EMBO/sd-character-level-ner huggingface dataset:
Figure 1Dox-induced prolongation of Nanog expression in Nanogtg embryos up to E9.5 results in lack of blood (left) and downregulation of erythropoietic gene expression. The center and right panels show whole-mount in situ hybridization for Hbb-bh1 (in embryos with intact yolk sacs) and for the long non-coding RNA Redrum. Asterisks mark the aorta-gonad-mesonephros region (AGM) and arrows the tailbud. Embryos of the same genotype but not treated with dox were used as controls (-dox). Scale bars, 500 µm. Endomucin staining of vessels in control (-dox) or treated (+dox) E9.5 Nanogtg embryos. On the right, higher magnifications of the boxed areas. Scale bar, 500 µm. Representative FACS plot of the distribution of the CD71 and Ter119 populations in dissected yolk sacs from untreated and dox-treated E9.5 Nanogtg embryos. Quantification of the CD71+ Ter119+ population in controls (-dox, black dots; n=8) and Nanog expressing (+dox, red dots; n=7) E9.5 yolk sacs. Each replicate contained a pool of 5 (-dox) or 8 (+dox) E9.5 Nanogtg embryos. ***P. < 0.0005; Student's t-test. Horizontal line represents mean values and error bars standard deviation (SD). Representative FACS plots showing the distribution of cKit and CD41 populations in yolk sacs from untreated controls (-dox) and Nanog expressing (+dox) E9.5 Nanogtg embryos. Quantification of different progenitor populations in yolk sacs from control (-dox, black dots; n=8) and Nanog expressing (+dox, red dots; n=7) E9.5 embryos. Each replicate contained a pool of 5 (-dox) or 8 (+dox) E9.5 Nanogtg embryos. Horizontal line represents mean values and error bars SD. Differences in the expression levels of Nanog and selected hematopoietic genes in the CD71+ Ter119+ population of control (-dox; n=7) and Nanog-expressing (+dox; n=4) E9.5 embryos. **P < 0.005, ***P < 0.0005; Student's t-test. Horizontal line represents mean values and error bars SD.
Fig.Figure 2(A) A plasmid system (inset) is used to control the expression of the non-required gene lacZ using the strain NQ1389. lacZ expression was induced using cTc to varying degrees at the moment of glucose depletion (time = 0 h) using the indicated range of cTc concentrations. Diauxic growth conditions with glucose and acetate, same as in Fig. 1. lacZ mRNA resulting from the different degrees of induction as well as aceB mRNA in the same cultures were measured by qPCR and plotted as fold-change increase compared to that in the absence of induction. The lag-times observed in panel A are plotted against the respective change in lacZ abundances (B) lacZ mRNA resulting from the different degrees of induction as well as aceB mRNA in the same cultures were measured by qPCR and plotted as fold-change increase compared to that in the absence of induction. , where the dashed line represents the 3.5h lag observed for the WT strain (no induction, Fig. 1A). Changes in lacZ and aceB mRNA levels are inversely related (C), lacZ mRNA resulting from the different degrees of induction as well as aceB mRNA in the same cultures were measured by qPCR and plotted as fold-change increase compared to that in the absence of induction. shown both as linear (left plot) and log2 (right plot) scales. The dashed line in the left plot shows a linear fit. aceB mRNA abundance is inversely related to the lag-time (D). Means of N=3 and N=5 biological replicates are shown for lag-times and expression levels respectively in panels B-D. Error bars denote SD.
Figure 5A, B. Flow cytometry analysis of distribution of CD45.1+ and CD45.2+ cells in indicated cell subsets purified from bone marrow (A) and lymph node (B) of sub lethally-irradiated Rag1-/- mice 8 weeks after reconstitution with equal numbers of bone marrow cells from Ahr+/+ (CD45.1+) and Ahr-/- (CD45.2+) mice. Dashed lines indicate 50% level. Representative data of n = 2 independent experiments; two-tailed paired t test. C. Host CD45.1 mice were co-transferred with a 1:1 mixture of HEL-specific Ahr+/+ CD45.1+CD45.2+ splenocytes isolated from SWHEL AhR+/+ mice and HEL-specific Ahr-/- CD45.2+ splenocytes isolated from SWHEL AhR-/- mice in the presence of SRBC-HEL or SRBC-mock. Readout at d7 post-challenge was distribution of Ahr+/+ CD45.1+CD45.2+ vs Ahr-/- CD45.2+ cells. D. Flow cytometry analysis of distribution of CD45.1+CD45.2+ and CD45.2+ cells harvested from host mice challenged as indicated above the dot plots. Representative data of n = 3 independent experiments. E. Flow cytometry analysis of distribution of Ahr+/+ CD45.1+CD45.2+ (black) and Ahr-/- CD45.2+ (white) cells harvested from host mice. Indicated distribution is quantified relative to transferred cells. X symbols indicate SRBC-mock-treated control. Dashed line indicates 50% threshold. Representative data of n = 3 independent experiments; two-tailed paired t test. F, G. Flow cytometry analysis of distribution (F) and cell numbers (G) of IgG1+ and IgG1- HEL3x-binding Ahr+/+ CD45.1+CD45.2+ (black) and Ahr-/- CD45.2+ (white) cells harvested from host mice. Representative data of n = 2 independent experiments; two-tailed paired t test.
Class switch recombination and proliferative potential of Dot1L-deficient B cells in response to T-cell dependent and -independent stimuli in vitro A.Figure 3A. Statistical analysis of IgG1 switching of Dot1L-proficient (WT) and -deficient (KO) B cells after four days of activation with anti-CD40 + IL-4. Results represent the data from one experiment and numbers represent biological replicates for each group (WT; n = 4, KO; n = 4). B. Number of cell divisions traced by CTV dilution of B cells stimulated for four days with anti-CD40 + IL-4. Data represent three biological replicates for each genotype. C. Statistical analysis of the percentage of IgG1 switched cells per generation of proliferating WT and KO B cells after four days of stimulation with anti-CD40 + IL-4. Results represent the data from one experiment and numbers represent biological replicates for each group (WT; n = 4, KO; n = 4). D-E. Statistical analysis of switching of WT and KO naïve B cells activated for four days with LPS alone (IgG3 switching) (D) or with LPS + IL-4 (IgG1 switching) (E). Results represent the data pooled from at least four independent experiments and numbers represent biological replicates for each group (D: (WT) n = 8, (KO) n = 9; E: (WT) n = 11, (KO) n = 13). F. Statistical analysis of IgG1 switching of naïve B cells after four days of activation with anti-CD40 + IL-4 in the presence of the DOT1L inhibitor Pinometostat or DMSO as a control. Results represent the data from one experiment and numbers represent biological replicates for each treatment (n = 4). G. Number of cell divisions traced by CTV dilution of B cells stimulated for four days with anti-CD40 + IL-4 either in the presence of DOT1L inhibitor Pinometostat or DMSO as a control. Data represents three biological replicates for each treatment. H. Statistical analysis of the percentage of IgG1 switched cells per generation of proliferating WT B cells after four days of stimulation with anti-CD40 + IL-4 either in the presence of DOT1L inhibitor Pinometostat or DMSO as a control. Results represent the data from one experiment and numbers represent biological replicates for each treatment (n = 3). I. Statistical analysis of IgG3 switching of naïve B cells after four days of activation with LPS alone in the presence of the DOT1L inhibitor Pinometostat or DMSO as a control. Results represent the data from one experiment and numbers represent biological replicates for each treatment (n = 4).
Figure 4(A) Representative confocal images of HEK293-AT1 cells, which show that treatment with OSW1 (20 nM), a drug that inhibits the cholesterol-PI4P transport protein, OSBP, causes accumulation of PI4P in the Golgi and in the endosomes (upper panels). Note that the accumulation is much reduced in the endosome but less so in the Golgi in the PI4K2A K/O cells. Scale bars: 20 µm (B) Quantification of these changes by BRET analysis. The PI4P-Rab7 BRET sensor construct was transfected into the respective cell lines and the cells treated with OSW1 (20 nM) for the indicated times. The BRET ratios were expressed relative to those of DMSO treated cells. Data information: Means ± S.E.M. are shown from three experiments performed in triplicates. (C) Detection of endosomal PI(4,5) P2 in cells pre-treated with OSW1 for 1 h in serum-free medium using confocal microscopy and the PLCδ1PH-GFP PI(4,5) P2 reporter. Angiotensin II (AngII, 100 nM) (a Gq and PLC-activating agonist) was used to liberate the PLCδ1PH-GFP PI(4,5) P2 reporter from the PM, which was necessary to detect the endosomal PI(4,5) P2. The signal is transient as the AngII receptors show desensitization. Note the reduced signal in the two PI4K2A K/O clones. Scale bars: 20 µm. (D) Quantification of the same changes by BRET analysis in wild-type cells using the PI(4,5) P2 sensor with Rab7-Q67L-targeting. Note the transient signal increase after AngII stimulation only in cells treated with OSW1. (E) This increase is barely detectable in the PI4K2A K/O cells. D Data information: Means ± S.E.M. are shown from three experiments performed in triplicates. The values in (D) and (E) were measured in the same 96-well plate at the same time.
Inhibition of miR-27a or miR-24 promotes somatic cell reprogramming to iPSC.Figure 5The schema of reprogramming MEFs to iPSCs by three transcription factors Oct4, Sox2 and Klf4 (OSK). Validation of the effect of miRNA inhibition in MEFs by q-PCR. Error bars indicate s.d. (n=3). AP staining of the iPSC colonies in reprogramming with OSK combined with scramble control, inhibitors of miR-23a, miR-27a, miR-24 and let-7c respectively. Fold increase in Oct4-GFP-positive cell colonies in the above cells. Data are shown as mean + s.d. (n=5); ** indicates P-value < 0.01; *** indicates P-value < 0.001. Morphology of typical Oct4-GFP-positive iPSC colonies that were reprogrammed by OSK combined with miRNA inhibitors. Detection of endogenous and exogenous Oct4, Sox2, Klf4 and Nanog expression in iPSCs, ESCs and MEFs by RT-PCR. Bisulfite genomic sequencing of the promoter regions of Oct4 and Nanog in iPSCs, ESCs and MEFs. Open circles indicated unmethylated CpG dinucleotides, while closed circles indicated methylated CpGs. Immunofluorescence staining of Oct4, Nanog and SSEA1 of the iPSC colonies. Q-PCR analysis of the expression of self-renewal (Oct4, Sox2, Nanog) and differentiation (Cdx2, Foxa2, Brachyury, Hand1, Nestin, FGF5) relative markers in EBs derived from the iPSCs. Error bars indicate s.d. (n=3). Hematoxylin and eosin staining of teratoma derived from the iPSC colonies. Neuronal rosette (Ectoderm), Cartilage (Mesoderm) and respiratory epithelium (Endoderm) are marked with black arrows.
HCV infection induces perinuclear clustering of mitochondria and Parkin translocation to mitochondria.Figure 1A) Representative confocal images showing perinuclear clustering of mitochondria and Parkin aggregates on the mitochondria in Huh7 cells infected with HCVcc. At 3 days post-infection, cells prestained with MitoTracker (Mito, red) were immunostained with anti-Parkin (green) and HCV core (orange) antibodies. Nuclei were stained with DAPI (blue). Infected (+) and uninfected cells (−) are marked. The illustrated images display typical distribution of mitochondria in uninfected cells and altered distribution of mitochondria in the perinuclear region of infected cells (gray). In the zoomed images, the white arrows indicate accumulation of endogenous Parkin recruited to the mitochondria in HCV-infected cells (yellow). (B) Ultrastructure of HCV-infected cells showing perinuclear clustering of damaged mitochondria. Control naïve Huh7 cells (left) and stable cells harboring HCV full-length replicon FLR-JFH1 (right) were examined by electron microscopy. In the zoomed images, typical ultrastructure of mitochondria in naïve cells and ultrastructural abnormalities of mitochondria in HCV replicon cells are shown. Organelle mark: N, nucleus; M and white arrow, mitochondria. Scale bar = 1 µM. Fluorescent images (below) indicate the expression of HCV core protein in HCV replicon cells. Cells were immunostained with anti-HCV core antibody (red). Nuclei were stained with DAPI (blue). (C) Quantification of the number of mitochondria in the perinuclear region (mean ± SEM; n≥5 cells; *p<0.05). (D) Quantification of fluorescence intensity of Parkin aggregates on the mitochondria in CCCP-treated (see Figure S1) or HCV-infected cells (A) and those treated with 3-MA or BafA1, respectively (see Figure 4A) (mean ± SEM; n = 10 cells; *p<0.01). P values were calculated by using an unpaired Student's t-test. (E) Western blot analysis showing endogenous Parkin recruitment to mitochondria in HCV-infected cells. Huh7 cells were infected with HCVcc. At 5 days post-infection, pure cytoplasm and mitochondria fractions were isolated by ultracentrifugation as described in Materials and Methods. Cellular fractions of HCV-infected cells were analyzed by immunoblotting using antibodies specific for the indicated proteins. Fractions: whole cell lysates, WCL; purified cytoplasm, pCyto; purified mitochondria, pMito. Organelle markers: VDAC1, mitochondria; Calreticulin, ER; GAPDH, cytoplasm.
Figure 3A. HEK293 Tet-off experiments showing the degradation of REL-OPT and REL-WT transcripts (left) as well as IL6-OPT, IL6-WT and IL6-DE transcripts (right), post-doxycycline addition. Data information: data is representative of 3 independent experiments each with 3 replicates. The data represents the mean ± SD for 3 replicates. A two-way ANOVA with Holm-Sidak multiple comparisons was performed. P-values are denoted as follows: p < 0.05 (*), p<0.01 (**) and p<0.001 (***). The half-lives of the respective transcripts are indicated in brackets. B. Representative immunoblot of FLAG-tagged REL-OPT and REL-WT in HEK293T cells transfected with either empty plasmids, plasmids bearing REL-OPT or REL-WT. The immunoblot is representative of 3 independent experiments. ACTB is shown as the loading controls. C. ELISA of secreted IL6 concentrations of IL6-OPT, IL6-WT and IL6-DE from HEK293T cells transfected with plasmids bearing IL6-OPT, IL6-WT and IL6-DE. Data information: In (C), the data is representative of 3 independent experiments each with 3 replicates. The data represents the mean ± SD for 3 replicates. A one-way ANOVA with Tukey's multiple comparisons was performed between samples where, p<0.01 (**) and p<0.001 (***). D. Fold changes of REL-OPT and REL-WT transcript levels (top) relative to their abundances from fraction 1 as detected by qPCR across polysome fractions (below). Data represents the mean ± SD for 3 biological replicates. Data information data is representative of 3 independent experiments each with 3 replicates. The data represents the mean ± SD for 3 replicates. A two-way ANOVA with Holm-Sidak multiple comparisons was performed. P-values are denoted as follows: p < 0.05 (*), p<0.01 (**) and p<0.001 (***). The half-lives of the respective transcripts are indicated in brackets.
LNS2SS2A+ structure to the Nxph1-LNS2SS2- structure and modulation of Nxph1-Nrxn1 LNS2 binding by alternative splicing.Figure 3Overall view and close-up of superimposed Nxph1-LNS2SS2- and Nxph1-LNS2SS2A+ structures in cartoon representation. Electron density maps comparing the Nxph1-LNS2SS2- and Nxph1-LNS2SS2A+ structures. These maps cover the same region as shown in the close-up in A. The 2mFo-DFc map for the Nxph1-LNS2SS2A+ structure is shown in gray at a contour level of 1 σ. The positive and negative Fo-Fo difference density maps (the difference between Nxph1-LNS2SS2- and Nxph1-LNS2SS2A+ structure factor amplitudes using phases from the Nxph1-LNS2SS2A+ model) are shown in green and red, respectively, at a contour level of 3 σ. Sequence alignment of mouse Nrxn1-3 LNS2SS2A/AB+ showing key regions involved in Nxph1 binding and SS2A and SS2AB inserts. Sequences are numbered in reference to the canonical UniProt mouse Nrxn1 sequence Q9CS84-1. Secondary structure elements are shown at the top and correspond to elements shown in the Nxph1-LNS2SS2A+ structure in A. Representative BLI traces showing increased binding of Nxph1 to Nrxn1 LNS2SS2A+ and Nrxn1 LNS2SS2AB+ compared to that of Nrxn1 LNS2SS2-. Replicate numbers are shown in E. Comparison of binding affinities for Nxph1 binding to mouse Nrxn1 LNS2 splice variants and splice-insert containing Nrxn1 LNS2 with the I401Q mutation; error bars represent SEM and replicate numbers are indicated in or above bars. Significance values were calculated using Welch's t-test, *P < 0.05, **P < 0.01, ****P < 0.0001.
Figure 1A, B. Quantification of oryzalin root growth assays in single (A) and double (B) mutants. DAG = days after germination. Graphs show mean ± SD of three biological replicates with at least 10 plants per genotype per replicate. Asterisks indicate a significant difference in root length in a two-way ANOVA followed by Tukey's multiple comparisons test (* P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001). C. Rosette pictures of 20-day-old Col-0 and cycb1;1 cycb1;2. Scale bar: 1 cm. D. Quantification of the rosette area using total leaf surface in Col-0 and cycb1;1 cycb1;2. Graph represents the single rosette area values and the horizontal lines indicates the mean value ± SD, n = 10 plants per genotype. Asterisks indicate a significant difference in rosette area using an unpaired t-test, t = 7.421, df = 18 (**** P < 0.0001). E. Silique pictures of cycb1 double mutant combinations. White arrowheads indicate aborted ovules and seeds. Scale bars: 500 μm. F, G. Quantification of aborted seeds in single (F) and double (G) mutants. Graphs represents the average seed abortion rate per plant ± SD of three biological replicates, n = 550-1029 seeds analyzed per genotype. Asterisks indicate significant differences in seed abortion rate in an ordinary one-way ANOVA test, followed by a Dunnett's multiple comparisons test (**** P < 0.0001).
Figure 3Ribbon representation of the Tle1 pseudoatomic model. Strands are in pink and helices in light blue. The inset shows the catalytic triad. Ribbon representation of the interaction site between the Tle1 finger domain in yellow green and the VgrG TTR domain in red. The site is shown in different orientations. Molecular surface representation of the interactions between Tle1 chain D and chains A-C of VgrG respectively (F, G and H).
Figure 1Cysteine conservation of AK2. AK2 contains four cysteines of which three (C40, C42 and C92) are highly conserved. AK2 exists in two isoforms that differ in the C-terminus with the "A" isoform containing C232 and the "B" isoform not. AK2 does not contain a mitochondrial targeting sequence. Logo plot was derived from alignments of AK2 from 163 species. Interaction analysis for MIA40 and AK2. Stable inducible cell lines that express either different variants of MIA40-Strep (wildtype and a redox inactive mutant (MIA40C53S,C55S), (Fischer et al., 2013) or AK2-HA were treated with N-ethyl maleimide (NEM) to stop thiol-disulfide exchange processes and lysed under denaturing conditions. Lysates were analysed by subsequent affinity purification against Strep (MIA40) and immunoblotting. Like classical MIA40 substrates, AK2 interacted with the active site of MIA40 (lane 6). 0.5% input was loaded as control. Redox state analysis of endogenous AK2 by inverse redox shift assay. HEK293 cells were lysed by TCA precipitation after NEM treatment, lysates were reduced using TCEP and then thiols modified using the alkylating agent mmPEG12. With this "inverse" method, previously oxidized cysteine thiols are modified with mmPEG12, which results in a slower migration on SDS-PAGE. Treatment of intact cells with diamide and consequently modification of all cysteines served as control. Likewise, absence of NEM treatment served as 'max' control. Treatment lysates with NEM instead of mmPEG12 served as minimal shift (min) control. A, B, isoforms of AK2. Both isoforms of AK2 are semi-oxidized in intact cells. Oxidation kinetics experiment to investigate maturation of endogenous AK2. HEK293 cells were pulse labeled for 10 min with [35S]-methionine and chased with cold methionine for the indicated times. The chase was stopped by TCA precipitation, and then the lysate was treated with mmPEG12 to determine the AK2 redox states, followed by IP against AK2. Eluates were analyzed by Tris-Tricine-PAGE and autoradiography. With this "direct" redox state determination method reduced AK2 was modified with either three (isoform B) or four (isoform A) mmPEG12. Semi-oxidized AK2 was modified with only one ("B") or two ("A") mmPEG12, respectively. Unmodified and fully reduced cell lysates served as controls. Endogenous AK2 becomes oxidized with a half time of less than 20 min. Oxidation kinetics experiment to investigate the influence of MIA40 on AK2 maturation. Experiment was performed as described in (D), except that 72 h before experiments cells were transfected with control siRNA or siRNA directed against MIA40. Endogenous AK2 oxidation depends on MIA40. Reported values are the mean of 3 independent experiments; error bars represent ±SD. Student's t-test was performed. * represents p<0.05, ** represents p<0.01 and *** represents p<0.001.
Figure 3Body weight loss of WT→WT, Ang-/-→WT and WT→Ang-/- chimera mice with (n = 7) or without (n = 5) 2.5% DSS treatment. DAI of WT→WT, Ang-/-→WT and WT→Ang-/- chimera mice with (n = 7) or without (n = 5) 2.5% DSS treatment. Colon length of colonic section from these chimera colitis mice on day 8 with (n = 7) or without (n = 5) 2.5% DSS treatment. serum FITC-Dextran level, of colonic section from these chimera colitis mice on day 8 with (n = 7) or without (n = 5) 2.5% DSS treatment. representative HE staining image, and (F) histopathological score of colonic section from these chimera colitis mice on day 8 with (n = 7) or without (n = 5) 2.5% DSS treatment. Ang mRNA expression in mouse lymphoid and myeloid cells; data represent two independent experiments (n = 2). Body weight loss of Angfl/fl and LyzMcre;Angfl/fl mice with (n = 7) or without (n = 3) 2.5% DSS treatment. DAI of Angfl/fl and LyzMcre;Angfl/fl mice with (n = 7) or without (n = 3) 2.5% DSS treatment. Colon length of colonic section from the Angfl/fl and LyzMcre;Angfl/fl mice on day 8 with (n = 7) or without (n = 3) 2.5% DSS treatment. serum FITC-Dextran level, of colonic section from the Angfl/fl and LyzMcre;Angfl/fl mice on day 8 with (n = 7) or without (n = 3) 2.5% DSS treatment. representative HE staining image, and (M) histopathological score of colonic section from the Angfl/fl and LyzMcre;Angfl/fl mice on day 8 with (n = 7) or without (n = 3) 2.5% DSS treatment.
Figure 2A. β-catenin-mediated reporter activity in HEK293T cells expressing WT RNF43 and oncogenic RNF43 (R519X) harboring mutations C290S/H292S in the RING domain (M1). Cells were treated with control medium (no Wnt3a) or Wnt3a-conditioned medium (Wnt3a) overnight. Average β-catenin-mediated reporter activities ±s.d. in n = 2 independent wells are shown. B. Schematic of RNF43 constructs in which the extracellular and transmembrane domains (ECD and TM) are replaced by those of the unrelated CD16 and CD7, respectively. CCR; CD16-CD7-RNF43. C. β-catenin-mediated reporter activity in HEK293T cells expressing the indicated RNF43 constructs. Cells were treated with control medium (no Wnt3a) or Wnt3a-conditioned medium (Wnt3a) overnight. Average β-catenin-mediated reporter activities ±s.d. in n = 2 independent wells are shown. D. β-catenin-mediated reporter activity in HEK293T cells co-expressing oncogenic RNF43 (R519X) and the Dishevelled DEP-C tail. Cells were treated with control medium (no Wnt3a) or Wnt3a-conditioned medium (Wnt3a) overnight. Average β-catenin-mediated reporter activities ±s.d. in n = 2 independent wells are shown. E. β-catenin-mediated reporter activity in HEK293T cells co-expressing oncogenic RNF43 (R519X) and dominant negative ∆N-TCF4. Cells were treated with control medium (no Wnt3a) or Wnt3a-conditioned medium (Wnt3a) overnight. Average β-catenin-mediated reporter activities ±s.d. in n = 2 independent wells are shown.