EMBO/sd-character-level-ner huggingface dataset:
Figure 7The FadA filaments were based on the crystal structure of mFadA (Nithianantham et al., 2009), with the addition of intercalating pre-FadA. The signal peptide pairs from two neighboring filaments (one clear bar and one shaded bar) bind to each other through hydrophobic interactions, serving as "hooks" crosslinking the filaments so that the filaments are stacked perpendicular to the fibril axis into fibrous sheets.
Figure.Figure 5(A) α1-Antitrypsin was incubated at varying molar ratios with the model protease bovine α-chymotrypsin, and the residual protease activity determined. The intercept of the regression with the abscissa reflects the number of molecules of α1-antitrypsin required to inhibit one molecule of chymotrypsin in the presence (+c) and absence (-c) of 50µM GSK716. Error bars reflect ±SEM of three independent experiments. (B) M α1-antitrypsin was incubated with different molar ratios of chymotrypsin and resolved by SDS-PAGE. The position of covalent α1-antitrypsin-chymotrypsin complex (cpx), native (nat) and cleaved (clv) is shown. (C) The inhibitory activity of α1-antichymotrypsin against chymotrypsin (ach+chym), neuroserpin against trypsin (neus+tryp), and antithrombin against thrombin (atiii+thrombin) was determined in the presence and absence of GSK716. Error bars reflect ±SD of two independent experiments.
Figure 6Schematic diagram of iron-dextran injections experimental plan. Prussian Perl blue staining in liver and skeletal muscle after iron injections. Representative western blot images and quantification of ferritin and tfr1 (indicated by arrowhead) to tubulin in skeletal muscles 24 h after iron injections. Insulin tolerance test 24 h after iron injections. Glucose tolerance test (GTT) 24 h after iron injections. Insulin tolerance test 24 h after iron injections. Quantification of area under curve in ITT (E). Glucose tolerance test (GTT) 24 h after iron injections. Quantification of area under curve for panel (F). Representative western blot images and quantification of phospho-IRS1 (Y612) and phospho-AKT (T308) to GAPDH in skeletal muscles 24 h after iron injection followed by i.p. insulin injection.
Figure 1(A) RT-qPCR analysis of NimB1, NimB2, NimB3, NimB4 and NimB5 transcripts, normalized to RpL32, from carcass, macrophages, fat body and gut of wild type (w1118) L3 wandering larvae. Data are represented as mean ± SD from three independent experiments. (B) Representative confocal imaging of NimB4-sGFP macrophages of embryo at stage 16; lateral (left) and ventral (right) views; Tissues were stained with anti-GFP (green, corresponding to NimB4-sGFP) and anti-SIMU antibodies (red, corresponding to macrophages). The arrows show the presence of NimB4-sGFP inside the embryonic macrophages. Scale bars = 20 μm (C) Representative live confocal imaging of NimB4-sGFP macrophages of L3 wandering larvae at 30 and 60 min after dissection. Scale bars = 10 μm. (D) Representative confocal imaging of NimB4-sGFP macrophages of L3 wandering larvae after fixation. Cells were stained with phalloidin (gray), DAPI (blue) and anti-GFP (green, corresponding to NimB4-sGFP). Scale bars = 10 μm.
Fig.Figure 4A-E Surface views (A-D) and z-sections (E) of third instar larval epidermis expressing the indicated fluorescent markers and RNAi or overexpression constructs. (A) Bazooka/Par3, normally seen at the apical adherens junction and in a perinuclear position, is lost or reduced in large areas under uncontrolled autophagy as are lateral membrane markers, (B) Fasciclin-III (FasIII), (C) neuroglian (Nrg) and (D) adherens junctions (DE-cad), whereas large bundles decorated with MRLC appear. (E) The baso-lateral transmembrane protein β-integrin is not lost; in extreme conditions, it is seen in both the apical and basal membranes (overexpression of Atg16B). Arrows in the z-sections point to high accumulation of integrin along the folded lateral membranes F Electron micrograph of a section through the larval epidermis to show the highly folded lateral junction between two epidermal cells (magenta and cyan). Left, cartoon; middle, false colouring; right, original image. C: cuticle; M: mitochondrion; BL: basal lamina; AJ: adherens junction; SJ: septate junction; GJ: gap junction, N: nucleus, Mv: apical microvilli connecting the cuticle to the cell.
Figure 6Snapshots of BrdU IP-Seq peaks at ARS305 from cells released into YPD medium containing HU for 45 min. The average BrdU IP-Seq read density around fired ACS from cells released into YPD medium containing HU for 45 min sites. Snapshots of BrdU IP-Seq peaks at ARS305 from cells released into YPD medium at 16°C for 72 min. The average BrdU IP-Seq read density around ACS sites from cells released into 16°C for 72 min. Rad52-GFP foci were counted in WT and rtt105∆ cells, the percentage of cells with Rad52-foci is reported. Error bars indicate one standard deviation from three independent experiments with at least 150 cells counted in each replicate. Rad53 phosphorylation was analyzed in WT and rtt105∆ cells by immunoblotting of protein extracts with anti-Rad53 antibodies. Ponceau S (Pon S) staining was applied as a loading control. Deletion of RTT105 leads to an increased rate of gross chromosomal rearrangements (GCRs). Wild-type (WT) and rtt105∆ mutant strains integrated with the yWSS439-5ori∆ yeast artificial chromosomes (YACs). GCRs rates in the evaluation of telomere marker loss were assayed Ten-fold serial dilutions WT or rtt105∆ cells in two different backgrounds were assayed on normal growth media (YPD) and on media containing the indicated DNA damaging agents, methyl-methane sulfonate (MMS), camptothecin (CPT), bleomycin (Bleo), and hydroxyurea (HU).
Fig.Figure 8(A) Epifluorescence microscopy analysis examining the subcellular localization of de novo made alcAp-AzgA-GFP and alcAp-FurA-GFP, after 6-8 hours transcriptional derepression in strains where the expression of Sec24, SedV, GeaA, HypB, RabE, AP-1σ and SsoA was repressed ab initio (o/n) by addition of thiamine. Images showing that sorting of AzgA or FurA to the PM does not require early (SedV, GeaA), late (HypB) or post-Golgi (RabE, AP-1σ ) key proteins. On the other hand, COPII coat protein Sec24 and PM t-SNARE SsoA are essential for proper localization of the two transporters to the PM. Scale bar: 5 μm. (B) Epifluorescence microscopy analysis of the localization of de novo made AzgA and FurA transporters (see text for details) in the absence or presence of Benomyl or Latrunculin B after derepression of the relevant genes for the time indicated. Notice that addition of Benomyl, has no effect on translocation of AzgA and FurA to the PM, whereas Latrunculin B abolishes their PM localization, as seen also for UapA. Scale bar: 5 μm.
CTD shortening hardly changes the transcriptome and RNA metabolism in human cells.Figure 1A- Schematic representation of the CTD variants expressed in U2OS cells used in this study. B- Western blot verifying the expression of the CTD variants RPB1-52R and RPB1-25R in U2OS cells in whole cell extracts. U1 snRNP was used as a loading control. C- Changes in RNA synthesis upon CTD shortening. MA plot showing RNA synthesis changes in TT-seq datasets upon CTD shortening in U2OS cells in steady state conditions. RPB1-52R cells are used as a control and the data was normalized using spike-in counts. 16214 expressed genes annotated in RefSeq were analyzed. Differentially expressed genes are in red. 473 genes were significantly upregulated (adjusted p value < 0.05, log2 fold change >=1) and 704 genes were significantly downregulated (adjusted p value < 0.05, log2 fold change ≤1). D- Box plot of estimated RNA synthesis rates of expressed RefSeq transcripts (12014 transcripts) based on TT-seq and RNA-seq datasets in RPB1-52R and RPB1-25R cells. Box limits are the first and third quartiles, the band inside the box is the median. The ends of the whiskers extend the box by 1.5 times the interquartile range. E- Box plot of estimated RNA degradation rates of expressed RefSeq transcripts (12014 transcripts) based on TT-seq and RNA-seq datasets in RPB1-52R and RPB1-25R cells. Box limits are the first and third quartiles, the band inside the box is the median. The ends of the whiskers extend the box by 1.5 times the interquartile range. F- Box plot of estimated RNA half-lives of expressed RefSeq transcripts (12014 transcripts) based on TT-seq and RNA-seq datasets in RPB1-52R and RPB1-25R cells. Box limits are the first and third quartiles, the band inside the box is the median. The ends of the whiskers extend the box by 1.5 times the interquartile range.
Transport of exosomes might cause upregulation of LncRNA CRNDE in GC cells .Figure 3(A) LncRNA CRNDE expression in MFC cells incubated with BMDMs-exo or M2-exo was measured by qRT-PCR. **P<0.01 vs. BMDMs-exo. Data information: The results shown are from three biological replicates. Data are expressed as mean ± SD. Student's t-test. (B) BMDM-polarized M2 macrophages were transfected with Cy3-labelled CRNDE and exosomes were isolated from the medium (Cy3-CRNDE-EXO). Then, MFC cells were incubated with Cy3-CRNDE-EXO. Left: Confocal microscopy was used to detect the Cy3-labelled CRNDE in MFC cells. The nuclei were stained with DAPI. Scale bar= 20 µm. Right: Flow cytometry was performed to quantitate the Cy3-positive cells. **P<0.01 vs. MFC cells incubated with the control EXO. Data information: The results shown are from three biological replicates. Data are expressed as mean ± SD. Student's t-test. (C) LncRNA CRNDE expression in SGC7901 cells incubated with monocytes-exo or M2-exo was measured by qRT-PCR. **P<0.01 vs. monocytes-exo. Data information: The results shown are from three biological replicates. Data are expressed as mean ± SD. Student's t-test. (D) Monocyte-polarized M2 macrophages were transfected with Cy3-labelled CRNDE and exosomes were isolated from the medium (Cy3-CRNDE-EXO). Then, SGC7901 cells were incubated with Cy3-CRNDE-EXO. Confocal microscopy was used to detect the Cy3-labelled CRNDE in SGC7901 cells. The nuclei were stained with DAPI. Scale bar= 20 µm. Right: Flow cytometry was performed to quantitate the Cy3-positive cells. **P<0.01 vs. SGC7901 cells incubated with the control EXO. Data information: The results shown are from three biological replicates. Data are expressed as mean ± SD. Student's t-test. (E) SGC7901 cells were incubated with chlorpromazine (CPZ, 50 μM), genistein (GEN, 200 μM), 5-(n-ethyl-n-isopropyl)-amiloride (EIPA, 100 μM), or methyl-β-cyclodextrin (MβCD, 10 mM), following by the incubation of Cy3-CRNDE-EXO. Confocal microscopy was used to detect the Cy3-labelled CRNDE in SGC7901 cells. The nuclei were stained with DAPI. Right: Flow cytometry was performed to quantitate the Cy3-positive cells. Data information: The results shown are from three biological replicates. Data are expressed as mean ± SD. one-way ANOVA. (F) SGC7901 cells were incubated with 0, 25, 50, 100 μM CPZ, following by the incubation of Cy3-CRNDE-EXO. Confocal microscopy was used to detect the Cy3-labelled CRNDE in SGC7901 cells. The nuclei were stained with DAPI. Right: Flow cytometry was performed to quantitate the Cy3-positive cells. Data information: The results shown are from three biological replicates. Data are expressed as mean ± SD. one-way ANOVA.
Figure 2Glycolytic flux of wildtype (WT) and TM6 strains strongly correlates with fructose-1,6-bisphosphate (FBP) concentration. The glycolytic flux is reported here as the flux between the metabolites fructose 6-phosphate (F6P) and FBP. Glycolytic fluxes were obtained on the basis of physiological and metabolome data, and via a novel method to estimate intracellular fluxes (Niebel et al, 2019). While on high glucose, the WT strain accomplishes a high glucose uptake rate (and thus glycolytic flux), the mutant strain (TM6) only generates a low glucose uptake (and thus glycolytic flux). On maltose, also the mutant strain achieves a high glycolytic flux, since maltose is transported by a separate transporter (Chang et al, 1989). To assess the linear correlation between the FBP concentration and the glycolytic flux across the studied conditions, we implemented Pearson's correlation analysis assisted by bootstrapping. Specifically, we used in total 53 FBP concentration measurements corresponding to six different metabolic conditions (combinations of strains and carbon sources), biological and technical replicates. We paired each of these FBP measurements with the mean and standard deviation of the model-derived glycolytic flux (A) in the corresponding metabolic condition. We assumed the normal distribution of the flux and growth rate with the given mean and standard deviation in every condition, and implemented ordinary nonparametric bootstrapping with 100,000 iterations by randomly sampling values with replacement from the 53 FBP measurements and flux or growth-rate distributions to calculate the correlation statistics. FBP concentration as a function of cellular growth rate shows weaker correlation. For metabolite levels and growth rates, error bars correspond to the standard deviation between three independent experiments; for glycolytic fluxes to the mean and standard deviations of the sampled flux solution space (cf. Methods). The carbon sources were used at a final concentration of 10 gL-1 and are indicated: Glucose (GLU); Galactose (GAL); Maltose (MAL) and Pyruvate (PYR). To assess the linear correlation between the FBP concentration and the growth rate (B) across the studied conditions, we implemented Pearson's correlation analysis assisted by bootstrapping. Specifically, we used in total 53 FBP concentration measurements corresponding to six different metabolic conditions (combinations of strains and carbon sources), biological and technical replicates. We paired each of these FBP measurements with the mean and standard deviation of the growth rate (B) in the corresponding metabolic condition. We assumed the normal distribution of the flux and growth rate with the given mean and standard deviation in every condition, and implemented ordinary nonparametric bootstrapping with 100,000 iterations by randomly sampling values with replacement from the 53 FBP measurements and flux or growth-rate distributions to calculate the correlation statistics.
Figure 3(A) VAMP2, SNAP-25 and Syntaxin-1 SNAREs were immunoblotted in WT and MPS-IIIA synaptosomal brain samples at indicated ages. Synapsin-1 was also blotted as a control. Quantitation of WB was shown. (B) Confocal microscopy images of WT and MPS-IIIA hippocampal neurons (DIV14) double labeled with anti-synapsin-1 (presynaptic marker; red) and either anti-VAMP2 or anti-SNAP25 antibodies (green). The merges (yellow) of confocal images are shown. SNAP25-synapsin-1 and VAMP2-synapsin-1 co-localizations were quantified using the Manders' Colocalization Coefficients (MCC) (ImageJ) and displayed as % (MCC x 100) of Synapsin-1 co-localizing with either SNAP25 or VAMP2 (means ± s.e.m. from 15 different images taken from 4-5 coverslips for each group). (C) VAMP2 and SNAP-25 protein levels were quantified by immunoblot analysis in WT and MPS-IIIA hippocampal neurons (DIV14) at different times after cycloheximide treatment and expressed as % of remaining protein at T0 (100 %). The proteasome was inhibited as indicated. (D) SDS-resistant complex levels were evaluated in WT and MPS-IIIA total brain samples at indicated ages by immunoblotting of non-boiled samples with VAMP2 or Syntaxin-1 antibodies. Quantitation of WB was shown. (E) Total brain lysates were immunoprecipitated with antibodies to Syntaxin-1 and co-immunoprecipitated VAMP2 and SNAP25 proteins were revealed by WB analysis. The levels of immunoprecipitated proteins were quantified. (F) α-Synuclein and CSPα were immunoblotted in WT and MPS-IIIA total brain lysates at indicated ages Total protein levels were quantified. (G) α-Synuclein and CSPα were immunoblotted in WT and MPS-IIIA synaptosomal fractions at indicated ages. Hsc70, an heat shock cognate protein, which forms together with CSPα and SGT (small glutamine-rich tetratricopeptide repeat domain protein) the "chaperon machine" was also blotted. Protein levels were quantified.
markedly impairs S-phase entry and progression.Figure 4A Quantification of percentages of BrdU-positive cells after induction of wild-type and KEN mutant E2F7/8 expression measured by flow cytometry. Bars represent mean ± sem (n=3). * P<0.05 versus vehicle. B Cellproliferation curves of cell lines with stable inducible wild-type E2F7-EGFP and E2F8-EGFP expression after doxycycline treatment. Dots represent average ± sem of two replicates. C Schematic overview of live microscopy using mCherry-tagged PCNA to monitor cell cycle progression. At the onset of S phase, nuclear dots are formed, which disappear when S phase is completed. PCNA leaks into the cytosol when cells become apoptotic. D Cumulative progression into S phase of HeLa cells expressing KEN mutant E2F7/8-EGFP, monitored by nuclear PCNA dot formation. P value indicates significant change of EGFP-positive versus negative cells from the same cell line.E Cumulative cell death of HeLa cells expressing KEN mutant E2F7/8-EGFP, monitored by cell blebbing and cytosolic PCNA-mCherry. F Schematic overview of synchronization experiment. Cells were arrested at the onset of S phase with 2 mM hydroxyurea (HU), and induction of E2F8WT or E2F8K/Kmut was started 12 hours prior to release from HU. G DNA content of synchronized cells described under (F) measured by flow cytometry. Green overlays indicate the fraction of E2F8-EGFP positive cells within each doxycycline-treated cell line. H Effect of CDH1 RNAi on percentages of BrdU-positive cells after induction of E2F7/8 expression, measured by flow cytometry. Bars represent mean ± s.e.m. (n=3). I Effect of CDH1 RNAi on cumulative S phase entry of cells with inducible expression of E2F7/8WT, measured by coexpressed PCNA-mCherry. P value indicates significant change of CDH1 RNAi versus control. J Effect of CDH1 RNAi on cumulative death of cells with inducible expression of E2F7/8WT. P values indicate significant change of CDH1 RNAi versus control.
LNA-miR-29 reduces atherosclerosis lesion size.Figure 1(A) Schematic representation of experimental mouse design. Briefly, 8 week old male ApoE-/- mice on western diet were injected with equal volume saline, or 4mg/kg of LNA-control, or LNA-miR-29 biweekly for 14 weeks. (A) Schematic representation of experimental mouse design. Briefly, 8 week old male ApoE-/- mice on western diet were injected with equal volume saline, or 4mg/kg of LNA-control, or LNA-miR-29 biweekly for 14 weeks. (B) H&amp;amp;E staining of representative atherosclerotic lesions in aortic root (upper panel-4x magnification) and brachiocephalic arteries (lower panel-10x magnification) at 14 week harvest. Lesion width of roots highlighted with black bars. (C) Quantification of root lesion size (left panel, *p=0.002), and of brachiocephalic lesion size (right panel, *p=0.001), both quantified as percent of total lumen area. Data in (C) are averaged lesion percentages from 4-6μm serial sections from each animal, n=9, 13, 15. All of the data represent the mean + SEM, *p<0.05 versus LNA-control tissue. Level of significance was determined using one-way ANOVA with Bonferroni's post test.
Figure 1A. Four full-thickness excisional wounds were produced on each of CD18-/- or WT mice by 6 mm biopsy punches. Each wound received intradermal injection of 2.5x105 AT-MSCs or PBS control. Each wound region was digitally photographed at the indicated time points, and wound areas were analyzed using Adobe Photoshop. Depicted are representative macroscopic pictures of PBS injected or AT-MSCs injected WT or CD18-/- wounds at days 0, 3, 5, 7 and 9 post-wounding. B. Quantitative analysis of 20 wound areas per group, expressed as percentage of the initial wound size at day 0. The line in each group represents the mean value of 20 wounds from 5 mice. *p<0.05, **p<0.01, ***p<0.001 by Student's t-test with Welch's correction.
Figure 7  Figure 7. Schematic of the proposed mechanism for prominin2 induction mechanism by lipid peroxidation. The lipid peroxidation metabolite 4HNE activates p38/MAPK to phosphorylate and activate the stress-responsive transcription factor HSF1. The consequent increase in prominin2 expression promotes ferroptosis resistance and this mechanism can be targeted to enable resistant cells to succumb to ferroptosis. Figure partially created using BioRender.
Figure 5Schematic for ssDNA curtain assay. Briefly, ssDNA curtains were formed by flowing 1 nM RPA-eGFP for 3 minutes. Then 5 nM RPA-mCherry pre-mixed with or without 5 nM Rtt105 wild-type (WT) or E171L172AA (EL) mutant proteins were injected into the flow cell. Data were acquired with a TIRF microscope (Nikon, Inverted Microscope Eclipse Ti-E). Lasers were shuttered at 2-second intervals, and the exposure time was 100 ms. Each kymogram with two channels, in which green and red represent eGFP and mCherry signal, respectively. Kymograms showing most ssDNA molecules two distinct stretching patterns of RPA-ssDNA molecules. Pattern 1, slow stretching; Pattern 2, fast stretching. The counts of the two patterns in each reaction were plotted in (C). Please note a fraction of individual ssDNA shows both patterns. Rtt105 increases the rate of ssDNA stretching upon RPA binding. Stretching rate was defined as the length change of ssDNA upon binding to RPA-mCherry in unit time as shown in (B, white arrow). Rtt105 increases the extent of ssDNA stretching upon RPA binding. Length increment = DNA length at 8 minutes / DNA length at 3 minutes.
Figure 1Geometric mean-centered, hierarchical cluster heat map from microarray data. 1077 annotated lncRNAs (p<0.05) were represented in liver CSC (CD13+CD133+) compared with non-CSC (CD13-CD133-) cells sorted from three HCC primary cells of three patients. Top ten upregulated lncRNAs in CSCs are shown. In situ hybridization of HAND2-AS1 in HCC tumor tissues. HAND2-AS1 staining is shown in tumor tissues (left panel). Quantitation of HAND2-AS1+ cells in liver tissues was examined in 10 high-power fields (HPF) of sections from ten different HCC patients (right panel). Results are shown as means ± SEM. Scale bar, 100 μm. Macroscopic appearance of livers in 12-month-old DEN-treated lncHand2+/+ and lncHand2 knockout (lncHand2-/-) mice (left panel). Black arrows indicate liver tumors. Quantitation of tumor foci numbers and areas in DEN-treated livers (right panel). Results are shown as means ± SD (n=12). Representative H&E and immunohistological staining with Ki67 antibody of liver sections of 12-month-old DEN-treated lncHand2+/+ and lncHand2-/- mice. Scale bar, 100 μm. Quantitation of Ki67-positive cells in non-treated and DEN-treated mice. Bars represent average percentages and SD of cells positively staining for Ki67 cells examined in 10 HPF of sections from five different mice. Data are shown as means ± SD. Representative immunofluorescence staining of livers in 5.5-month-old DEN-treated and untreated lncHand2RFP reporter mice for indicated molecules (lower panel). Scale bar, 100 μm. Upper panel: scheme of targeting strategy for IRES-RFP knockin allele.