EMBO/sd-character-level-ner huggingface dataset:
Figure 4(A) A SYPRO Orange-based thermal stability assay, which reports the transition of M and Z α1-antitrypsin from the native to an intermediate state over a 1°C/min thermal ramp, in the presence and absence of 50µM GSK716. (B) M (above) and Z (below) α1-antitrypsin, at a concentration of 0.2mg/ml in PBS+5% v/v glycerol, were heated at a range of temperatures between 48°C-65°C for 4 hours in the presence and absence of GSK716, as indicated. The oligomerisation state was determined by non-denaturing PAGE electrophoresis; the lane denoted 'M' contains the unheated monomeric control. (C) M and Z α1-antitrypsin were subjected to equilibrium unfolding into different concentrations of guanidine hydrochloride (gdnhcl) in the presence and absence of GSK716, with bis-ANS dye added to report the presence of the unfolded intermediate. The normalised fluorescence intensity data were fitted with an equation describing a three-state unfolding curve. Values shown are the mean of three independent experiments and the error bars represent ±SEM. (D) Top panel, the association of GSK716 induced a marked quenching and blue-shift of the α1-antitrypsin intrinsic tryptophan fluorescence spectrum with respect to unbound protein (inset graph). The association of 10µM GSK716 with four α1-antitrypsin variants that vary in their propensity to polymerise, ranging from inert (M), to mild (S), moderate (Baghdad denoted 'B') and severe (Z). Representative progress curves of the change in intrinsic tryptophan fluorescence at 330nm for 0.2mg/ml protein are shown. Bottom panel, the half-time of association calculated from three such independent experiments (error bars are ±SEM) show a correspondence with the polymerisation propensity of the four variants. The change in Z α1-antitrypsin fluorescence was faster than the dead-time of the apparatus (~10s) and scaling was with reference to unbound intensity. (E) Non-denaturing PAGE characterisation of the conformational state of M and Z α1-antitrypsin, after rapidly refolding by snap dilution from 6M urea in the presence or absence of GSK716 at the molar ratios indicated. The migration of polymers (pol) and monomers (mon) are shown. Arrows indicate the misfolded by-product M* arising at sub-stoichiometric concentrations of compound.
PIN4 interaction in vivo a) Summed 2D projections of PLA interactions (left) and co-localisation (right) (n=3) between PIN1 and PIN4 laterally bisected 2D-projected heat map.Figure 4a) Summed 2D projections of PLA interactions (left) and co-localisation (right) (n=3) between PIN1 and PIN4 laterally bisected 2D-projected heat map. Treatments as indicated. b) Quantification of positive PLA interactions. n.t (no treatment) and 10µM NPA, n=4; 10µM IAA and 10µM IAA+ 10µM NPA; n=3. Error bars indicate standard deviation.
Figure 8(A) Representative western blots showing the levels of SQR, TST, ETHE1 and SUOX protein in the kidney of three wild-type (WT) and three mutant (Mut) mice. (B-E) Relative levels of proteins normalized to Vinculin.
Figure 4A Upper panel: representative images of in-gel enzymatic activity of mitochondrial and cytosolic aconitase (mAco and cAco, respectively). The lower part of the gel was cut, stained with Coomassie blue, and a protein band was used as loading control (Loading). Lower panel: quantification of mAco and cAco enzymatic activity by densitometry. B Upper panel: Western blot analysis of mitochondrial and cytosolic aconitases. Lower panel: quantification of mAco and cAco by densitometry. C Hemequantification by absorbance at 400 nm of the soluble cell lysates. D Upper panel: western blot analysis of transferrin receptor (TfR1) and ferritin (FtH). Lower panel: quantification of TfR1 or FtH normalized on actin by densitometry. All the data are presented as means + SEM of at least three independent experiments (unpaired, two-tailed t-test).
Figure 2 Figure 2. MGC present dysregulation in inos expression A BMMΦ were stimulated with live BCG (MOI 15), with heat fixed (HI) BCG, or with LP, 1µg/ml of mono-, di- or tri-acylated lipomannan from M.bovis BCG for 6 days. Then, cells were stained for intracellular iNOS and quantification by flow cytometry. Depicted are means ± SEM from 3 independent experiments. B, C BMMΦ were stimulated with LP and transcript levels of tnfα and inos relative to gapdh were quantified by qRT-PCR at indicated timepoints as fold change to unstimulated. Depicted are means ± SEM from 4 independent experiments. B, C BMMΦ were stimulated with LP and transcript levels of tnfα and inos relative to gapdh were quantified by qRT-PCR at indicated timepoints as fold change to unstimulated. Depicted are means ± SEM from 4 independent experiments. D BMMΦ were stimulated with LP and with repeated LP stimulation on day 3 where indicated and transcript levels of inos relative to gapdh were quantified by qRT-PCR at indicated timepoints as fold change to unstimulated. Depicted are means ± SEM from 3 independent experiments.  E, F BMMΦ were stimulated with LP and DNA-methylation of CpG sites -192 and -196 (D) or in the promoter/enhancer region and control site -845 (E) were analyzed by pyrosequencing at indicated timepoints. Depicted are means ± SEM of 4 independent experiments. *p < 0.05, ***p < 0.001, ****p < 0.0001 (two-tailed unpaired t-test).
Figure 3A Phase contrast images of embryoid bodies (EBs) from 2a2iL-RH6 cells that differentiated into neurons. Scale bars: 200 and 100 µm in the inset. B qRT-PCR analysis of 20 day-EBs from pRH6 and 2a2iL-RH6 for markers of different embryonic germ layer derivatives. *P<0.05, **P<0.01, ***P<0.001 (t-test). Data presented as mean ± SD (n=3). C Immunofluorescence images of differentiated 2a2iL-RH6 cells that depict expression of markers for neuron-like cells (TUJ-1), cardiac-muscle-like cells (CTNT), and hepatocyte-like cells (SOX17). Nuclei were stained with 4′, 6-diamidino-2-phenylindole (DAPI). Scale bar: 100 µm. D 2a2iL-RH6 cells form teratomas that consist of three embryonic germ layer-derived tissues. (i) Rosette structures (ectoderm), (ii) cartilage (mesoderm), and (iii) primitive gut (endoderm; scale bar 50 µm). E From left to right: Phase contrast and GFP representation in 2a2iL-RH6-eGFP; presence of injected 2a2iL-RH6-eGFP in the mouse blastocyst and integration into the inner cell mass (ICM) after 3, 24 and 48 h incubation periods. Scale bars: 200, 100, and 50 µm in the inset. F Fluorescence images of tissue-sections from human-mouse chimeras in E10.5 showed cells positive for GFP (top) and human nuclear antigen (HNA) (bottom) in different parts of the embryos, including the head (i), trunk (ii), and tail (iii) with the higher magnification in the right panels. Scale bars: 200 and 100 µm in the inset. G Genomic PCR analysis for a human specific mitochondrial sequence (391 bp) in human-mouse chimeras. H Immunofluorescence staining against NANOG shows lack of expression in E10.5 human-mouse chimera tissue sections. Scale bars: 200 and 100 µm in the inset. I Immunofluorescence staining against TUJ1, BRACHYURY (BRA), and SOX17 in E10.5 human-mouse chimera tissue sections represent differentiation-derivatives of 2a2iL-RH6. Scale bars: 200 and 100 µm in the inset.
Figure 8Representative electron micrographs of random ultrathin sections from IHC ribbon synapses of p15 mutants lacking different endophilin-A alleles. Scale bars: 200 nm. Schematic illustration depicting the analysis criteria for random section electron micrographs (not drawn to scale). Synaptic vesicles (SVs) were categorized as ribbon-associated SVs (RA-SVs, green) or membrane-proximal SVs (MP-SVs, yellow). Reduced numbers of RA-SVs in IHCs of endophilin mutants with the strongest reduction observed in 1/2-DKO mice. One-way ANOVA followed by Tukey's post-hoc test; *p < 0.05; **p < 0.01; ***p < 0.001. Reduced ratio of RA-SVs in the distal half of the ribbon over RA-SVs in its proximal half in 1/2-DKO and 1/3-DKO IHCs. K-W followed by Dunn's test; **p < 0.01; ***p < 0.001. Reduced numbers of MP-SVs (D) in IHCs of endophilin mutants with the strongest reduction observed in 1/2-DKO mice. One-way ANOVA followed by Tukey's post-hoc test; *p < 0.05; **p < 0.01; ***p < 0.001. Representative 3D reconstructions of EM tomograms of 250 nm sections from Wt and endophilin DKO mutants. Please note the accumulations of ELVs in IHCs of endophilin 1/2-DKOs. Analysis was performed on 8 tomograms from 2 animals per genotype. Scale bars: 100 nm. Schematic illustration depicting the analysis criteria for tomograms (not drawn to scale). SVs were categorized as RA-SVs (green) and as MP-SVs (yellow). ELVs (light blue) were counted if the smallest distance between ELV and ribbon was less than 200 nm. Significantly reduced numbers of RA-SVs in 1/2-DKO IHCs (one-way ANOVA followed by Tukey's post-hoc test; *p = 0.0229). Significantly reduced numbers of MP-SVs in 1/2-DKO and a strong trend (p = 0.0511) towards less MP-SVs in 1/3-DKO IHCs (one-way ANOVA followed by Tukey's post-hoc test; *p = 0.0138). Increased SV diameter in 1/2-DKO, but unchanged SV diameter in 1/3-DKOs (one-way ANOVA followed by Tukey's post-hoc test; *p = 0.0154). Accumulation of ELVs with a minimal distance to the ribbon of less than 200 nm in 1/2-DKOs (one-way ANOVA followed by Tukey's post-hoc test; ***p = 0.0002).
Figure 1A Concentrations of amylase, lipase, CRP, and ANGPTL4 in the sera of patients (n = 90) diagnosed with pancreatitis. The box plots show a typical display consisting of a median value depicted by the line in the center of the box; an interquartile range (IQR; 25th to the 75th percentile) depicted by the box; and the maximum (Q3+1.5*IQR) and minimum (Q1-1.5*IQR) values depicted by the whisker. Statistical significance of Mann-Whitney U-tests is indicated (** P < 0.01 and *** P < 0.001). Exact P values are shown in Appendix Table S2. ANGPTL4 and CRP concentration from pancreatitis patients were determined by sandwich ELISA. The correlation between ANGPTL4 and CRP is depicted with Pearson correlation coefficient (R). Each dot represents the levels of ANGPTL4 and CRP from an individual patient (n = 90). B Pancreatitis and adjacent normal regions (n = 6) were stained by immunofluorescence for amylase and ANGPTL4. Scale bar represents 30 µm C H&E staining, ANGPTL4 and amylase expression by immunofluorescence analysis after the induction of mild (AP) and severe acute pancreatitis (SAP) for 2 h (n = 15, each group). Scale bar represents 50 µm. D Amylase, lipase, and ANGPTL4 were assessed in the serum from AP and SAP animal models and ANGPTL4 protein expression were determined in the blood and pancreas (n = 15, each group).
Ca2+ influx is sufficient to drive a P-EMT phenotype and increased migration and invasion Representative 20X brightfield and fluorescent images of murine YFP+ tumor cells treated for 72h with vehicle control (DMSO), 2.Figure 2Representative 20X brightfield and fluorescent images of murine YFP+ tumor cells treated for 72h with vehicle control (DMSO), 2.5μM Ionomycin, or 10ng/ml TGFβ and co-stained for E-cadherin (Red) and DAPI (Blue). Scale bar is 100μm. Representative flow cytometry analysis of surface ECAD (Left), MFI of surface ECAD (Middle), and percentage of surface ECAD negative cells (Right) in murine PDA cells, after treatment with DMSO, 2.5μM Ionomycin (IONO), and 10ng/ml TGFβ for 72h. Representative of 3 independent murine PDA cell lines run in triplicate. Statistical analysis by ANOVA (****, p<0.0001; non-significant (NS); ±SD). Top: Relative mRNA expression of Ecad after treatment with 2.5μM ionomycin or 10ng/ml TGFβ for denoted time. Bottom: Western blot analysis of total E-cadherin and Vimentin protein for denoted time. Experiment was run in duplicate with three different murine PDA cell lines. Statistical analysis by ANOVA (***, p<0.001; non-significant (NS); ±SEM). Normalized MFI of surface E-cadherin in PDA cells treated with DMSO, 2.5μM Ionomycin (IONO), and 10ng/ml TGFβ for 7 days, followed by withdrawal of Ionomycin or TGFβ for 7 days as indicated on the x-axis. Treated cells were analyzed at day 2, 3, and 7. Withdrawn cells were analyzed on days 1, 2, and 7. Experiment was performed in duplicate in two different murine PDA cell lines. Error bars indicate SD. Quantification of live cellular migration over 4h after treatment with DMSO, 2.5μM Ionomycin (IONO), or 10ng/ml TGFβ for 48hrs. (n=16 cells per condition. Experiment was run in triplicate in two murine PDA different cell lines) (Right). Quantification of transwell migration after treatment with DMSO, 2.5μM Ionomycin (IONO), or 10ng/ml TGFβ for 72h (n= 2 cell lines, 3 replicates per cell line with 3 20X images taken per transwell) (Left). Statistical analysis by ANOVA (*, p<0.05; ****, p<0.0001; non-significant (NS); ±SD).
Mitosis‐selective cell surface localization of PCDH7Tubulin and PCDH7 immunostaining of unsynchronized HeLa cells: tubulin, green; PCDH7, red; DNA, blue.Figure 3A Tubulin and PCDH7immunostaining of unsynchronized HeLa cells: tubulin, green; PCDH7, red; DNA, blue. PCDH7 localizes to the mitotic cell surface (arrows). PCDH7 staining is barely detectable in the surrounding interphase cells. Scale bar, 5 μm.B Quantification of PCDH7 signal on the cell surface of immunostained cells in mitosis (n = 6, red) and interphase (n = 7, blue). Bars show mean ± SEM. C PCDH7‐EGFP‐transfected unsynchronized U2OS cells, stably expressing mCherry‐tubulin, at mitosis (top) and interphase (bottom). PCDH7, green; tubulin, red; DNA, blue. Scale bar, 10 μm. D Immunostaining of HeLa Kyoto cells stably expressing PCDH7‐LAP‐tagged BAC transgenes (green) probed for tubulin (red) and DNA (blue). Top panel shows mitotic cell those were treated with 5 μM STC for 10 h, and bottom panel shows interphase cells. Scale bar, 10 μm.
Optical detection of glutamate release at individual IHC synapses: low and variable voltage threshold.Figure 1Maximum intensity projections of the organ of Corti from the right ear of a P17 mouse injected at P6 with 1-2 µl of AAV9.hSyn.iGluSnFR suspension, immunolabeled for iGluSnFR (GFP), IHCs, OHCs and SGNs (parvalbumin) and synaptic ribbons and nucleus (CtBP2/RIBEYE). Close-up (right panel) highlights IHCs innervated by several iGluSnFR-expressing SGN boutons. Step sizes are 0.6 (left) and 0.5 µm (right), respectively. (Scale bars: 10 µm) Simultaneous perforated patch-clamp and imaging of IHCs (1.3 mM [Ca2+]e). IHCs were patch-clamped from the pillar side, with a patch pipette containing TAMRA-conjugated CtBP2-binding peptide. Towards the end of the recording, the membrane patch was ruptured to fill the IHC with the peptide, which stains synaptic ribbons and nucleus. (Left) Two exemplary confocal sections of IHCs showing baseline fluorescence of iGluSnFR-expressing afferent boutons from both modiolar (IHC1) and pillar (IHC2) side of the cell. Glutamate release from IHCs was evoked upon step depolarizations and detected as fluorescence change (ΔF) of iGluSnFR signal located on the SGN membrane (See B'). (Right) Overlaid images of the IHC1 and IHC2, displaying the boutons (iGluSnFR) and the synaptic ribbons (CtBP2), after the recording. (*: transduced afferent boutons, >: synaptic ribbons) (Scale bar: 2 µm) (see Appendix Fig S2 for the iGluSnFR ROI detection routine) The stimulus protocol of the example IHCs from B, displaying the voltage stimulation (Vstim), whole-cell Ca2+ influx (ICa) and single iGluSnFR responses. IHCs were stimulated by 10-ms-long step depolarizations to -22 mV from a holding potential of -87 mV (1.3 mM [Ca2+]e), and iGluSnFR fluorescence was recorded at 50 Hz. Average ΔF/F0 iGluSnFR traces in response to 10-ms-long step depolarizations from the holding potential (-87 mV) to a voltage within the physiologically relevant range of receptor potentials: from -62 mV to -22 mV (applied in pseudo-randomized order, step-size 5 mV, perforated patch-clamp, 1.3 mM [Ca2+]e, n = 29 boutons, N = 11 IHCs from 9 mice). Shaded areas show ± SD. The voltage threshold of glutamate release was low and variable (-47.11 ± 4.09 mV, mean ± SD). The area under the curve of the iGluSnFR signal (top; AUC(ΔF/F0)10ms) from (C) and corresponding whole-cell QCa (bottom) (mean ± SD). Detectable signals were defined here if the peak iGluSnFR signal was 2 times higher than baseline SD. All synapses had detectable signals in response to depolarizations ≥ -42 mV. (See also Figs. EV1-2) * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001
a) Rag acts through TORC1 to regulate S6K phosphorylation.figf5(a) Rag acts through TORC1 to regulate S6K phosphorylation. HEK293 cells were transfected with constructs as indicated. Co-expression of of mTORKD construct (600 ng) or rapamycin treatment (rapa, 20 nM, 30 min) abolished the effect of RagAQ66L and RagCS75N on S6K phosphorylation. The protein level of mTORKD was determined by immunoblotting with anti-mTOR antibody. (b) RagA/RagC and TSC1/TSC2 independently regulate S6K phosphorylation. HEK293 cells were transfected with 200 ng of each Rag and/or ′ constructs as indicated. Amino acid starvation for 1 h (-AA) is indicated. Phosphorylation and protein levels of the transfected proteins were determined by immunoblotting with appropriate antibodies, as indicated. (c) TSC2 and RagA/B independently affect S6K phosphorylation. HA-S6K (20 ng) was transfected into HeLa cells with or without RNAi against human TSC2, RagA and RagB as indicated. (d) RagAT21N and RagCQ120L do not block Rheb-induced S6K phosphorylation. RagAT21N and RagCQ120L (200 ng each) were transfected into HEK293 cells with or without Rheb construct (20 ng). S6K was included in the co-transfection. Phosphorylation and protein levels of the transfected proteins were determined by immunoblotting with appropriate antibodies, as indicated. Full scans of blots are provided in Supplementary Information, Fig. S6.
A.Figure 4A. CHX assay with S309A. N2a cells transfected with either S309A or S309A+4D were treated with CHX for 0, 4 or 8 hrs before harvest. Lysates were subjected to immunoblotting with anti-Atoh1 or β-actin. Quantification of relative levels of ATOH1 to the β-ACTIN (right panel). Error bars are representing as Mean ± SEM from three trials of each condition. B. Lysates of N2a cells transfected with either WT or a phospho-mimic form of ATOH1 (S309D) were immunoblotted with anti-Atoh1 or β-actin. Quantification of relative levels of ATOH1 to the β-ACTIN (right panel). Mean± s.e.m. Student T-test between WT and S309D was performed. ***p < 0.001. Three slots of each experimental condition represent the three biological replicants of each condition. C. GST-pull down assay of WT and WT with 4D. GST-Atoh1 plasmids were transfected with or without Ccnd1 and Cdk4 plasmids (4D) in N2a cells. Cell lysates were pull-downed with glutathione-conjugated beads after a 6 hr treatment of 10µM MG132 and then immunoblotted with anti-poly Ubiquitin. Quantification of relative levels of poly-ubiquitin (right panel). Bars and error bars are representing as Mean ± SEM from three trials of each condition. Student T-test between WT with MG132 and WT+4D with MG132. **p < 0.01. D. Lysates of N2a cells transfected with either WT or S309D were immunoblotted to detect Atoh1 S328 phosphorylation (p-S328-Ab), Atoh1 or β-actin. Quantification of relative levels of phosphorylated ATOH1 at S328 to the total ATOH1 (right panel). Mean± s.e.m. Student T-test between WT and S309D was performed. **p < 0.01. Three slots of each experimental condition represent the three biological replicants of each condition. E. Lysates of N2a cells transfected with either WT or S328A were immunoblotted with anti-Atoh1 or β-actin. Quantification of relative levels of ATOH1 to the β-ACTIN (right panel). Bars and error bars are representing as Mean ± SEM from three trials of each condition. Student T-test between WT and S328A was performed. Three slots of each experimental condition indicated the biological replicants. Rescue experiment of Atoh1 KD with WT or S309A form of Atoh1. (F-I) Scramble (Control), Atoh1 shRNA (Atoh1 KD), shRNA and WT (KD + WT) or shRNA and S309A (KD + S309A) plasmids were introduced to P5 mice EGL with H2B-EGFP plasmid and immunostaining for NEUROD1(magenta) was performed at P7. Nuclei of electroporated cells were detected by GFP (green). Arrows indicate the cells of interest which GFP signals are not co-localized with NEUROD1 (AT+GCPs). Rescue experiment of Atoh1 KD with WT or S309A form of Atoh1. (J) Proportions of NEUROD1- cells (AT+GCPs) among the electroporated cells. Rescue experiment of Atoh1 KD with WT or S309A form of Atoh1. (K) Proportions of KI67+ cells (total GCPs). Tukey-Kramer test was performed among the control (N=3), Atoh1 KD (N=3), Atoh1 KD +WT (N=3) and Atoh1 KD +S309A (N=3) mice. ***p < 0.001, *p < 0.05, N.S.: Not Significant. Bars and error bars are representing as Mean ± SEM. Number of cells: 138, 147 and 124 from N=3 mice (Control), 82, 75 and 117 from N=3 mice (KD), 127, 269 and 347 from N=3 mice (KD+WT), 195, 71 and 111 from N=3 mice (KD+S309A).
Figure 5(A) left panel: effect on expression of the single point mutation compared to the consensus sequence (indicated as dots whose size scales with the loss of expression after mutation). Middle and right panels: comparison of the XXmotif logos with the expression-based activity logos for INR, TATA-Box, INR2, DRE and Ohler7. Expression-based activity logos show an overall lower specificity. IC, information content. (B) Effect of motif positional shifts. log2 expression of native promoters (cyan dots) and promoters with motifs shifted relative to their original locations (red dots), for INR, MTEDPE, TATA-Box in cas, and DRE, Ohler7 in RpL36A. (C) Motif occurrence around TSS (at position 0) discovered in the genome-wide analysis by XXmotif. The blue rectangular boxes indicate the -20 to 20 bp region surrounding the original positions of the motifs in the tested core promoters (strictly positioned INR, MTEDPE, TATA-Box in cas; broadly distributed DRE, Ohler7 in cas).