EMBO/sd-character-level-ner huggingface dataset:
Filamentation in mutant DHFR strains is associated with strong SOS response.Figure 3(A) recA (B) recN genes measured by quantitative PCR when WT and mutant strains are grown in M9 medium with or without supplementation of amino acids or dTMP. WT and mutant strains were grown for 4 hours of growth in the indicated medium at 37°C, while WT treated with different concentrations of Tmp were grown for 4 hours at 42°C. Brown bars (M9+AA) in the gray shaded area correspond to filamentation conditions and these are associated with pronounced upregulation of all three SOS genes. On the other hand, conditions with loss of filamentation (with dTMP or no supplementation) show much less expression. Error bars represent SD of 2-3 biological replicates (see Methods). sulA genes measured by quantitative PCR when WT and mutant strains are grown in M9 medium with or without supplementation of amino acids or dTMP. WT and mutant strains were grown for 4 hours of growth in the indicated medium at 37°C, while WT treated with different concentrations of Tmp were grown for 4 hours at 42°C. Brown bars (M9+AA) in the gray shaded area correspond to filamentation conditions and these are associated with pronounced upregulation of all three SOS genes. On the other hand, conditions with loss of filamentation (with dTMP or no supplementation) show much less expression. Error bars represent SD of 2-3 biological replicates (see Methods). Treatment of WT E. coli cells with sub-MIC concentration of Tmp (0.5μg/ml) leads to filamentation at 42°C when grown in amino acid supplemented medium. However, a recA knock-out strain under similar condition shows no elongation, indicating the role of SOS pathway in filamentation. A sulA knock-out continues to elongate, indicating the role of sulA-independent pathways. An E. coli strain deleted for the e14 prophage region however showed no filamentation upon Tmp treatment, indicating that sfiC gene in the e14 region might be one such sulA independent player. The central band in the box plots represents the median of the distribution, the box ends represent the 25th and 75th percentile, the whiskers represent the 10th and 90th percentile, while the dots represent the 5th and 95th percentile. Data was usually obtained from 2-3 biological replicates. The number of cells used to derive the boxplot distributions in the different panels range usually between 100 to 200 (please refer to Figure 3 source data for exact number of cells for each dataset).
Vesicle accumulation test and kinetic analysis of prAPI import in the temperature conditional Apg9p mutant.Figure 9Vesicle accumulation test and kinetic analysis of prAPI import in the temperature conditional Apg9p mutant. A, Morphological analysis of apg9Δ indicates an import defect at an early step in the pathway. Wild-type (YW5-1B) and apg9Δ (CTD1) cells were grown in YPD (vegetative) or transferred to SD(−N) (starvation) for 3 h in the presence of PMSF (starvation) and examined by DIC (Nomarski) microscopy (Zeiss Axioplan). PMSF inhibits the degradation of autophagic bodies. Under starvation conditions, wild-type cells accumulate autophagic bodies when vesicle breakdown is blocked. However, under the same conditions, apg9Δ cells do not accumulate autophagic bodies, indicating that Apg9p is required for a step before vesicle fusion and release of the autophagic body into the vacuolar lumen. The apg9Δ strain transformed with the APG9 plasmid shows the same result as wild-type (data not shown). B, The apg9ts strain is tightly blocked for prAPI import at nonpermissive temperature. Wild-type (SEY6210) and apg9Δ (JKY007) cells transformed with the apg9ts centromeric plasmid were incubated at 24 and 38°C for 5 min, pulse-labeled for 10 min, and then subjected to nonradioactive chase reactions for the indicated times. Samples at each time point were immunoprecipitated with antiserum to API and resolved by SDS-PAGE as described in Materials and Methods. API-immunoprecipitated bands were quantified by a Molecular Dynamics STORM PhosphorImager and the results are presented in the graph. The percent mature API was determined by dividing the mature API value over the sum of the precursor and mature API values for each time point.
Figure 6Figure 6. Helix-7 mutants display reduced target-binding rates. A) Ago2-miR122 complexes were mixed with a 32P-labeled seed matched target RNA; bound and free RNAs were then separated using a filter-binding apparatus at various times. Representative time course for target RNA (0.1 nM) binding to wild type, MI-AA, and Δhelix-7 Ago2 (1 nM). Average values from at least three independent experiments ± SD are plotted. B) Observed binding rates (kon, obs) plotted as a function of Ago2-miR122 concentration. Average values ± SD are plotted. C) Arrival time distribution of single Ago2-miRNA complexes binding immobilized targets in a microfluidic chamber. The value is the average of three independent measurements.
AMPK suppresses HSF1 activation through physical interaction.Figure 2A-C Reporter cells were treated with and without 1 μM A-769662 for 3 h followed by heat shock at 43°C for 30 min and recovery at 37°C for 5 h. Hspa1a and Hspb1 mRNA levels were quantitated by qRT-PCR (mean ± SD, n = 3, Student's t-test, ***P < 0.001). ACC phosphorylation was detected by immunoblotting (C). D-F Primary MEFs were derived from Ampkα1fl/fl; Ampkα2fl/fl embryos and transduced with either adenoviral GFP or Cre to delete Ampkα1/2 in vitro. AMPKα levels were detected 4 days after transductions (D) Following viral transductions, Hsp mRNA levels were quantitated by qRT-PCR in MEFs with and without 10 mM metformin treatment overnight followed by 43°C heat shock for 30 min and recovery for 4 h (mean ± SD, n = 3, one-way ANOVA, n.s., not significant, *P < 0.05, **P < 0.01, ***P < 0.001). G Transcriptional activities of HSF1 were measured by a dual reporter system consisting of two plasmids: the HSF1-dependent reporter pHSE-SEAP, in which ideal HSEs drive the expression of secreted alkaline phosphatase (SEAP), and the control reporter pCMV-Gaussia Luc, in which a CMV promoter drives the expression of secreted Gaussia luciferase. In HEK293T cells, either LacZ or GST-AMPKα1CA plasmid was co-transfected with the two reporter plasmids. After 24 h, the transfected cells were heat-shocked at 44°C for 45 min. The culture supernatants were collected to measure SEAP and luciferase activities 24 h after heat shock. SEAP signals were normalized to Gaussia luciferase signals (mean ± SD, n = 5, Student's t-test, **P < 0.01, ***P < 0.001). H, I Following treatment with 10 mM metformin overnight, endogenous AMPKα and HSF1 proteins were co-precipitated using anti-AMPKα agarose conjugates from lysates of immortalized Hsf1+/+MEFs (H). HC: heavy chain. Endogenous HSF1-AMPKα interactions in immortalized Hsf1+/+ and Hsf1−/− MEFs were visualized in situ by PLA (I). Experimental details are described in Materials and Methods. Scale bars: 50 μm.
Figure 4(A) Representative confocal images of APP-DKO cells expressing GFP-tagged C99WT or C99MUT (in green) and fluorescent markers of mitochondria (MitoDsRed, in red) and ER (Sec61β-BFP, in blue), and treated with DAPT to prevent C99 cleavage. Note the different distribution of C99WT or C99MUT forms. Scale bar = 10 µm. Insets show 5X amplifications of individual (C99 in green, and mitochondria in red) and merged images. Black and white bottom panels represent the areas where C99 and mitochondria signals colocalize. Upon thresholding each channel, a mask for mitochondria was generated and the C99 channel was superimposed on the mitochondria mask, so the positive pixels found in both channels are shown in black. (B) Quantification of the degree of colocalization of C99WT or C99MUT forms with mitochondria or ER measured by ImageJ. Note the significantly decreased colocalization between C99MUT and mitochondria, when compared to that of C99WT. Unpaired t-test. (20-30 cells/condition from 3 independent experiments and 4-5 images/experiment; * p<0.05; n.s., not significant). (C) Representative confocal images of APP-DKO cells expressing GFP-tagged C99WT or C99MUT and ER and mitochondria markers as in (A) (upper panel). Individual zoomed single channels (middle panel) as well as areas of ER-mitochondria colocalization (bottom panel) are shown for each condition. Scale bar = 10 µm. (D) Quantification of ER-mitochondria colocalization per cell analyzed. Upon thresholding each channel, a mask for ER was generated and the mitochondria channel was superimposed on the ER mask, so the positive pixels found in both channels were quantified and referenced over the total area of the ER mask. The number of cells analyzed is indicated in white text within each column. Levels of ER-mitochondria colocalization in APP-WT cells are shown for reference as a dashed line. EV, empty vector. One-way ANOVA (n=3 independent experiments and 5-8 images/experiment; * p< 0.05).
Figure 2Cells were pretreated with the indicated concentrations of aprotinin (A) and SB412515 (B), which are inhibitors of TMPRSS2 and cathepsin L, respectively. Infection of Calu-3, Caco-2, A549*, and Vero cells with SARS-CoV-2 at MOIs of 0.3, 0.4, 0.2, and 0.3, respectively, was achieved in the continuous presence of the drug. Infected cells were quantified by flow cytometry as described in Fig. 1D, and data were normalized to samples where inhibitors had been omitted. n = 2-4 biological replicates. SARS-CoV-2 particles were bound to A549* and Vero cells (MOIs 0.2 and 0.3, respectively) (C) or Calu-3 and Caco-2 cells (0.6 and 0.5, respectively) (D) on ice for 90 min and subsequently warmed rapidly to 37°C to allow infectious penetration. SB412515 (10 µM, C) or aprotinin (30 µM, D) was added at different times post warming to block further proteolytic activation. Infection was analyzed by flow cytometry, and data were normalized to samples where protease inhibitors had been omitted. n = 2.
Effect of syk deletion on B‐cell stimulation A Flow cytometric analysis of IgM, CD19 and CD23 expression of splenic B cells from Tam‐treated mb1‐CreERT2 control (dashed line) and mb1‐CreERT2;Sykfl/fl mice (solid line).Figure 3A Flow cytometric analysis of IgM, CD19 and CD23 expression of splenic B cells from Tam‐treated mb1‐CreERT2 control (dashed line) and mb1‐CreERT2;Sykfl/flmice (solid line). B Flow cytometric analysis of tyrosine phosphorylation (pY) in splenic B cells from Tam‐treated mb1‐CreERT2 and mb1‐CreERT2;Sykfl/flmice stimulated with medium (dotted line) or 50 μM pervanadate (dashed and solid lines). C, D Flow cytometric analysis of the intracellular Ca2+ influx in purified splenic B cells derived from Tam‐treated mice. mb1‐CreERT2 or mb1‐CreERT2;Sykfl/fl were treated with (C) 10 μg/ml anti‐Kappa F(ab′)2 fragments or (D) 1 μM ionomycin.
Figure 2Sagittal section of adult mouse brain showing lacZ expression driven by EE2. HC, hippocampus. EE2-driven expression in rostral migratory stream (black arrow) and in subventricular zone (white arrow). Scale bar 760 µm. Adult mouse spinal cord, EE2 and (D) EE3 expression pattern. Dorsal horns, laminae I-III (black arrows) and central canal (arrowheads). LacZ staining. Dashed lines in (C) indicates the region of dorsal horn shown in (D), (F) and (G). Inlets in (C) and (E) show POLG immunohistochemistry of dorsal horn and central canal (grey arrow), respectively. Scale bars: 100 µm if not indicated otherwise. (F)-(G) Calbindin (red) and β-Gal (green) expression in interneurons of the dorsal horn laminae I-III of (F) EE2 and (G) EE3 transgenic adult mice. Immunofluorescence. Scale bars 20 µm.
regulates neurogenesis through TDG (a) GO analysis of 215 significantly downregulated genes (Log2 fold change < -1, -P value < 0.Figure 6(a) GO analysis of 215 significantly downregulated genes (Log2 fold change < -1, -P value < 0.05) (b) GO analysis of 197 significantly upregulated genes (Log2 fold change > 1, -P value < 0.05) (c) Heat map of significantly different genes. (d) TDG is significantly decreased after TCF20 deletion. (e) Volcano map showing that TDG is significantly decreased. (f, g) Electroporation of GFP at E13.5 to E16.5 in TCF20 KO mice impairs cell distribution. n=3 brains. Scalebar 25 μm Data information: Error bars represent the means ± S.E.M. Two tailed unpaired T-tests were used to analyze the data, n.s. (no significant difference), p<0.05(*), p<0.01(**). (h) Chip-qPCR of TCF20 binding to the TDG promoter. n= 4 biological repeats. Data information: Error bars represent the means ± S.E.M. Two tailed unpaired T-tests were used to analyze the data, n.s. (no significant difference), p<0.05(*), p<0.01(**). (i) Combining RNA-seq and TDG ChIP-seq data, TCF-4 is a downstream target gene.
OCRL deficiency leads to perinuclear lysosome positioningA NHF, Lowe 1676 and Lowe 3265 cells were grown in complete media or 5 h serum starvation, then immunostained with LAMP1 antibody.Figure 1A NHF, Lowe 1676 and Lowe 3265 cells were grown in complete media or 5 h serum starvation, then immunostained with LAMP1 antibody. Dashed lines in images outline cell boundaries. Scale bar, 10 μm. C Quantitative analyses of lysosome perinuclear distribution in the experiments shown in A. The intracellular distribution of LAMP1 positive vesicles was quantified as described in the Methods. n = 3 per group in which 30 cells were counted per condition per experiment. Data information: data are presented as mean ± SEM. * ≤ 0.05, ** ≤ 0.01, ***≤ 0.001, ****P ≤ 0.0001, n.s., not significant, calculated by two-tailed Student's t-test. B Wildtype (Ocrl+/y) and Ocrl-/- MEF cells were grown in complete media or 5 h serum starvation, then immunostained with indicated antibodies. Dashed lines in images outline cell boundaries. Scale bar, 10 μm. D, E Quantitative analyses of lysosome and mTOR perinuclear distribution in the experiments shown in B. The intracellular distribution of mTOR positive vesicles was quantified as described in the Methods. n = 3 per group in which 30 cells were counted per condition per experiment. Data information: , data are presented as mean ± SEM. * ≤ 0.05, ** ≤ 0.01, ***≤ 0.001, ****P ≤ 0.0001, n.s., not significant, calculated by two-tailed Student's t-test. F, Representative images of the trajectory of GFP signals from NHF (n= 4) and Lowe 1676 (n= 4) cells transfected with lysosome-GFP. Trajectories were obtained as described He et al., 2016() and labeled in different colors with the same colored circles showing the start of the track. G. Measured MSD (⟨∆r2 (t)⟩) as a function of delay time t for the mobile lysosome trajectories taken at F. H Diffusion coefficient of lysosome in the experiments shown in G. Means ± SEM of n = 4 cells per group. I Snap images of WT (Ocrl+/y), OCRL knockout (Ocrl-/-) and IOB MEFs transfected with lysosome-GFP. After transfection, WT, OCRL knockout and IOB MEFs were left in serum starvation for 8 h. Scale bar, 5 μm. J Quantitative analyses of tubulated lysosomes in the experiments shown in H. n = 3 per group in which 10 cells were counted per condition per experiment. D Data information: data are presented as mean ± SEM. * ≤ 0.05, ** ≤ 0.01, ***≤ 0.001, ****P ≤ 0.0001, n.s., not significant, calculated by two-tailed Student's t-test.
Autophagy evaluation in motor cortex and lumbatr spinal cordA) Analysis of LC3-II and P-mTOR by western-blot in the motor cortex of control and treated animals.Fig 3A) Analysis of LC3-II and P-mTOR by western-blot in the motor cortex of control and treated animals. Proteins were detected by Western blotting using β-tubulin for calibration of sample loading. The protein bands were quantified by Image Multigauge v3.0 software. Immunoblots are representative of one of three experiments with similar results. These results were quantitated in arbitrary units and were represented in the accompanying graphs as mean ± SEM. *, ** Indicate differences of p-value <0.05 <0.01 with the control group, respectively. #, ### indicate differences of p-value <0.05 <0.001 with the group of animals treated with L-BMAA, respectively. B) Analysis of LC3-II and P-mTOR by western-blot in the lumbar spinal cord of control and treated animals. Proteins were detected by Western blotting using β-tubulin for calibration of sample loading. The protein bands were quantified by Image Multigauge v3.0 software. Immunoblots are representative of one of three experiments with similar results. These results were quantitated in arbitrary units and were represented in the accompanying graphs as mean ± SEM. *, *** Indicate differences of p-value <0.05 <0.001 with the control group, respectively. ## Indicate differences of p-value <0.01 with the group of animals treated with L-BMAA.
TREM2 vs.Figure 4Effects of sTREM2 on AV1451-PET-assessed tau pathology stratified by Braak-stages shown as boxplots (central band = median, boxes = Quartiles, whiskers = 1.5 * interquartile range).
Figure 7Representative IF images showing the average intensity of endogenous RNF168 at UV-A micro-laser-induced DNA damage tracks in U2OS cells. (Scale bar; 10µm).   Graph represents the quantification of (A). (***P<0.001; unpaired t-test, n=3, mean +SEM, more than 50 nuclei were analysed).   Representative IF images showing the average intensity of endogenous 53BP1 at UV-A micro-laser-induced DNA damage tracks in U2OS cells. (Scale bar; 10µm).   Graph represents the quantification of (C). (ns P>0.05, ***P<0.001; unpaired t-test, n=2, mean +SEM, at least 80 nuclei per condition and experiment).   Western blot analysis of Flag-histone H1 denaturing-IP, showing ubiquitination pattern of histone H1 after 30 min of IR (10 Gy) treatment under indicated siRNA-depleted conditions. Representative IF images showing the average intensity of endogenous BRCA1 at UV-A micro-laser-induced DNA damage tracks in U2OS cells. (Scale bar; 10µm).   Graph represents the quantification of (F). (ns P>0.05, ***P<0.001; unpaired t-test, n=2, mean +SEM, at least 100 nuclei per condition and experiment).
Figure 2Mice received daily intraperitoneal (IP) injections of A151 (300 μg) or an equal volume of vehicle for 3 consecutive days after MCAO induction. (A) The mRNA expression levels of the cGAS and STING after MCAO were determined using qRT-PCR. n = 6 mice per group. ** (B) The expression levels of cGAS, STING, and NF-κB proteins in the brains of indicated groups were measured using Western blot analysis. (C) Bar graph shows the relative protein expression levels of cGAS, STING, and NF-κB in the brains of indicated groups. n = 6 mice per group. (D) The mRNA expression levels of the AIM2 inflammasome components (AIM2, ASC, and caspase-1), effector of pyroptosis GSDMD, IL-1β, and IL-18 after MCAO were determined using qRT-PCR. n = 6 mice per group. (E) The expression of AIM2 inflammasome components, GSDMD, and IL-1β in the brains of indicated groups was measured using Western blot analysis. (F) Bar graph shows the relative protein expression levels of AIM2 inflammasome components, GSDMD, and IL-1β in the brains of indicated groups. n = 6 mice per group.
Figure 1(A) The expression of AR was determined using Western blots (overexpressing AR in J82 cells and knocking down AR in UMUC3 cells). (B) Relative expression of the top 20 up-regulated circRNAs from the GEO dataset GSE92675 (BCa tumor tissues compared to para-tumor tissues) in response to AR modulation in J82 and UMUC3 cells. The # indicates an RNA with a relatively low expression (qRT-PCR cycle number > 35), thus the circRNA was excluded for further analysis. (C) Schematic illustration showing the 4 circRNAs identified that could be regulated by AR. (D) Schematic illustration showing the position of the targeting shRNA to knock down the circRNAs (sh-circ) by targeting the specific 5'-3' splice junction. (E) Real-time quantitative PCR (qRT-PCR) results show the knock down efficiency for these 4 circRNAs in J82 oe-AR (J82-AR) cells. (F) Transwell invasion assays showed that knock down of hsa_circ_0084171 (circ_oo84171), but not the other circRNAs, can partly reverse oeAR-increased invasion in J82 cells. Scale bar, 100 μm. (G) MTT assays showed that shcirc_0084171 can partly reverse oeAR-increased cisplatin chemo-resistance (2 μg/ml) in J82 cells. To calculate the mean, the value of 0 µg/ml cisplatin groups were set as 1. (H) The qRT-PCR results showed that the overexpression (oe) efficacy for hsa_circ_0084171 (oecirc_0084171) in UMUC3-shAR cells. (I) Transwell invasion assays showed that oecirc_0084171 can partly rescue shAR induced invasion deduction in UMUC3 cells. Scale bar, 100 μm. (J) MTT assays showed that oecirc_0084171 can partly overcome shAR effect for cisplatin chemo-resistance (2 μg/ml) in UMUC3 cells.
Figure 7(A) Volcano plot of all detected proteins from proteomics of TGFβ-treated naïve (WT) and COUP-TFII overexpressing (COUP-TFII-OE) cells. PDK4 expression is significantly decreased in OE cells (n=2). (B) Overexpression of COUP-TFII inhibited, and knockout of COUP-TFII increased, mRNA of PGC1α and PDK4 (n=6). ***p<0.001, ****p<0.0001 by one-way ANOVA, mean ± SD. (C) Chip-qPCR analysis on C3H10T1/2 cells in vitro revealed binding of COUP-TFII on the promoter of PGC1α, but not αSMA or PDK4 (n=2). **p <0.01, mean ± SD. (D) Overexpression of PGC1α was achieved through adenovirus transduction in C3H/10T1/2 cells (WT) and confirmed by qRT-PCR (n=6). Noted TGFβ1 significantly decreased PGC1α expression. ***p<0.001, ****p<0.0001 by one-way ANOVA, mean ± SD. (E) PGC1α OE significantly increased expression of Cpt1 and PDK4, both are involved in key steps of FAO. However, overexpression of PGC1α did not abrogate the TGFβ1 up-regulated genes in glycolysis and αSMA (n=6). *p<0.05; **p<0.01; ****p<0.0001 by one-way ANOVA, mean ± SD. (F) PGC1α OE did not decrease αSMA protein neither without nor with TGFβ1 in COUP-TFII OE cells by WB (n=3). (G) Schematic model of the role of COUP-TFII in metabolic reprogramming during myofibroblast differentiation and fibrosis formation after injury.
Figure 5A. Protein sequences were extracted from UniProt accession number BP06_BPT4 for Enterobacteria phage T4 (gp6; Bacteriophage T4) and the Pseudomonas Genome Database (TssA1; reference strain PAO1). Conserved positions are shown in black and grey background. The secondary structural elements corresponding to the 3D structure of gp6 (PDB code 3H2T) are shown above the alignment. Regions with significant sequence conservation are indicated with a corresponding block number. B. Cartoon representations of the dodecameric gp6_334C structure (PDB code 3H3W). Chains A and B of gp6 dimers are shown in magenta and green respectively, and the conserved blocks mapped onto the assembled dodecameric gp6 structure are shown in orange. A close up view showing the conserved blocks present in regions of gp6 involved in ring assembly, is shown on the right.
A) Assessment of the detection limit of synthetic SARS-CoV-2 by the CRISPR-based assay, inferred from fluorescence values after 30 min.Figure 2.A) Assessment of the detection limit of synthetic SARS-CoV-2 by the CRISPR-based assay, inferred from fluorescence values after 30 min. Background subtracted fluorescence represents sample minus control fluorescence without target. Detection of synthetic SARS-CoV-2 RNA using commercially available paper strips (lateral-flow) in buffer (B) Detection of synthetic SARS-CoV-2 RNA using commercially available paper strips (lateral-flow) in saliva (C).