EMBO/sd-character-level-ner huggingface dataset:
(A) Boyden chamber experiment to measure attraction and retention of CD8 T cells towards factors in the lower chamber.Figure 4(A) Boyden chamber experiment to measure attraction and retention of CD8 T cells towards factors in the lower chamber. Cells in the lower chamber were measured by flow cytometry (attraction) or on the matrix coatings (retention) by counting Dapi stained nuclei 5 hours after plating. Attraction and retention were determined towards coatings with collagen I (COL), fibronectin (FN) or TNC in the presence of CXCL10, CCL21, CXCL12 (B, C) 3 independent experiments in duplicates, Mann-Whitney test, ns > 0.05; * p <0.05; **p < 0.01; ***p < 0.005. Mean ± SEM. retention were determined towards coatings with collagen I (COL), fibronectin (FN) or TNC in the presence of CXCL12 and, upon AMD3100 (AMD, 5 µg/mL) (D) CXCL12 (indicated concentrations) was added overnight at 4°C during the coating followed by washing (2 times with PBS) and plating CD8 T cells for 5 hours before fixation and counting Dapi stained nuclei. 3 independent experiments in duplicates, Mann-Whitney test, ns > 0.05; * p <0.05; **p < 0.01; ***p < 0.005. Mean ± SEM. retention were determined towards coatings with collagen I (COL), fibronectin (FN) or TNC in the presence of CXCL10 and, upon AMD3100 (AMD, 5 µg/mL) 3 independent experiments in duplicates, Mann-Whitney test, ns > 0.05; * p <0.05; **p < 0.01; ***p < 0.005. Mean ± SEM. Attraction were determined towards coatings with collagen I (COL), fibronectin (FN) or TNC in the presence of CM from shC cells (CM:shC) and, upon AMD3100 (AMD, 5 µg/mL) 3 independent experiments in duplicates, Mann-Whitney test, ns > 0.05; * p <0.05; **p < 0.01; ***p < 0.005. Mean ± SEM. retention were determined towards coatings with collagen I (COL), fibronectin (FN) or TNC in the presence of CM from shC cells (CM:shC) and, upon AMD3100 (AMD, 5 µg/mL) 3 independent experiments in duplicates, Mann-Whitney test, ns > 0.05; * p <0.05; **p < 0.01; ***p < 0.005. Mean ± SEM.
ORP5 and ORP8 targeting to ER-mitochondria contact sites and interaction with the mitochondrial protein PTPIP51.Figure 2(A) Confocal images of HeLa cells transfected with EGFP-ORP5, EGFP-ORP8, or GFP-Sec61β together with PTPIP51-HA and immunostained using HA antibody to detect PTPIP51. Scale bar, 10 μm. (B) Electron microscopy of ultrathin cryosections of HeLa cells co-transfected with EGFP-ORP5 and PTPIP51-HA and immunogold stained with anti-GFP (15 nm gold) and anti-HA (10 nm gold) showing EGFP-ORP5 localization at ER-mitochondria contacts (arrows). Arrowheads indicate the highly expanded ER membrane juxtaposed to mitochondria. Scale bar 200 nm. (C) Quantification of the IEM labelling for EGFP-ORP5, EGFP-ORP8 and GFP-Sec61β in HeLa cells co-transfected with PTPIP51-HA. Results are presented as the percentage of the total number of gold particles (800 per condition, n=30-35 cells) for ORP5, ORP8 and Sec61β in the indicated compartments (ER, reticular ER; ER-mito MCS, ER-mitochondria membrane contact sites; ER-PM MCS, ER-PM membrane contact sites). % gold particles ±SEM. Data are representative of three independent replicates. **P<0,001. (D) Confocal images of HeLa cells transfected with EGFP-ORP5 or EGFP-ORP8 together with PTPIP51-HA and RFP-Sec61β and immunostained using HA antibody to detect PTPIP51. Scale bar, 10 μm. (E) Representative EM images of ER-mitochondria contacts in the indicated overexpression conditions. (F) Quantification of the percentage of ER-mitochondria contact sites (ER-mito contact) per mitochondria of HeLa cells transfected with HRP-KDEL (Ctrl) or co-transfected with PTPIP51-HA and EGFP or PTPIP51-HA and EGFP-ORP5 or PTPIP51-HA and EGFP-ORP8 (n=30 cells and 1082-1223 mitochondria). % ER-mitochondria contact sites ±SEM. Data are representative of three independent replicates. **P<0,001, *P<0,05. (G) EGFP, EGFP-ORP5 or EGFP-ORP8 were immuno-precipitated from lysates of HeLa cells co-expressing PTPIP51-HA and treated with control (siCtrl) or VAPA and VAPB (SiVAPA/B) siRNAs and analyzed by western blot using antibodies against GFP (ORPs), HA (PTPIP51), VAPA, VAPB and tubulin.
a) Structure of the GBA1 gene on chromosome 1 and schematic overview of gene correction using ZFNs.f1(a) Structure of the GBA1 gene on chromosome 1 and schematic overview of gene correction using ZFNs. Black boxes represent exons. (b) Mutated and gene-corrected (GC) iPSCs after sequencing (upper lanes: iPSCs with heterozygous mutations; lower lanes: iPSCs with homozygous wt-GBA1 sequences as indicated by arrows).
Overall genome organization is preserved upon RNase digestion A) Experimental scheme of RNase A digestion of K562 cells before and after formaldehyde (FA) crosslinking (termed as bXL and aXL, respectively) followed by the Hi-C assay.Figure 1A) Experimental scheme of RNase A digestion of K562 cells before and after formaldehyde (FA) crosslinking (termed as bXL and aXL, respectively) followed by the Hi-C assay. B) Agarose gel electrophoresis of RNA extracted from RNase treated or control K562 cells. C) Experimental scheme of ActD treatment of K562 cells followed by Hi-C analysis. D) Agarose gel electrophoresis of RNA extracted from control, 4hr and 24hr ActD treated K562 cells. E) Barplots showing qRT-PCR gene expression levels (mean +/- S.D.) of PTEN, FNDC3B, and STAM genes in control, 4hr and 24hr actinomycin D treated K562 cells. p-value: two-tailed t-test, (*** p < 0.05). The results represent data from 3 technical replicates from 3 independent biological preparations.
Figure 2(A-F) Oral and intraperitoneal glucose tolerance testing in Abca12tm1d mice at 6 (A,B), 8 (C, D) and 12 weeks (E,F) (mean±SEM, n=4-9 mice per group, * p=<0.05, **p<0.01 Students t-test). (G) Insulin tolerance in 12 week old mice (mean±SEM, n=4-9 mice per group). (H) Whole blood Hb1Ac levels in 16 week old Abca12tm1d mice (mean±SEM, n=4-9 mice per group, **p<0.01 Students t-test). (I) Acute phase insulin secretion after intraperitoneal injection of glucose (mean±SEM, n=4-9 animals per group, white/grey/black = Abca12+/+, cre/+ and Abca12tm1d respectively, mice at 8 weeks of age, **p<0.01 Students t-test). (J) Silencing of ABCA12 in MIN6 cells as assessed by Western blot of ABCA12 immuno-precipitate (top panel) or confocal microscopy in cells transfected with Dy547-siRNAABCA12 (lower panel, scale bar = 10μm). (K) Glucose stimulated insulin secretion (GSIS) from MIN6 cells after silencing of ABCA12 (mean±SEM, n=4 replicates from assays in quadruplicate, ***p<0.0001, Students t-test). (L) Cellular insulin content in MIN6 cells after silencing of ABCA12 (mean±SEM, n=4 replicates from assays in quadruplicate).
Defects in NOD2 sensing of PGN promote gut bacterial translocation to adipose tissue and diet-induced adipose inflammation and dysfunction.Figure 2A Transcript levels of NOD2 in various tissues of chow-fed (n = 5, all tissues in both genotypes) and 16 week HFD-fed (n = 6) WT and NOD2−/− mice, *P = 0.005, **P = 0.001, and ***P = 0.0004. B H&amp;amp;E histology and IHC of the macrophage marker F4/80 in adipose tissue from HFD-fed WT and NOD2−/− mice (representative of n = 5 WT and n = 6 NOD2−/− mice). Scale bar equals 50 μm. C, D Quantification of immune cell and inflammatory markers (C) and pro-inflammatory cytokines (D) in adipose tissue of chow-fed (n = 5 in both genotypes) or HFD-fed (n = 11 in both genotypes) WT and NOD2−/− mice, *P = 0.04, **P = 0.02, ##P = 0.004, and #P = 0.04. E Quantification of serum non-esterified fatty acids (NEFA) in fasted and clamped HFD-fed WT (n = 4) and NOD2−/− (n = 3) mice, *P = 0.0001. F Quantification of insulin-stimulated pAktSer473 in gonadal adipose tissue at the end of the clamp in HFD-fed WT (n = 5) and NOD2−/− mice (n = 5), *P = 0.04. G The number of ampicillin-resistant E. coli cfu per gram of visceral adipose tissue was determined in WT (n = 5) and NOD2−/− (n = 5) mice after oral administration of 109 cfu of WT or mutant (ΔamiA) DsRed-labeled E. coli, *P = 0.02. H Quantification of GFP-positive E. coli in the adipose tissue stromal vascular fraction (SVF) from WT mice with no oral bacterial administration (CON,n = 3) or after administration of 109 cfu WT GFP-positive E. coli (n = 4) or ΔamiAGFP-positive E. coli (n = 4), *P = 0.03.
Figure 2Representative images (A) and cfu quantification (B) of Shigella WT or ΔipaB mutant strain bound to HeLa cells pre-treated or not with arsenite. Ruffle formation induced by Shigella WT in panel (A) is indicated by white arrowheads Cfu quantification of Shigella WT or ΔipaB mutant strain bound to HeLa cells pre-treated with anisomycin or DMSO (control) Representative images (D) and cfu quantification (E) of Shigella WT or ΔipaB mutant bound to HCT-8 cells pre-treated or not with arsenite
Figure 3(A, B) Co-IP analysis of the interaction between NRF2 and KEAP1 in absence and presence of TRIM16. (C) Co-IP analysis of the interaction between NRF2 and p62 in absence and presence of TRIM16. (D) Co-IP analysis of the interaction between KEAP1 and p62 in absence and presence of TRIM16. (E) Graphical representation of data presented
Figure 2A) GC cells (SGC7901 and MGC803) were transfected with (A) shUHMK1-#1,-#2, -#3, or control shRNA lentiviral vector, or (B) with a shRNA-resistant expression construct, UHMK1Δ. Western blotting was used to measure the levels of UHMK1. NIH ImageJ software was used to quantify the band intensity. Western blotting assay were conducted for three replicates. SGC7901 cells were transfected with or without shUHMK1-#1 or the shRNA-resistant expression construct UHMK1Δ. CCK-8 assays were conducted. Scale bars=5mm. Data information: **P< 0.01, ***P< 0.001, # marked no significance. SGC7901 cells were transfected with or without shUHMK1-#1 or the shRNA-resistant expression construct UHMK1 colony formation assays were conducted. Scale bars=5mm. Data information: **P< 0.01, ***P< 0.001, # marked no significance. (D and E) The volumes of subcutaneous gastric tumors derived from SGC7901 cells in NOD/SCID mice were determined at different time points. Tumors and representative bioluminescence images are also shown. UHMK1 silencing in the mouse model was confirmed by western blotting (three biological replicates). Data were presented as mean ± standard deviation from shcontrol and shUHMK1#1 mice (n = 10 mice/group). Unpaired, two-tailed statistical significance was assessed by Student's t‐test. Scale bars=1cm.. Data information: **P< 0.01, ***P< 0.001, # marked no significance. (F, G) UHMK1 silencing (F) and overexpression (G) affected GC cell abdominal metastasis. The quantitative number of metastatic nodules (left panel) in nude mice (n = 10 mice/group). Representative images of abdominal metastases are also shown (right panel). Scale bars=1cm. Data information: **P< 0.01, ***P< 0.001, # marked no significance. (H) Heatmap of the top 500 genes upregulated in GC patients with high UHMK1 expression. (I) Gene ontology enrichment analysis was used to analyze the top 20 enriched biological processes among GC patients with high UHMK1 expression.
Figure 3(A) Levels of Nlrp3 expression (relative quantitation, RQ) in Ctrl (Npm1F/FVav1Cre-) and cKO (Npm1F/FVav1Cre+) mice (4 months old, n = 4 mice per group). (B) Quantification of IL-1β, Il-6, IFNγ, and IL-27 inflammatory cytokines in serum from Ctrl and cKO mice (4 months old, n = 8-10 mice per group). (C) Representative flor plot (top) and quantification (bottom) of myeloid fractions (CD11b+Gr1+) and myeloid fraction subpopulation related to malignancy (CD11b+Gr1mid) in the bone marrow of Ctrl and cKO mice (n= 5-8 mice per group). (D) Representative flow plot (top) and quantification (bottom) of myeloid progenitors: common myeloid progenitor (CMP), Lin-Sca1-c-Kit+CD16/32lowCD34+; granulocyte-macrophage progenitor (GMP), Lin-Sca1-c-kit+CD16/32highCD34+; megakaryocyte-erythroid progenitor (MEP), Lin-Sca-1-c-Kit+CD16/32low/negCD34low/neg in the bone marrow of Ctrl and cKO mice (n = 4 mice per group). (E) In vitro colony-forming capacity of progenitor cells. Sorted KSL cells were cultured in semisolid medium. Counting and classification of colonies were performed in independent littermate pairs (n = 3 mice per group). GEMM, Colony-forming unit-granulocyte, erythroid, macrophage, and megakaryocyte; GM, Colony-forming unit-granulocyte and macrophage; M, Colony-forming unit-macrophage; E, Burst-forming unit-erythroid. (F) Representative peripheral blood (PB) smears. Insets show the differentiated granulocytes (a, b) in control mice, and anisocytosis (c), dysplastic myeloid cells (d-g) in Npm1 cKO mice (Npm1F/FVav1Cre+). Npm1 cKO mice show dysplastic erythroid cells (polychromatophilic, blue arrowhead; schistocyte, blue-outlined arrowhead; tear drop, Howell-Jolly bodies, black arrowhead; black-outlined arrowhead), dysplastic monocytes (black arrow), and dysplastic neutrophils (d-f). Scale bars, 10 μm. (G) Representative pictures (right) and weights (left, n = 6-8 mice per group) of spleens of 4-month-old Ctrl and Npm1 cKO mice. (H) c-Kit positivity in myeloid fraction in the spleen (n = 5 mice per group).
Figure 5A. Protein levels in cells grown on glucose and galactose. Cell lysates were analyzed by quantitative mass spectrometry (n = 4 biological replicates Levels of antioxidative enzymes (especially peroxiredoxins, PRDX in blue) are not changed except for cytosolic thioredoxin reductase (TXNRD1). B. Protein levels in cells grown on glucose and galactose. Cell lysates were analyzed by immunoblot (n = 2 biological replicates). Cytosolic thioredoxin reductase (TXNRD1) is increased by more than two-fold in cells grown on galactose compared to glucose-grown cells.The dotted line depicts 1, meaning no change in protein levels. C. Mechanisms of H2O2 degradation by peroxiredoxins. Auranofin inhibits thioredoxin reductase and thus the reductive half reaction in the detoxification of H2O2. Arrows indicate the "flux" of electrons. D,E. Response of HyPer7 (D) and roGFP2-scPrx1 (E) probes targeted to indicated compartments to treatment of mtDAO-expressing HEK293 cells with 2 mM D-Ala (cells generated with the Flp-In T-REX-system). HEK293 cells were incubated either with 1 µM auranofin (red curve and data points) or DMSO (black curve and data points). Solid line represents average, points colored in the lighter version of the respective color are the corresponding ratios measured in individual cells. As most of the data were not normal distributed, instead of a t-test, a Wilcoxon/Mann-Whitney-U-test was performed and samples were compared in pairs.***, p ≤ 0.001
Figure 3A. SIM of exponential B. subtilis cells. RNase J1 was fused to GFP (green), RNAP to mCherry (red). The graph shows relative fluorescence intensities at the cell midsection (along the long axis); SIM of the cell is below. Yellow indicates colocalization of the two proteins. B. Pull-down with RNase J1-8xHis tag and RNAP-10xHis tag and detection of the proteins with antibodies. Lanes 1, 2 - RNase J1-8xHis was used to pull down RNAP; lanes 3, 4 - RNAP-10xHis was used to pull down RNase J1; lanes 5, 6 - purified proteins were used as markers; lanes 7, 8 - strains without His-tagged proteins were used as negative controls to demonstrate the specificity of the interaction. M, molecular size marker; EX, exponential phase; STA, stationary phase. C. Pull-down with RNase J1-8xHis tag from stationary phase cells The samples then either were (lane 9) or were not (lane 10) treated with RNase A to detect whether the interaction was via RNA. Lanes 11, 12 - purified proteins were used as markers. The experiment was performed three times (biological replicates) with the same result.
Interferon signaling in L.Figure 6A Western blot analysis of STAT1 tyrosine phosphorylation. Bone marrow derived macrophages (BMDMs) of wild type (WT), Stat1Y701F (YF) and Stat1-/- (S1) mice were infected with L. monocytogenes (LO28, MOI 10) for 5 or 6 h. Whole cell extracts were collected and tested in western blot for levels of total STAT1 and phosphorylation of STAT1 on tyrosine (Y701). The blots are representative of more than 3 independent experiments. B Impact of Stat1Y701F mutation, or of deletion of ISGF3 subunits on the expression of the IFNβ gene. BMDMs of wild type (WT), Stat1Y701F, Stat1-/-, Stat2-/- and IRF9-/- mice were infected with L. monocytogenes (LO28, MOI 10) for 4, 8, 12, 24 or 48 h. Levels of Ifnβ gene expression were determined by Q-PCR. Bars represent mean values of three independent experiments. Error bars represent standard error of mean (SEM).
Microtubule length and numbers determine sporozoite curvature and infectivity A.Figure 7Quantitative analysis of salivary gland sporozoite lengths from the different lines. n indicates numbers of investigated sporozoites. Box plots represent 50% (boxes) and 95% (bars) of data with horizontal bar showing the median. *** and **** indicate p<0.001, and p<0.0001, respectively; ns: not significant; Kruskal-Wallis-test; scale bar: 1 µm. Quantification of SiR-tubulin fluorescence from 69-127 salivary gland derived sporozoites. Note the decrease in the fluorescence intensity of sporozoites expressing α1-tubulin without introns as well as the decreased intensity and length of the chimeras. Linear correlation of sporozoite length and microtubule length reveals a R2=0.94. *, **, and **** indicate p<0.05, p<0.01, and p<0.0001, respectively; ns: not significant; Kruskal-Wallis-test. Shorter sporozoites exhibit stronger curvatures and hence tighter circular motility on glass. Scale bar: 20 µm. Box plots represent 50% (boxes) and 95% (bars) of data with horizontal bar showing the median. * and **** indicate p<0.05, and p<0.0001, respectively; ns: not significant; Kruskal-Wallis-test. Sporozoites with fewest microtubules (α2+++) show a motility defect. n indicates numbers of investigated sporozoites; Numbers on grey bars indicate percentage of partially and persistently moving sporozoites. Sporozoites from lines with fewer microtubules move more frequently in apparent clockwise (CW) direction. Numbers on black bars indicate percentage of CW moving sporozoites; numbers in movie stills indicate time in seconds. CW moving sporozoites move at lower speed than CCW moving ones. Salivary gland sporozoites contain at least 10 microtubules as revealed by TEM of α2++ infected salivary glands. Green arrowheads point to microtubules. Scale bar: 0.2 µm. Error bars show the full range and boxes 50% of values. Red crosses indicate the mean, red lines the median. Fisher's exact test (two-tailed). Infectivity of sporozoites to mice decreases with decreasing numbers of microtubules and shorter microtubules. The prepatency is prolonged in parasites with a reduced sporozoite length and gliding circle diameter. NMRI mice were exposed to ten mosquito bites of the respective parasite line. Numbers indicate infected mice / mice exposed to mosquito bites (left) and infected mice / mice injected with 10.000 sporozoites (right). Cartoon illustrating the various consequences of different microtubule numbers and lengths during Plasmodium sporozoite formation and tissue invasion. Sporozoites with more than 10 microtubules can enter salivary glands while salivary glands sporozoites with shorter microtubules are least infectious.
Myosin VI depletion leads to p53 activation and cell cycle arrest.Figure 3(A) Growth curve of hTERT-RPE1 cells transfected with myosin VI siRNA. A representative plot of three independent experiments is shown. (B) Growth curve of BJ-hTERT cells transfected with myosin VI siRNA. A representative plot of two independent experiments is shown. (C) Analysis of DNA content in hTERT-RPE1 cells stably expressing a myosin VI shRNA. After 10 days of doxycycline induction, the cells were stained with Propidium Iodide (PI) and analysed by FACS. Ctrl = control cells, not induced. (D) Senescence-associated β-gal assay (SA-β-gal) of cells treated as in C. Scale bar, 100 μm. (E) IB analysis of hTERT-RPE1 cells transfected with myosin VI siRNA, with anti-myosin VI, anti-p53 and anti-p21 antibodies. Anti-GAPDH was used as loading control. (F) Growth curve of hTERT-RPE1 cells transfected with the indicated p53 and myosin VI siRNAs. A representative plot of three independent experiments is shown. (G) Representative bright field images of cells treated with the indicated p53 and myosin VI siRNAs. Scale bar, 200 μm. (H) IB analysis with anti-myosin VI, anti-p53 and anti-p21 antibodies of hTERT-RPE cells treated with Nutlin-3, or not treated as control. Anti-GAPDH was used as loading control. (I) Quantification of OFD1 intensity at the mother or daughter centrioles. hTERT-RPE1 cells treated or not with Nutlin-3, were incubated with nocodazole for 1 hour (6 μg/ml) and immunostained with anti-OFD1, anti-centrin1 and anti-Cep164 antibodies. Mother centrioles were identified by the coincident staining of centrin1 and Cep164, while daughter centrioles were centrin1-only stained. Results are expressed as fold change with respect to mock average intensity. Bars represent mean ±SD. Mother centrioles: Mock, n=148 cells; Nutlin-3, n=169 cells, from four independent experiments. Daughter centrioles: Mock, n=101 cells; Nutlin-3, n=122 cells, from three independent experiments. ns, not significant by Mann-Whitney test. (J) Quantification of Cep164 intensity at the mother centrioles in hTERT-RPE1 cells treated as in (I). Results are expressed as fold change with respect to mock average intensity. Bars represent mean ±SD. Mock, n=195 cells; Nutlin-3, n=196 cells, from four independent experiments. ns, not significant by Mann-Whitney test.
Figure 6A. Schematic illustration of 2-DG uptake assay protocol for control and NICDiOE-EC mice. B. Blood glucose levels of control and NICDiOE-EC mice during the 2-DG uptake assay. n=5, data represent mean ± SEM, unpaired t-test. C. Plasma insulin levels of control and NICDiOE-EC mice during the 2-DG uptake assay. n=5, data represent mean ± SEM, unpaired t-test. D. 2-DG uptake levels in skeletal muscle of control and NICDiOE-EC mice. n=5, data represent mean ± SEM, unpaired t-test. E. 2-DG uptake levels in visceral white adipose tissue (vWAT) of control (n=4) and NICDiOE-EC (n=5) mice. Data represent mean ± SEM, unpaired t-test. F. Schematic illustration of 2-DG uptake assay protocol for control and RbpjiΔEC mice. G. Blood glucose levels of control and RbpjiΔEC mice during the 2-DG uptake assay. n=5, data represent mean ± SEM, unpaired t-test. H. Plasma insulin levels of control and RbpjiΔEC mice during the 2-DG uptake assay. n=5, data represent mean ± SEM, unpaired t-test. I. 2-DG uptake levels in skeletal muscle of control and RbpjiΔEC mice. n=4, data represent mean ± SEM, unpaired t-test. J. 2-DG uptake levels in vWAT of control and RbpjiΔEC mice. n=5, data represent mean ± SEM, unpaired t-test.