates (rep.). U6 was probed as an endogenous control. Relative U6-normalized band-intensity quantifications are indicated for each tissue in -SO4 conditions. Stem-loop RT-qPCR-based analysis of rootstock/scion ratio of miR395a levels in plants grafted with hyl1-2 rootstocks onto scions of indicated genotypes (top) and RT-qPCR analysis of APS4miR395 mRNA levels in the corresponding hyl1-2 rootstocks (bottom), under -SO4 conditions. Error bars: SD. t-test (top) and Welch's t-test (bottom) p-values are indicated. n=4. miR160 northern analysis in Meselect-separated leaf vasculature and epidermis in WT versus hst-1 plants, in three biological replicates (rep.). U6: as in (A). Relative U6-normalized band-intensity quantifications are indicated. Stem-loop RT-qPCR-based analysis of epidermis/vasculature ratio of miR160 levels (top) and RT-qPCR analysis of ARF17miR160 mRNA levels (bottom) in the samples in (C). Error bars: SD. t-test (top) and Tukey's multiple comparisons test (bottom) p-values are indicated. n=3. Basic fuchsin staining of protoxylem (unfilled arrowheads) and metaxylem (filled arrowheads) in WT, shr-2 or hst-1 roots. *: protoxylem gap. Scale bars: 10 µm. miR165/166 northern analysis in WT versus hst-1 roots. U6: as in (A). Average relative U6-normalized band-intensity quantifications are indicated. RT-qPCR analysis of pri-miR165a/-miR166a, SHR, SCR, PHBmiR165/166 and PHVmiR165/166 mRNA levels in WT versus hst-1 roots. Error bars: SD. t-test p-values are indicated. n=4.
Figure 4A, B, % IDR (A) and propensity for LLPS (B) in TSN2_NS and TSN2_HS interactomes versus corresponding GFP-TAPa controls (C_NS and C-HS) using IUPred and PSPredictor algorithms, respectively. Upper and lower quartiles, medians and extreme points are shown. The number of protein sequences included to the analyses was 566, 277, 995 and 149 for C_NS, TSN2_NS, C_HS and TSN2_HS, respectively. P values denote statistically significant differences for comparisons to controls (two-tailed t-test). C, Disorder profiles of TSN2 generated by PONDR-VLXT, PONDR-VL3, PONDR-VSL2, IUPred-short, IUPred-long and PONDR-FIT and a consensus disorder profile (based on mean values of six predictors). SN, staphylococcal nuclease region composed of four N-terminally situated SN domains. C-terminally situated Tudor region is composed of the domain of the same name and a partial SN domain. D, BiFC between cYFP-TSN2 (full-length), cYFP-SN or cYFP-Tudor and nYFP-TSN-interacting proteins in N. benthamiana protoplasts after HS (39ºC for 40 min). Scale bars = 10 μm. Boxplots show quantification of the reconstituted YFP signal. AU, arbitrary units. Upper and lower box boundaries represent the first and third quantiles, respectively, horizontal lines mark the median, and whiskers mark the highest and lowest values. Three independent experiments, each containing seven individual measurements, were performed. ***P < 0.001 vs Tudor (one-way ANOVA).
Atg16L1Δ(230–300) mutant is impaired in isolation membrane targeting and partially restores the autophagic defects of Atg16L1 KO MEFs.Figure 4(A) WT MEFs, Atg16L1 KO MEFs or Atg16L1 KO MEFs stably expressing either full‐length Atg16L1(1-588), Atg16L1(1-230) or Atg16L1Δ(230-300) were cultured in regular DMEM or starvation medium in the presence or absence of 100 nM BafA1 for 2 h. * indicates nonspecific immunoreactive bands. (B, C) Atg16L1 KO MEFs stably expressing GFP-ULK1 and either full‐length Atg16L1(1-588), Atg16L1(1-230) or Atg16L1Δ(230-300) were cultured in regular DMEM (C) or starvation medium (B,C) for 1 h. Cells were fixed and analysed by immunofluorescence microscopy using anti‐GFP, anti‐Atg16L1 and anti‐LC3 antibodies. Arrowheads indicate the perinuclear localization of Atg16L1(1-230). The number of dots was quantified from more than 30 randomly selected cells from three independent samples as described in the Methods. Data represent mean±s.e. (*P0.001, analysis of variance followed by Bonferroni/Dunn post hoc test). Scale bar, 10 μm, and 2 μm in inset. (D, E) Cells stably expressing GFP-LC3 were cultured in regular DMEM, starvation medium or starvation medium in the presence of 0.2 μM WM or 100 nM BafA1 for 6 h. Total cellular GFP-LC3 signals were analysed by flow cytometry. Representative FACS data were shown (D). The geometric mean of fluorescence intensity was determined. Values are expressed as a percentage of the mean of control cells cultured in regular DMEM. Data represent mean±s.e. (*P0.05) (E). BafA1, bafilomycin A1; GFP, green fluorescent protein; KO, knockout; MEFs, mouse embryonic fibroblasts; NS, not significant; WM, wortmannin; WT, wild‐type.
Figure 8(A) Representative AP trace of a TECRLHom-hiPSC-CM in the presence of NA alone or NA and flecainide. (B) Effect of flecainide on AP parameters of hiPSC-CMs. (C-D) Addition of 5 μM of flecainide decreased the susceptibility to triggered activity in hiPSC-CMs. Note that the effect of flecainide on triggered activity of TECRLHet- and TECRLHom-hiPSC-CMs is more pronounced than its effect on their spontaneous activity.
Figure 5A. Western blot (WB) analysis of specified proteins in small intestine of Pkd2wt/wt and Pkd2ki/ki male mice after one week of HFD. Quantification of the bands for each protein normalized to loading control and relative to Pkd2wt/wt. n=3. B. WB of ApoliproteinA4 in jejunum of Pkd2wt/wt and Pkd2ki/ki male mice after one week of HFD. Quantification of bands normalized to loading control and relative to Pkd2wt/wt. n=3. C. WB of ApoliproteinA4 in jejunum of Pkd2flox/flox and Pkd2gutΔ/Δ male mice after one week of HFD. Quantification of bands normalized to loading control and relative to Pkd2flox/flox n=7. D. WB analysis of specified apolipoproteins in 10µL of serum of overnight fasted, 2 hours refed male animals after 1 week in HFD. Quantification of bands relative to Pkd2wt/wt. n=4. E. WB of 20µL of basal medium of Caco2 cells expressing shscramble or shPkd2 in transwell system after 12 hours stimulation with oleic acid. n=6. F. Electron microscopy pictures of chylomicrons obtained by ultracentrifugation of plasma from Pkd2wt/wt and Pkd2ki/ki male mice as specified in methods. G. Quantification of the diameter the chylomicrons shown in panel F. n=3. H. WB with specified antibodies after an in-vitro kinase assay.