EMBO/sd-character-level-ner huggingface dataset:
AtNTR1 mutation results in reduced seed dormancy, low DOG1 expression and a tendency towards downstream splice site selection A, B Photographs (A) and quantification (B) of seed dormancy tests.Figure 1A, B Photographs (A) and quantification (B) of seed dormancy tests. The chart represents the average percentage of germinated seeds without stratification after 4 days of growth in LD. The error bars represent ± SE (n = 3). Tests were performed on freshly harvested seeds, with or without 3 days of stratification growth in LD. C qPCR of DOG1 expression in siliques (16 days after pollination). The graph represents the average ratio of DOG1 to UBC, normalised to Col‐0 (WT). The error bars represent ± SE (n = 3). D DOG1 splicing was assessed by RT-PCR combined with capillary electrophoresis. The graph represents the mean relative contribution of the mRNA forms found in the total pool of amplified products. The black and grey bars represent the data for Col‐0 and atntr1‐1, respectively. The error bars represent ± SD (n = 3). To the right of the charts, the structures of the examined transcripts are shown (black boxes, constitutive exons; white boxes, alternative regions; black lines, introns). The black arrows show the locations of primers. Downstr. and upstr. stand for downstream and upstream splicing event, respectively.E Directionality of splice site selection in atntr1. Splicing was analysed in 14‐day‐old MS grown plants. For each type of alternative splice event, the black and white boxes show the contributions of opposite direction splicing events. The numbers represent the percentage of splice events supporting the direction of the splice site event change (also shown on horizontal axis). The numbers on the right‐hand panel represent the number of affected splicing events versus total number of splicing events analysed. The white bars represent distal 3′ and downstream 5′ splice site selection (3′SS/5′SS), exon skipping (ES) and intron retention (IR), while the black bars represent 5′ and 3′ splice site selection (3′SS/5′SS), exon inclusion (ES) and intron splicing (IR).
a) Rapamycin (Rap) treatment or nutrient deprivation attenuated the expression of the Wnt reporter TopFlash in HEK-293T cells.figf1a) Rapamycin (Rap) treatment or nutrient deprivation attenuated the expression of the Wnt reporter TopFlash in HEK-293T cells. (n = 3; two asterisks, P 0.01). DMSO, dimethylsulphoxide. (b) Expression analysis of Wnt target genes by quantitative RT-PCR after 4 h of treatment with rapamycin. (n = 3; asterisk, P 0.05; two asterisks, P 0.01). Wnt3a CM, Wnt3a conditioned medium. (c) Knockdown of LC3 or Beclin 1 potentiated Wnt signalling. (n = 3; asterisk, P 0.05). (d) Atg5 knockout enhanced Wnt signalling. TopFlash reporter activity induced by Wnt3a CM in Atg5−/− or wild-type MEFs. Results are means ± s.d. after normalization to Renilla luciferase activity. (n = 3; asterisk, P 0.05).
Figure 5A. Heatmap showing motif activity inferred from differential chromatin accessibility of stage 10½ - AC -DMZ. B. Heatmap showing motifs identified based on regulatory regions closest to cluster-specific genes, and regression to cluster gene expression. C. Heat map of transcription factor (TF)-motif combinations showing cluster-specific motif activity (z-score, color) and gene expression (size of dot). Motifs and the Motif-TF combinations were hierarchically clustered.
a) Upregulation of the levels of LC3-II and Thr 145-phosphorylated EndoB1 in the cerebellum and striatum of α-synucleinA53T transgenic mice.figf6(a) Upregulation of the levels of LC3-II and Thr 145-phosphorylated EndoB1 in the cerebellum and striatum of α-synucleinA53T transgenic mice. Right panels show quantified data as means ± s.e.m.; n=3. b) Overexpression of α-synucleinA53T mutant upregulates LC3-II level and Thr 145-phosphorylated EndoB1 in cultured neurons. Lower panel shows quantified data as means ± s.e.m.; n=3. (c) Knockdown of EndoB1 or Cdk5 inhibits α-synucleinA53T mutant-induced autophagy induction in neurons. Data are means ± s.e.m.; n=7. (d) Cdk5 mediates α-synucleinA53T mutant-induced Thr 145 phosphorylation of EndoB1 and the increase of LC3-II in neurons. (e) Overexpression of EndoB1T145A attenuates α-synucleinA53T mutant-induced autophagy in neurons. Data are means ± s.e.m.; n=7. Uncropped images of blots are shown in Supplementary Fig. S8.
Correlation of root Na+/K+ ratio with salt resistance during tomato domestication.Figure 1A Distribution of 369 accessions, including PIM (36 S. pimpinellifolium accessions), CER (118 S. lycopersicum var. cerasiforme accessions), and BIG (215 S. lycopersicum accessions). B The Na+ and K+ contents and Na+/K+ ratio in roots and shoots of three groups of 369 accessions after treatment with 150 mM NaCl for 1 d. C Linear regression of root Na+/K+ ratio under salt stress (150 mM NaCl for 1 d) and fruit weight in 195 accessions from three groups. R2, coefficient of determination. n is the accession number of the three groups. D The Na+/K+ ratios in roots of TS-21, TS-422, TS-577, and TS-670 after treatment with 150 mM NaCl for the indicated time (n = 3 biological repeats). E, F Salt-resistance assay of two PIM accessions (TS-21 and TS-422) and two BIG (TS-577 and TS-670). 20-day-old seedlings grown in soil were treated with 200 mM NaCl for 3 weeks (E). The survival rates were obtained from at least 12 plants in three repeated experiments (F).
PGS1 depletion rescues mitochondrial fragmentation in OPA1-deficient human and mouse fibroblasts (A) Representative confocal images of control (CTL-1) and OPA1S545R patient fibroblasts treated with OPA1, DNM1L, PGS1, and non-targeting (NT) siRNAs or indicated combinations for 72 hours.Figure 3Representative confocal images of control (CTL-1) and OPA1S545R patient fibroblasts treated with OPA1, DNM1L, PGS1, and non-targeting (NT) siRNAs or indicated combinations for 72 hours. Mitochondria (anti-TOM40, green) and nuclei (DAPI, blue). Scale bar=20μm. Passages number between P10-15. Mitochondrial morphology quantification of (A) using control fibroblasts with fragmented (OPA1 siRNA), normal (non-targeting NT siRNA), and hypertubulated (DNM1L siRNA) mitochondria. Data represent mean ± SD of three independent experiments, One-way ANOVA (905-3695 cells per cell line), (% fragmented); ; ****p < 0.0001, ns; not significant.. Representative confocal images of wild type (WT) and Opa1Crispr MEFs treated with NT or Pgs1 siRNA for 72 hours. Live imaging of mitochondria (mitoYFP, green) and nuclei (NucBlue, blue). Scale bar=10μm. Mitochondrial morphology quantification of (C) using WT MEFs treated with Opa1 siRNA (fragmented), NT siRNA (normal), or Dnm1l siRNA (hypertubulated) ground truth training sets. Data represent mean ± SD of three independent experiments, One-way ANOVA (6613-8758 cells per cell line), (% fragmented) ; ****p < 0.0001, ns; not significant. Representative confocal images of WT, Opa1Crispr MEFs complemented with pLenti-Opa1, Opa1CrisprPgs1Crispr MEFs and Pgs1Crispr MEFs complemented with pLenti-Pgs1 by lentiviral delivery. Live imaging of mitochondria (mitoYFP, green) and nuclei (NucBlue, blue). Scale bar=10μm. Supervised ML mitochondrial morphology quantification of (E) using WT MEFs treated with Opa1 siRNA (fragmented), NT siRNA (normal), or Dnm1l siRNA (hypertubulated) training sets. Data represent mean ± SD of three independent experiments, One-way ANOVA (691-3990 cells per cell line), (% fragmented) ; ****p < 0.0001, ns; not significant.. (G) Equal amounts of protein extracted from MEFs were separated by SDS-PAGE, immunoblotted with anti-OPA1 antibody, and quantified (H) by densitometry relative to Stain-Free. Data represent mean ± SD of three independent experiments, One-way ANOVA; *p < 0.05, ***p < 0.001, ****p < 0.0001, ns; not significant..
a) HCT116.figf4(a) HCT116.vector and HCT116.UVRAG cells were labelled with LysoTracker and subjected to flow cytometry analysis. HCT116.vector, HCT116.UVRAG, and HCT116.UVRAG ΔCCD cells were treated with or without wortmannin (1 μM) for 2 h and their lysates were used to measure the lysosomal acid phosphatase enzymatic activity. Data represents mean ± s.d. of three experiments. (b) UVRAG induces autophagosome formation in NIH3T3 cells. After transfection with GFP-LC3 vector, NIH3T3.vector and NIH3T3.UVRAG cells were maintained under normal conditions, starved for 3 h in the absence or presence of 10 mM 3-methyladenine or treated with or without 2 μM rapamycin or rapamycin + 10 mM 3-methyladenine. Data represents mean ± s.d. of three experiments.
Hypothetical models describing the formation of double membrane vesicles and their possible role in viral RNA replication.Figure 9(A) By analogy to flaviviruses [9] HCV proteins induce invaginations of the ER membrane. Extensive invagination leads to a local 'shrinking' of the ER lumen. This model assumes that enzymatically active HCV replicase (green dots) reside in the lumen of the invagination and remain active as long as the vesicle is linked to the cytosol. Upon closure of the DMV, the replicase would become inactive (grey dots). Alternatively, closed DMVs might be connected to the cytosol via proteinaceous channels. (B) HCV proteins might induce tubulation of ER membranes that undergo secondary invagination and thus double membrane wrapping. These DMVs could initially be open to the cytosol, but might close off as replication/infection progresses. The resulting DMV might stay connected to the ER via a stalk or be released as a 'free' DMV (left or right drawing, respectively). (C) Induction of DMVs follows the same pathway as described for panel B, but the viral replicase remains on their cytosolic surface as discussed e.g. for the poliovirus [28], [29]. (D) HCV RNA replication might occur on SMVs in close proximity of DMVs. In this case, DMVs might be an epiphenomenon or serve some other purpose for the HCV replication cycle. For each model, structures identified in the 3D reconstructions are shown next to or below the corresponding schematic drawing. For further details see text.
A) Immunoblot analysis of BAG1, BAG3, Hsc/Hsp70 and Hsp90 in young and old I90 cells.Figure 1(A) Immunoblot analysis of BAG1, BAG3, Hsc/Hsp70 and Hsp90 in young and old I90 cells. For detection of BAG proteins, an antibody directed against the conserved BAG domain was used. (B) Real‐time PCR analysis of human BAG family members and Hsc70 and Hsp90 in young and old I90 cells. Depicted is the expression ratio (log2) of target genes in old cells relative to young cells. *P0.05 and **P0.01 versus young, n=3. (C) Co‐IP analysis of Hsc/Hsp70 interaction with BAG1 and BAG3 in young (Y) and old (O) I90 cells. Upper panel shows relative amounts of proteins in cell lysates used for Co‐IP (Input). Middle panel shows levels of BAG1 and BAG3 found in Hsc/Hsp70 immunoprecipitates. Lower panel shows levels of Hsc/Hsp70 co‐sedimented upon immunoprecipitation of BAG3. (D, E) 293 cells were treated with the indicated agents for 8 h followed by immunoblot analysis of indicated proteins.
Figure 1Schematic representation of the strategies for fluorescence-activated cell sorting (FACS) and transcriptomic analyses. During inducing hematopoietic differentiation, human embryonic stem cells (hESCs, H1) on day 0, FACS-purified lateral plate mesodermal APLNR+ cells on day 2, purified CD31+CD34+ endothelial progenitor cells (EPCs) on day 5, and purified CD43+ hematopoietic stem and progenitor cells (HSPCs) on day 8 were collected for RNA-Seq, respectively. STAR Cufflinks, MISO, DESeq2 and rMATS were used to analyze the expression abundance of genes and transcripts, alternative splicing events, differentially expressed genes and transcripts, and differential splicing events, respectively. n = 3 technical replicates. Cumulative distribution curves of Log2(FPKM) of splicing factors (n=235) in hESCs APLNR+, CD31+CD34+, and CD43+ cells. The upper and lower dotted lines represent cumulative scores of 1 and 0, respectively. n=3 technical replicates. The P-value was calculated by a two-tailed Wilcoxon signed-rank test. The heatmap illustrates the row normalized expression of components within the major spliceosomal machinery (n=182) using unsupervised hierarchical clustering. The heatmap was scaled with Z-Score using the log2(FPKM) expression of components within the major spliceosomal machinery. n=3 technical replicates. The boxplots depict the expression of splicing factors in cluster 1 (n=115) and cluster 2 (n=67) identified in (C) at distinct differentiation stages. The central band indicates the median level of expression. Boxes presents 25%-75% of genes expression. The whiskers indicate the lowest and highest points within 1.5× the interquartile. n=3 technical replicates. P-values were calculated by a two-tailed Wilcoxon signed-rank test. The heatmap shows the dynamic expression of genes in the SF3A/3B complex (E) and SR family (F) at each indicated differentiation stage. Both heatmaps were row normalized. The heatmaps were scaled with Z-Score using the log2(FPKM) expression of indicated components. n=3 technical replicates. The mRNA expression of splicing regulator SRSF2 and splicing factor PCBP2 during hematopoietic differentiation were measured by RT-qPCR. The ACTB gene was used as a control. Results given are mean ± standard deviation (SD). P-values were determined by an unpaired two-tailed Student's t-test. n ≥ 3 biological replicates. The number of differentially expressed transcripts at the isoform level (fold change (FC) > 2 & FDR < 0.01), but not at the gene level (FDR > 0.01), by comparing two consecutive differentiation stages (hESC vs APLNR+ cells, APLNR+ cells vs CD31+CD34+ cells, and CD31+CD34+ cells vs CD43+ cells).
Celf2a repression in GS∆44 is due to epigenetic silencing.Figure 2A iPSCs obtained from a control (WT#1; (Lenzi et al, 2015), GS∆44 (GS∆44#2, GS∆44#8) and GSM (GSM#1) were differentiated into myocytes by MYOD/BAF60c overexpression. Cells were collected before the induction (iPSC) and after 9 days in DM (Myo) and RNA was analysed by RT-PCR for CELF2a, CELF2b, CELF2c and DMD expression. GAPDH was used as loading control. Representative results are shown (n=3). B Representative immunofluorescence for Myosin Heavy Chain (MHC in red) in combination with DAPI staining (in blue) of iPSCs obtained from WT#1, GS∆44 (GS∆44#8) and GS∆44 edited clones (GS∆44#8.2) differentiated for 9 days into myocytes by MYOD/BAF60c overexpression. Scale bar 100 μm. C Histogram represents fusion index quantification. At least 7 randomly chosen microscope fields of two independent biological samples were analysed (n=2). Data are presented as mean ± SD of the biological replicates. D RNA extracted from myocytes obtained by differentiation of WT#1, GS∆44 (GS∆44#8) and GS∆44 edited clones (GS∆44#8.2 ∆CELF2a) was analysed by RT-PCR for CELF2a, CELF2b, CELF2c and DMD exon 45 skipping. GAPDH was used as loading control. The arrow indicates the DMD exon 45 skipped band. Representative results are shown (n=3). E Western blot on proteins (40 μg) extracted from myocytes obtained by differentiation of WT (WT#1), GS∆44 (GS∆44#8) and edited GS∆44 (GS∆44#8.2 ∆CELF2a) iPSC clones probed with antibodies against dystrophin (DMD). Actinin (ACTN) was used as a loading control. For the WT 5% sample, 2 μg of proteins were diluted in 38 μg of proteins belonging from GS∆44#8 to reach the total amount of 40 μg. Representative results are shown (n=3).
Figure 4(A, B) (A) RNA-Seq was performed in WT and MCK-KO soleus muscle at rest. The scatter plots show differentially expressed genes in MCK-KO soleus muscle at sedentary (A) and after a bout of low-intensity exercise for 50 min (B) (FDR<0.05, p < 0.05). (C) Proposed model for ROS regulation during loss of Dnmt3a. Loss of DNMT3A increases ALDH1L1 expression, thus leading to the increase in NADPH levels and increased activity of NADPH oxidase, leading to increased ROS levels. The increased oxidative stress contributes to mitochondrial dysfunction and muscle fatigue. (D) Q-PCR validation of Aldh1l1 at sedentary and after exercise for 50 min (n = 6, means ± SEM, * p < 0.05, two-tailed student's t-test). (E, F) ALDH1L1 protein expression and the quantification in WT vs. MCK-KO soleus, GA and EDL muscles at sedentary and normalizing to GAPDH using ImageJ. (G) The map of CpG-rich promoter regions of Aldh1l1 and MeDIP and ChIP primers (P1-P5) that cover the CpG rich regions. The numbers correspond to the position from the transcriptional start site of Aldh1l1. (H) MeDIP-qPCR was performed in WT and MCK-KO soleus muscle to assess differential methylation using primer sets from (G) (n = 4, means ± SEM, * p < 0.05, two-tailed student's t-test). (I) DNMT3A ChIP-PCR was conducted in WT and MCK-KO soleus muscles using primer sets from (G) (n = 3, means ± SEM, * p < 0.05, two-tailed student's t-test). (J) H3K27ac ChIP-PCR was conducted in WT and MCK-KO soleus muscles using primer sets from (G) (n = 3, means ± SEM, * p < 0.05, two-tailed student's t-test).
Figure 3 (A) Expression of RagC WT, 3A, or 3E was induced by overnight addition of tetracycline in HeLa cells followed by cell size analysis using a particle size counter (Horizontal lines, box limits and whiskers represent the median, the 25th percentile ot the 75th percentile and the minimum to the maximum respectively. * p<0.05, # no significant difference, One-way ANOVA with Bonferroni multiple comparisons, n=4)  (B) Phospho-RagC positively regulates wing compartment size in Drosophila. Over-expression (OE) of dRagC WT, 3A, or 3E in wing posterior compartment using en-Gal4 driver and the posterior compartment sizes were measured in each genotype ((Horizontal lines, box limits and whiskers represent the median, the 25th percentile ot the 75th percentile and the minimum to the maximum respectively. ** p<0.01, *** p<0.001, **** p<0.0001, One-way ANOVA with Bonferroni multiple comparisons, n ≥ 63 flies per condition)  (C) Empty vector (EV), RagC WT, 3A or 3E were transiently expressed in stable RagC knockdown HEK-293E cells followed by cell size analysis using a particle size counter (Horizontal lines, box limits and whiskers represent the median, the 25th percentile ot the 75th percentile and the minimum to the maximum respectively. * p<0.05, ** p<0.01, **** p<0.0001, # no significant difference, One-way ANOVA with Bonferroni multiple comparisons, n=2 for C2 and n=4 for other conditions)  (D) Empty vector (EV), HA-RagC WT, 3A or 3E were expressed in HEK-293E cells followed 48 hr later by incubation with DMEM in the presence or absence of serum for 4 hr. Lysates were analyzed by Western blot for phosphorylation of ULK1 and total ULK1  (E) Empty vector (EV), HA-RagC WT, 3A or 3E were expressed in HEK-293E cells followed 48 hr later by incubation with DMEM in the presence or absence of serum for 4 hr. Chloroquine (100μM) was added to media 30 min prior to harvest. Lysates were analyzed by Western blot for levels of LC3-II and 14-3-3.Graphs show mean ± S.E.M. of quantitative analyses of LC3-II blotting after normalization for 14-3-3. * p<0.05, two tailed Student's t test, n=4  (F) GFP-LC3 was co-expressed with empty vector, RagC WT, 3A or 3E in HEK-293E cells followed 48 hr later by serum deprivation for 4 hr. Chloroquine (100μM) was added to media 30 min prior to harvest. LC3 and RagC were monitored by confocal fluorescence microscopy. Scale bar, 10 µm  (G) Quantitative analysis of GFP puncta (F) (**** p<0.0001, two tailed Student's t test; n ≥ 21). Mean value is shown as a horizontal bar
The MSP domain of MOSPD2 binds the FFAT motif A, B: (A) Structural alignment of the MSP domains of MOSPD2 (PDB ID: 1WIC; green) and VAP-A in complex with the FFAT motif of ORP1 (PDB ID: 1Z9O; orange).Figure 2(A) Structural alignment of the MSP domains of MOSPD2 (PDB ID: 1WIC; green) and VAP-A in complex with the FFAT motif of ORP1 (PDB ID: 1Z9O; orange). The FFAT peptide is in blue (B) FFAT-MSP binding interface: the critical residues K94, M96 in VAP-A, and R404, L406 in MOSPD2 are shown as sticks  C: Sequence alignment of the MSP domains of MOSPD2, VAP-A, and VAP-B from human (Hs) and mouse (Mm). Numbers refer to amino acid positions. Red stars indicate the position of residues interacting with the FFAT motif in VAP-A [27]. Filled stars indicate the position of the two critical residues in VAP proteins (K94 and M96 in VAP-A; K87 and M89 in VAP-B) for FFAT interaction. Sub-regions with high homology between the three proteins are indicated with grey boxes (A-G)  D: Schematic representation of MOSPD2 showing the position of the RD/LD mutation of the MSP domain  E: Coomassie blue staining of recombinant wild-type and mutant MSP domains of MOSPD2, VAP-A, and VAP-B on SDS-PAGE. Note that the mutant MSP domains of MOSPD2, VAP-A and VAP-B displayed a slowed migration, likely resulting from the negative charges introduced by the mutations  F: Coomassie blue staining of proteins pulled-down with the control or the FFAT peptide. Representative illustration of at least two independent experiments
a) Graph representing fold change in mRNA levels in cells treated with Shh against untreated cells.f6(a) Graph representing fold change in mRNA levels in cells treated with Shh against untreated cells. Each circle represents an autophagy gene; those with largest fold-changes are indicated in red. The central line represents no changes in expression; above the central line, genes whose expression is increased: below, those with reduced levels. Grey lines indicate 2-fold increase or decrease. (b) Table showing the 10 genes with higher fold changes in expression and the values obtained. (c) Levels of PERK protein were assessed by western blotting in HeLa cells treated with 10 μM purmorphamine for 24 h. Actin was used as a protein loading control. (d) HeLa cells were treated with purmorphamine for 24 h and either left in rich media or in HBSS for the last 4 h. Levels of S51 phoshorylation and total levels of eIF2α were detected by western blotting using LI-COR infra-red imager. (e,f) Wild-type and PERK−/− (e) or eIF2α S51A/S51A MEFs (f) were treated with DMSO or 10 μM purmorphamine for 24 h and, where indicated, cells were treated with bafA1 for the last 4 h. tubulin-II and tubulin or tubulin, as a loading control, were detected and a representative blot is shown. HBSS, Hank's balanced salt solution.
Figure 4A-D Representative single particle tracks of cells expressing MIC60- (A) and MIC10-SNAP (B) in WT Hela cells and MIC60- (C) and MIC10-SNAP (D) in MIC13 KO Hela cells, stained with silicone-rhodamine dye, and imaged at a rate of 33 ms/frame. Single tracks were colour-coded according to temporal appearance. Scale Bar 5 µm. Insets in each image show zoomed-in images of few representative tracks. E Cumulative frequency of tracks having corresponding values of instantaneous diffusion coefficients (insD) in WT and MIC13 KO Hela cells expressing MIC60- and MIC10-SNAP stained with silicone-rhodamine. Number of tracks analysed (obtained from 2 independent experiments): n = 2541, 2540, 3560 and 1441 in WT Hela cells expressing MIC60-SNAP and MIC10-SNAP, MIC13 KO Hela cells expressing MIC60-SNAP and MIC10-SNAP, respectively. (****P ≤ 0.0001 for all possible comparisons, unpaired Student's t-test). F Percentage of subtracks having directed motion in WT and MIC13 KO Hela cells expressing MIC60- and MIC10-SNAP and in WT and MIC60 KO HAP1 cells expressing MIC10-SNAP. Number of corresponding subtracks analysed (obtained from 2 independent experiments): n = 2561, 2443, 3402, 1360, 561 and 1110. Data are mean ± SEM. *P ≤ 0.05, ***P ≤ 0.001, ****P ≤ 0.0001, unpaired Student's t-test.