EMBO/sd-character-level-ner huggingface dataset:
Figure 5(A) Left, atomic model of RAG TCC. One RAG1-RAG2 dimer omitted to allow visualization of target and RSS DNA. Right, expanded view surrounding flipped C-t-2 base with M848 in configuration 1. C-t-2 is potentially stabilized by hydrogen bonds with D600 and D708. Blue sphere, integration site phosphate; yellow sphere, RSS 3'-OH. (B) Diagram depicting the different configurations of M848 in the RAG TCC. In configuration 1, M848 would clash with C-t-2 in an intrahelical position. This clash is resolved when M848 adopts configuration 2. (C,D) Local structures of top and front views of RAG TCC (M848 configuration 1) near integration site, depicted as in Fig 3C, D. (E) Speculative model of DNA configurations in the RAG TCC. Because M848 can adopt two different configurations, C-t-2 has the possibility of assuming either an intrahelical or flipped configuration. (F) Schematic of the different amino acids side chains tested for in vitro transposition activity at RAG1 position 848. The relative transposition efficiency of each mutant compared with wild-type (Arg) is shown above the diagram of each residue (data derived from (Zhang et al., 2019)). Strongest activity was observed for methionine.
PC1-CTF/PC2_AA heteromeric channel has a different ion permeability to that of the PC2_AA homomeric channel.Figure 5A. Representative I-V curves of the PC2_AA and PC1-CTF/PC2_AA channels in divalent ion-free bath solutions containing 100 mM of indicated ions, showing the differences in reversal potential. B. Bar graphs showing the markedly different reversal potentials of PC2_AA channel and the PC1-CTF/PC2_AA channel in bath solutions containing 100 mM of the indicated ions. Oocyte numbers are indicated in parentheses. Data are presented as mean ± SD (n.s.: not significant, ***P < 0.001, Student's t-test). Bar graphs showing the permeability ratios of the indicated ions to that of Na+ (Px: PNa) (C) These ratios are strikingly different between the two channels. The ratios of the particular ions, which are the values of the bars, are indicted on top of the bars. Bar graphs showing the permeability ratios of the indicated ions to that of NMDG+ (Px: PNMDG) (D). These ratios are strikingly different between the two channels. The ratios of the particular ions, which are the values of the bars, are indicted on top of the bars.
A prolonged G1 arrest following CDK4/6 inhibition in RPE1 cells causes defects in the next cell cycle.Figure 1(A) Top panel displays structure of each CDK4/6 inhibitor tested. Bottom panel shows dose response curves with these inhibitors displaying percentage of G1-arrested RPE1-FUCCI cells. To obtain dose response curves, the number of mKO-Cdt1 positive (G1-arrested) cells were calculated following 24hr drug addition (dark blue solid lines) or 24h after subsequent drug washout (light blue dotted lines). Cmax values observed in patients (taken from (He et al., 2017; Klein et al., 2018)) are represented on each graph with red dotted lines. Graphs display mean data -/+ SEM from 3 experiments, with at least 500 cells counted per condition per experiment. (B) Percentage of G1-arrested RPE1-FUCCI cells, calculated as in panel A, but using a fixed concentration of CDK4/6 inhibitor for different durations of time, as indicated. Note, this is a fixed assay that quantifies the percentage of G1-arrested cells prior to, or 24 hrs following, CDK4/6 inhibitor washout. Each bar displays mean data + SEM from 3 experiments, with at least 500 cells counted per condition per experiment. ( (C) Cell cycle profile of individual RPE1-FUCCI cells (each bar represents one cell) after washout from 1 (top panel), 4 (middle panel) or 7 (bottom panel) days of treatment with CDK4/6 inhibitor, at the indicated doses (same concentration used in panel B). STLC (10 μM) was added to prevent progression past the first mitosis. 50 cells were analysed at random for each repeat and 3 experimental repeats are displ­­ayed (150 cells total). Underneath the single cell profiles are quantifications of the observed cell cycle defects and G1 durations. Note, G1 length is estimated by mKO-Cdt1 expression, and G1 lengths of less than 6h could not be quantified since movies were only stated after the 6h drug washout period. These are indicated on the graphs as ≤6h. Bar graphs show mean + SD. In the violin plots, horizontal lines display the median, and error bars show 95% confidence intervals. (D) Representative images and quantification of colony forming assays of RPE1 cells treated with CDK4/6 inhibitor for 1, 4 or 7 days and then grown at low density without inhibitor for 10 days. Bar displays mean data + SEM from 3 experiments.
No evidence for miRNA-dependent destabilization of cytosolic lncRNAs.Figure 2(A) Schematic representing 4sU metabolic labelling of conditional Dicer knockout and wildtype cells experiment. (B Correlation (pearson) between degradation rates (log10) obtained after 10 (x-axis) and 15 (y-axis) minutes of 200 µM 4sU labelling in wildtype (WT) and DICER null (KO) cells. C) Correlation (pearson) between degradation rates (log10) obtained after 10 (x-axis) and 15 (y-axis) minutes of 200 µM 4sU labelling in wildtype (WT) and DICER null (KO) cells. (D) Volcano plot showing the adjusted p-value (y-axis) as a function of the fold-change in degradation rate estimates, based on the 10 minutes pulse, between KO and WT cells (x-axis) for protein-coding genes (red), cytosolic (blue) and nuclear (grey) lncRNAs. Each point represents a transcript and the horizontal dashed line represents the significance cut-off. (E) Cumulative distribution plot of the density of AGO2 clusters in the 3'unstralated regions of AGO2 bound mRNAs (AGO2 cluster>0) whose degradation rates were either significantly (n=711, red) or not significantly changed (n=1127, black) between KO and WT cells, based on the 10 minutes pulse estimates. Density of clusters presented in this analysis was estimated based on data from (Leung et al., 2011). (F) Distribution of the fold-change (FC) in degradation rates of mRNAs (n=29900, red), cytosolic (n=474, blue) and nuclear (n=2348, grey) lncRNAs, in 4-OHT treated (KO) relative to ethanol treated (WT) cells after 8 days of treatment (estimated based on the 10 minutes 4sU pulse), horizontal dashed line represents a KO/WT FC in degradation rate of 1 Statistics: NS-p-value>0.05 anband of boxplot represents median box depicts quantiles distribution and whiskers represent the>th and 95th quantiles of the distribution. (two-tailed Mann-Whitney U test).
Figure 4Interaction between MITOL and IRE1α. Lysates of HEK293 cells were subjected to immunoprecipitation with anti-IRE1α antibody, followed by immunoblotting with indicated antibodies (A). Interaction between MITOL and IRE1α. Lysates of HEK293 cells co-transfected with indicated vectors were subjected to immunoprecipitation, followed by immunoblotting with indicated antibodies (B). Interaction between MITOL and IRE1α. GST-pull-down assay of IRE1α or MITOL was performed on lysates of MEFs, followed by immunoblotting with indicated antibodies (C, D). N-ter: 1-90 aa of MITOL, Loop: 158-210 aa of MITOL, C-ter: 241-278 aa of MITOL, 465-977 aa of IRE1α. MITOL ubiquitylated IRE1α. Lysates of HEK293 cells immunoprecipitated with indicated antibody, followed by immunoblotting with indicated antibodies. MITOL ubiquitylated IRE1α. Lysates of HEK293 cells transfected with indicated vectors, were immunoprecipitated with indicated antibody, followed by immunoblotting with indicated antibodies. MITOL ubiquitylated IRE1α. Lysates of MEFs (G) transfected with indicated vectors, were immunoprecipitated with indicated antibody, followed by immunoblotting with indicated antibodies. MITOL adds lysine 63-linked polyubiquitin chains to IRE1α. Lysates of HEK293 cells transfected with indicated vectors were immunoprecipitated with indicated antibody. IRE1α ubiquitylation was detected MITOL adds lysine 63-linked polyubiquitin chains to IRE1α. Lysates of HEK293 cells transfected with indicated vectors were immunoprecipitated with indicated antibody. IRE1α ubiquitylation was detected
Fig.Figure 5(A) [γ-32P]ATP radioactive in vitro kinase assay of TF-SGN1 against GST-N-terminal cytoplasmic domains of RBOHD or F. Autoradiograph is shown on top. Coomassie stained-gel below illustrates presence and equal loading of recombinant proteins. Experiments were done independently three times with similar results. (B) HEK293Tcell based NOX activation assay. Cells were transfected with the indicated plasmid combinations. The phosphatase inhibitor Calyculin A was added directly before the start of the measurements. Each data point represents the mean of six wells analyzed in parallel, bars indicate S.D.. Experiments were repeated three times
RNA-seq reveals strong global upregulation of NMD-sensitive targets upon UPF3 dKO in HEK293 cells A nVenn Diagram showing the overlap of up- or downregulated genes in the UPF3 dKO cell lines 1 and 2.Figure 5A nVenn Diagram showing the overlap of up- or downregulated genes in the UPF3 dKO cell lines 1 and 2. Log2 fold change < -1 (downregulated) or > 1 (upregulated) and adjusted p-value (padj) < 0.05. DGE = Differential Gene Expression. B Fraction of expressed genes (genes with non-zero counts in DESeq2) were calculated which exhibit individual or combinations of differential gene expression (DGE), differential transcript usage (DTU) and/or alternative splicing (AS) events in the indicated conditions using the respective computational analysis (cutoffs are indicated). AS and DTU events were collapsed on the gene level. For DGE, p-values were calculated by DESeq2 using a two-sided Wald test and corrected for multiple testing using the Benjamini-Hochberg method. For DTU, p-values were calculated by IsoformSwitchAnalyzeR using a DEXSeq-based test and corrected for multiple testing using the Benjamini-Hochberg method. For AS, p-values were calculated by LeafCutter using an asymptotic Chi-squared distribution and corrected for multiple testing using the Benjamini-Hochberg method. C Volcano plots showing the differential transcript usage (via IsoformSwitchAnalyzeR) in various RNA-Seq data. Isoforms containing GENCODE (release 33) annotated PTC (red, TRUE), regular stop codons (blue, FALSE) or having no annotated open reading frame (grey, NA) are indicated. The change in isoform fraction (dIF) is plotted against the -log10 adjusted p-value (adj.p-value). Density plots show the distribution of filtered isoforms in respect to the dIF, cutoffs were |dIF| > 0.1 and adj.p-value < 0.05. P-values were calculated by IsoformSwitchAnalyzeR using a DEXSeq-based test and corrected for multiple testing using the Benjamini-Hochberg method. D Read coverage of SRSF2 from the indicated RNA-seq sample data with or without UPF3A siRNA treatment shown as Integrative Genomics Viewer (IGV) snapshot. The canonical and NMD-sensitive isoforms are schematically indicated below. Quantification of gene expression and isoform usage by IsoformSwitchAnalyzeR (right).
Pharmacological induction of autophagy during AICD results in cell death inhibition independently from DRP1-dependent mitochondrial shape changes.Figure 5A. Apoptotic cells were detected at indicated time points after AICD induction in hPBT cells by flow cytometry as AnnexinV/PI double positive cells. Where indicated hPBT cells were pre-incubated with 100nM rapamycin for 24h before AICD induction. Data represent mean ± SE of six independent experiments. B. AICD was induced in Jurkat cells and where indicated 100nM rapamycin was added at time 0h of AICD induction. Apoptotic cell death analysis was carried out as in (A), except that cells were analysed 32h after AICD induction. Data represent mean ± SE of six independent experiments. C. Morphometric analysis of mitochondrial shape 24h after AICD induction. Jurkat cells were transfected with mtYFP 24h before AICD induction and, where indicated, 100nM rapamycin was added. Data represent mean ± SE of four independent experiments (n = 30 cells per condition in each experiment). D. Representative electron micrographs of cells subjected to AICD untreated (upper panel) or treated with 100nM rapamycin (lower panel). 24h after AICD induction cells were fixed and processed for electron microscopy, Scale bar, 2μm. E. Jurkat cells were transfected with mtYFP and treated as indicated. 32h after AICD induction cells were fixed and immunostained with an anti-cytochrome c antibody. Cytochrome c localization index was calculated from 30 randomly selected images per condition. Data represent mean ± SE of four independent experiments. F. Jurkat cells were transfected with DRP1S637A-GFP, 24h after transfection AICD was induced. 32h after AICD induction viability was determined cytofluorimetrically as the percentage of GFP-positive, annexin-V-PE-negative cells. Data represent mean ± SE of three independent experiments. G-H. Jurkat cells were co-transfected with mt-RFP and the indicated plasmids, 32h after AICD induction cells were fixed and immunostained with an anti-cytochrome c antibody. Cytochrome c localization index (G) and mitochondrial morphology (H) were calculated from 30 randomly selected images. Data represent mean ± SE of three independent experiments. I. Jurkat cells were transfected with DRP1K38A, 24h after transfection AICD was induced. 32h after AICD induction viability was determined cytofluorimetrically as the percentage AnnexinV/PI double negative cells. Data represent mean ± SE of three independent experiments.
Figure 7Under normoxic conditions, the ER and mitochondria remain in close contact. Small amounts of DRP1 are recruited to mitochondria to maintain the balance of mitochondrial fission and fusion. A small proportion of FUNDC1 associates with the ER membrane protein Calnexin at the sites where ER and mitochondria are tethered. At the early stage of hypoxia, FUNDC1 increasingly accumulates at the MAM by interacting with Calnexin mediated by an unknown intermediate protein. As mitophagy proceeds, FUNDC1 and Calnexin dissociate, and DRP1 is recruited by FUNDC1 to the MAM to initiate mitochondrial constriction. Mitochondria are further constricted by self-assembled DRP1 and fragment at the later stage of hypoxia. Meanwhile, the most upstream ATG complex (ULK1 complex) phosphorylates FUNDC1 to promote engulfment and clearance of damaged mitochondria by the autophagy pathway (Wu et al, 2014a).
Figure 5(A) Phosphorylated Myosin-II Regulatory Light Chain (p-MLC; purple) detected by antibody staining reveals high levels around the cortex of rounded mitotic cells (pH3, red) in wild-type wing discs, and aurA homozygous mutant wing discs, but not upon inhibition of both AurA and AurB by treatment with VX-680. Note increased numbers of mitotic cells in aurA homozygous mutant wing discs, due to delayed mitotic progression. Scale bars ~20µm (low mag.) and ~1µm (high mag.). n > 15 independent biological replicates. (B) Live-imaging of MyoII-GFP reveals delayed mitosis in aurA homozygous mutant wing discs, while inhibition of both AurA and AurB by treatment with VX-680 leads to complete loss of mitotic rounding and MyoII-GFP membrane localisation. Asterisks indicate a single mitotic cell. Scale bars ~1µm. Quantification shown on the bottom right (n = 5 independent samples per genotype). (C) Live-imaging of E-cad-GFP reveals delayed mitosis in aurA homozygous mutant wing discs, cytokinesis failure in UAS.aurB-RNAi expressing wing discs, and complete failure of mitotic rounding and E-cad-GFP downregulation upon inhibition of both AurA and AurB by treatment with VX-680. Asterisks indicate a single mitotic cell. Scale bars ~1µm. Quantification shown on the bottom right (n = 9 independent samples per genotype).
Fig.Figure 5(A) WT (SCY62), tag∆ste∆ (H1246), tag∆ (H1226) and ste∆ (H1112) cells expressing GFP−Atg8 were grown to mid-log phase in YPD and shifted to SD-N for the indicated time periods. Cells were lysed and subjected to SDS−PAGE, followed by western blot analysis using anti-GFP antibodies. (B) Quantification of the GFP/GFP−Atg8 ratio. Error bars represent the s.e.m. of three independent experiments. *P < 0.05 (Student's t-test). ( . (C) WT (SCY62), tag∆ste∆ (H1246), tag∆ (H1226) and ste∆ (H1112) cells expressing GFP-Atg8 were grown to mid-log phase in YPD and shifted to SD-N for 2 h. GFP−Atg8 was visualized by fluorescence microscopy. Scale bar, 5 μm. ( (D) WT (SCY62), tag∆ste∆ (H1246), tag∆ (H1226) and ste∆ (H1112) cells were grown as in (A). Lysates were subjected to SDS−PAGE in urea gel, followed by western blot analysis using anti-Atg8 and anti-Pgk1 antibodies. Atg8 I, non-lipidated Atg8; Atg8 II, lipidated Atg8. DIC, Differential interference contrast; SD-N, nitrogen-starvation medium; WT, wild type.
Figure 5(A) (Top) Illustration of the HIST2H2AA3 reporter construct. (Bottom) FISH to detect the distribution of the HIST2H2AA3 mRNA in HeLa cells depleted of Cntl, ALYREF, THOC2 (THO) and UAP56. DAPI staining served as a nuclear marker. N and C indicate nuclear and cytoplasmic FISH signals, respectively. N/C ratios were determined for 20 cells in each experiment. Data information: Error bars represent standard deviations from biological repeats (n=3 Statistical analysis was performed using Student's t-test. *P < 0.05, **P < 0.01, ***P< 0.001. (B) Distribution of the endogenous HIST2H2AA3 mRNA was detected with the transcript-specific probe in HeLa cells depleted of Cntl, ALYREF, THOC2 (THO) and UAP56. N and C indicate nuclear and cytoplasmic FISH signals, respectively. N/C ratios were calculated Data information: Error bars represent standard deviations from biological repeats (n=3 Statistical analysis was performed using Student's t-test. *P < 0.05, **P < 0.01, ***P< 0.001. Western analysis to examine the purities of nuclear and cytoplasmic fractions (C) in Cntl and UAP56 KD cells. MTR4 and Tubulin were used as the nuclear and cytoplasmic marker, respectively. RT-qPCRs to examine N/C distribution of each histone mRNA (D) in Cntl and UAP56 KD cells. Data information: Error bars represent standard deviations from biological repeats (n=3 Statistical analysis was performed using Student's t-test. *P < 0.05, **P < 0.01, ***P< 0.001. (E) Graphic displays that TREX promotes histone mRNA export. (F) Relative NXF1 iCLIP read populations on histone mRNAs in Cntl, ALYREF and SLBP KD cells. Data information: Error bars represent standard deviations from biological repeats n=2 Statistical analysis was performed using Student's t-test. *P < 0.05, **P < 0.01, ***P< 0.001. (G) NXF1 iCLIP signals along the histone mRNA. The histone mRNA is divided into 10 fractions based on their mRNA size. Each fraction represents 10% of each histone mRNA. The tenth fractions that contains the SL is boxed by green dashed lines.
Figure 5(A) Genotype of Ts65Dn/DSCR1+/- mouse was confirmed by detecting markers for Ts65Dn and DSCR1 KO (B) Expression of the DSCR1 mRNA in Ts65Dn mouse was increased about 2.5 folds compared to that of wild type. However, Ts65Dn/DSCR1+/- mouse showed normal level of DSCR1 mRNA transcript. Values are shown as mean ± SEM and tested for statistical significance by one-way ANOVA followed by Bonferroni post hoc test. N=3 for each condition, *P< 0.05. As expected, the levels of TET1 are decreased in Ts65Dn mouse, however Ts65Dn/DSCR1+/- mouse restores the TET1 mRNA expression. Values are shown as mean ± SEM and tested for statistical significance by one-way ANOVA followed by Bonferroni post hoc test. ß-actin was used for normalization. N = 3 (Diploid), 3 (Ts65Dn), 4 (Ts65Dn/DSCR1+/-) mice, *P< 0.05, **P< 0.01. As expected, the levels of miR-124 are decreased in Ts65Dn mouse, however Ts65Dn/DSCR1+/- mouse restores the miR-124 expression. Values are shown as mean ± SEM and tested for statistical significance by one-way ANOVA followed by Bonferroni post hoc test. ß-actin was used for normalization. N = 3 (Diploid), 3 (Ts65Dn), 4 (Ts65Dn/DSCR1+/-) mice, *P< 0.05, **P< 0.01. (E, F) The number proliferating progenitor neurons identified by BrdU and Ki-67 double staining is restored in the SGZ of Ts65Dn/DSCR1+/- mouse compared to that of Ts65Dn mouse. The white box area is magnified in the lower panels: DAPI (blue), Ki67 (green), and BrdU (red). Arrow heads indicate BrdU and Ki-67 double positive cells in the SGZ. Scale bars: 100 μm in the large image and 10 μm in magnified image. Each hippocampal section was 40 μm in thickness, and total 6 hippocampi were used for analysis. Values are shown as mean ± SEM and tested for statistical significance by one-way ANOVA followed by Bonferroni post hoc test. N = 6 (control), 5 (Ts65Dn), 3 (Ts65Dn/DSCR1+/-) animals, *P< 0.05. (G-J) Ts65Dn has learning and memory defects, but Ts65Dn/DSCR1+/- mouse clearly rescues learning and memory defects to control levels. Values are shown as mean ± SEM, and tested for statistical significance by one-way ANOVA followed by Bonferroni post hoc test. N = 8 (control), 5 (Ts65Dn), 10 (Ts65Dn/DSCR1+/-) animals, *P< 0.001 (G, H). *P< 0.05 (I), **P< 0.01 (I). (J) Open-field analysis shows no defects in movement of tested animals. Values are shown as mean ± SEM, N = 6 (control), 5 (Ts65Dn), 3 (Ts65Dn/DSCR1+/-) animals.
TIM23 but not TIM22 dependent import is impaired in TIMM50 mutant fibroblasts A In organello import of the precursors of TFAM and AAC1 into isolated mitochondria from control and TIMM50 mutant fibroblasts in the absence or presence of the uncoupler FCCP.Figure 3A In organello import of the precursors of TFAM and AAC1 into isolated mitochondria from control and TIMM50 mutant fibroblasts in the absence or presence of the uncoupler FCCP. The radiolabeled precursors were incubated with isolated mitochondria for the indicated times followed by trypsin treatment only in the case of AAC1. Coomassie Blue (CB) staining was used as loading control and the radiolabeled protein (TNT) as reference for quantification. The position of the precursor (empty arrowheads) and the mature protein (filled arrowhead) are also shown. Western blot for TIMM50 is shown as cell line control. B Quantification of TFAM and AAC1 import from control and TIMM50 mutant fibroblasts expressed as fraction of the input radiolabeled precursor converted into mature protein and normalized to Coomassie Blue signal. Data are shown as mean ± SD, n = 3 biological replicates; ∗p < 0.05, ∗∗p < 0.01, ∗*∗p < 0.001 grouping both controls together. C In organello import of the precursors of TFAM and AAC1 into isolated mitochondria from control and TIMM50 mutant fibroblasts transduced with the empty vector (EV) or with wild-type TIMM50 in the absence or presence of the uncoupler FCCP The position of the precursor (empty arrowheads) and the mature protein (filled arrowhead) are also shown. D Quantification of TFAM and AAC1 import from control and TIMM50 mutant fibroblasts transduced with the empty vector (EV) or with wild-type TIMM50 expressed as fraction of the input radiolabeled precursor converted into mature protein and normalized to Coomassie Blue signal. Data are shown as mean ± SD, n = 3 biological replicates; ∗p < 0.05.