EMBO/sd-character-level-ner huggingface dataset:
The TIP30/eEF1A1 ratio decreases in human failing and hypertrophic cardiomyopathy hearts.Figure 6Quantification of Tip30 and eEF1A1 mRNA transcript abundance in human failing hearts (A), N=6 to 8 hearts/group A ratio of Tip30 and eEF1A1 expression was calculated Quantification of Tip30 and eEF1A1 mRNA transcript abundance in patients with Hypertrophic Cardiomyopathy (HCM; B, N=4 to 8 hearts/group) A ratio of Tip30 and eEF1A1 expression was calculated Quantification of Tip30 and eEF1A1 mRNA transcript abundance in 6 months old mdx mice or WT mice (C), N=4 mice/group. A ratio of Tip30 and eEF1A1 expression was calculated (D) Schematic representation of AAV-study design in mdx (MDX) mice. (E) Echocardiographic diastolic left-ventricular wall-thickness in MDX mice at the age of 2 months at baseline, and 1-9 months (mo) after injection of AAV9-Con or AAV9-TIP30 (TIP; N=6 to 7 mice/group). (F) Quantification of HW/TL ratio of 9mo old MDX mice, N=5 to 6 mice/group.
Silencing the R3hdml gene ameliorates C2C12 cell differentiation.Figure 3A R3hdml siRNA or non-targeting siRNA was transfected into C2C12 cells. Then, C2C12 cells were differentiated into myotubes. RNA was extracted from the cells after induction of differentiation at the indicated time points. Closed bars, control; open bars, silenced R3hdml gene; ** p < 0.01; * p < 0.05; Student's t-test. B Immunofluorescence after C2C12 differentiation. MyHC is shown in green and the nuclei in blue (counterstained with Hoechst). Representative images of MyHC immunofluorescence of C2C12 myocytes transfected with the siRNA or after addition of R3hdml protein. Scale bar = 100 µm. C The number of nuclei per MyHC+ cell were calculated on day 5 after differentiation. ** p < 0.01; one-way ANOVA followed by Bonferroni post-hoc test. D The distribution of nuclei per myotube were calculated on day 5 after differentiation. ** p < 0.01; * p < 0.05; one-way ANOVA followed by Bonferroni post-hoc test.
Figure 10We calculated the ΔΨCr-IBM between neighboring cristae over time as a surrogate measure of cristae individuality and functional independence, using live-cell Airyscan imaging. Stability of cristae ΔΨCr-IBM over time. Representative images from time series of HeLa cells co-stained with MTG and TMRE. Top sequence shows merged green (MTG) and red (TMRE) channels. Bottom sequence shows color-coded LUT of TMRE signal of the same sequence shown above. White arrowheads in the image point to specific cristae that were measured over time. Scale bar = 500 nm. N = 5 independent experiments. Quantification of A, showing average ΔΨCr-IBM of Cristae "A-D" over time. Note that Cristae C and D maintain more than double the ΔΨCr-IBM of Cristae A and B. Error bars indicate STDEV. Quantification of A, showing standard deviation in ΔΨCr-IBM (mV) of individual cristae over time compared to the standard deviation in ΔΨCr-IBM (mV) among different cristae along a single mitochondrion. N = 5 independent experiments. Note that there is less variability in ΔΨCr-IBM of a single crista over time within the same mitochondrion compared to the variability of ΔΨCr-IBM among cristae over time, indicating cristae possess unique bioenergetic properties. We calculated the ΔΨCr-IBM between neighboring cristae over time as a surrogate measure of cristae individuality and functional independence, using live-cell Airyscan imaging. Representative live-cell Airyscan image from time series showing merged green (MTG) and red (TMRE) channels. Area marked by dashed box is shown in the time series in E. Scale bar = 500 nm. Time series of cropped region from D, showing tip of mitochondrion (arrowhead) maintaining ΔΨm while neighboring region of same mitochondrion depolarizes. The capacity of some cristae to maintain polarity, while adjacent cristae transiently depolarize, indicates cristae can exhibit a significant amount of electrochemical autonomy. N = 3 independent experiments. (See Movies EV 3, 4 for time series.) Zoomed-in LUT of mitochondrion from E (~2 sec), showing some cristae remaining polarized (red) while adjacent cristae depolarize (blue). (See Movie EV 5 for time series.) Quantification of D-F, showing that during a flickering event some cristae maintain their membrane potentials while other cristae depolarize, resulting in large differences in ΔΨCr-IBM along the individual mitochondrion. N = 3 independent experiments.
DOR moves out of the nucleus in response to the activation of autophagy.Figure 1(A) The intracellular distribution of DOR is modified by starvation. HeLa cells were transiently transfected with DOR and incubated for 1 h with DMEM (basal), HBSS (starvation), 50 μM chloroquine or 10 mM 3MA. The intracellular localization of DOR was analysed by immunofluorescence and is shown in red. The nuclei are shown in blue (DAPI staining). Scale bars, 10 μm. (B) Endogenous DOR leaves the nucleus under starvation. C2C12 myoblasts were incubated for 30 min at 37°C with DMEM (basal) or HBSS (starvation). DOR is shown in red and nuclei are in blue (DAPI staining). Scale bars, 5 μm. (C,D) De‐activation of autophagy returns DOR to the nucleus. HeLa cells were transiently transfected with DOR and incubated for 1 h with DMEM (basal) or HBSS (starvation). Cells were then incubated with normal DMEM for an additional hour (starvation+GM). The intracellular distribution of DOR was determined by immunofluorescence. DOR is shown in red and the nuclei are in blue. Scale bars, 5 μm. Cell lysates were also obtained and western blot assays were performed with specific antibodies. 3MA, 3‐methyladenine; DAPI, 4′,6‐diamidino‐2‐phenylindole; DMEM, Dulbecco's modified Eagle medium; DOR, diabetes‐ and obesity‐regulated gene; HBSS, Hank's balanced salt solution; LC3, microtubule‐associated protein 1A/1B‐light chain 3.
Figure 2A The kinetics of LM1 (n = 10), LM9 (n = 4), LM1 Nfib KO (n = 4), and LM9 Nfib KO (n = 4) tumour growth upon orthotopic injection of 250 x 103 cells into NOD/SCID mice. Curves show means of tumour volume ± s.d., two-way ANOVA group analysis of the times to reach 250 mm3 (dashed line). B Kaplan-Meier plot depicting metastatic onset after tumour removal in mice injected orthotopically with LM1 (n = 5), LM9 (n = 4), LM1 Nfib KO (n = 3), or LM9 Nfib KO (n = 4) cells, two-tailed log-rank test. C Nfib knockout abrogates metastasis in the orthotopic LM1 model. Representative bioluminescence images (left panel) and bar plot quantification (right panel) of lung metastases at 25 (LM1) and 40 (KO) days after primary tumour removal; n = 3 mice, means ± s.d., two-tailed Student's t-test. D Nfib knockout impairs experimental metastases formation in the LM models. Kaplan-Meier plot showing metastatic incidence of animals inoculated i.v. with LM1 (n = 4), LM9 (n = 4), LM1 Nfib KO (n = 3), or LM9 Nfib KO (n = 5) cells, two-tailed log-rank test. E Representative bioluminescence images (left panel) and bar plot quantification (right panel) of lung metastases 16 days after i.v. injection of LM1 or LM1 Nfib KO cells, n = 3 mice, means ± s.d., two-tailed Student's t-test.
Figure 1Design concept. Bacteria are engineered to exhibit collective survival. Bacteria confined in the microbial swarmbot can maintain a high local density and survive. Cells escaping the swarmbot will have a reduced density due to a larger extra-capsule environment. If their density drops below their survival threshold, they will die, leading to safeguard control. Collective survival. A population survives only when its initial density is >NCT. Simulation of collective survival. The collective survival can be described by a simplified model: , where Modulating safeguard with VR. The effectiveness of safeguard can be quantified by the area under the curve (AUC) from density differences between the microbial swarmbot and the surrounding environment. The swarmbot dynamics can be described by a two-compartment model: , and , where the transport of cells between the two compartments is described by the final term in each equation. Modeling predicts that AUC increases with VR, and once VR becomes sufficiently large, AUC approaches infinity, indicating high level of safeguard. As the initial conditions, the parameters are set as: NMSB = 0.1 at t = 0, μ = 1.75, Nm = 1, d = 2, K = 0.01, α = 4, fN = 0.1, VR = variable from 1 to 105. Insets demonstrate the simulated growth dynamics for two representative VR values.
a, Heat map of H4K16ac ChIP-seq performed in U1810 cells without or with 8 h rapamycin treatment.f2a, Heat map of H4K16ac ChIP-seq performed in U1810 cells without or with 8 h rapamycin treatment. Data are shown as log2 values of tag counts in the 3,422 regions defined as peaks in the control sample. b, We analysed de novo detection of transcripts using GRO-seq in 8 h rapamycin-treated U1810 cells and compared them with untreated U1810 cells. GRO-seq data visualized as 'MA' plots (log ratio versus abundance). The plot shows GRO-seq gene expression for pairwise comparison between rapamycin-treated versus control cells. The red points denote the differentially expressed genes. c, Autophagy-related genes identified as regulated by rapamycin in the GRO-seq data analysis and in the ChIP-seq data analysis.
A minimal 19-amino-acid subdomain between amino acids 263281 holds the autophagic activity of TMEM59.Figure 3(A, B) Ability of serial C‐terminal deletions of TMEM59 to activate LC3. 293 cells were transfected with the indicated TMEM59 C‐terminal deletions, HA-LC3A (A) or HA-LC3B (B) and GST, and lysed for western blotting against the indicated molecules. (C) Amino acids 263-281 retain the full potential of TMEM59 ID to promote HA-LC3 conversion. 293 cells were transfected with the shown CD16:7 chimeras, HA-LC3A or HA-LC3B (as indicated) and GST, and subjected to anti‐CD16 aggregation before lysing them for western blotting. The lower panels show control WBs (unaggregated samples). D Amino acids 263-281 suffice for GFP-LC3 activation and retain the full potential of TMEM59 ID to promote GFP-LC3 activation. 293 cells were transfected with the indicated CD16:7 chimeras and GFP-LC3A or GFP-LC3B (as indicated), and subjected to anti‐CD16 aggregation before fixing them for microscopy. The graph shows percentages of transfected cells exhibiting redistributed GFP-LC3. Quantification and data expression were done as in Figure 2H. (E) Amino acids 282-323 of TMEM59 lack LC3 activation potential. 293 cells were transfected with the shown chimeras, HA-LC3A or HA-LC3B and GST, and processed for western blotting against the indicated molecules. Control WBs of unaggregated samples are shown in the lower panels. (F) Surface expression levels of CD16:7 chimeras. Procedures and data expression were as in Figure 2E. The figure shows that the functional differences observed between CD16:7 chimeras (C-E) are not caused by differential surface expression. (G) Scheme of TMEM59 showing amino‐acid positions and the minimal active subdomain (amino acids 263-281). ED, extracellular domain; TM, transmembrane domain.Source data for this figure is available on the online supplementary information page.
Figure 2(A) Plants after pressure infiltration with Pst DC3000, at 4 dpi. The experiment was repeated three times (each time 9 - 12 plants) with similar results. (B) Pst DC3000 growth in ANAC032 transgenics and WT plants 3 dpi after pressure infiltration. Two independent experiments were performed with three replications per experiment, each replicate consisting of three plants grown in individual pots (six measurements in total). The graph shows data points of the two individual experiments (I and II) along with their mean (Avg). (C) Disease status of plants sprayed with Pst DC3000, 5 dpi. The experiment was repeated four times with similar results. (D) Disease severity index (1, small chlorotic lesions; 5, large lesions) scored 5 dpi after spraying with Pst DC3000. Data are from three independent experiments with at least six plants per genotype in each. Means are shown ± SD. Asterisks indicate significant (*p < 0.05 and **p < 0.005) differences between transgenic and WT plants in chi-square analysis. (E) Heat map showing the fold change (log2 basis) in the expression ratio of defense-/stress-related differentially expressed genes in anac032-1 and 35S:ANAC032 compared to WT after spraying with Pst DC3000 (6 hpi) normalised to their respective controls. Blue, downregulated; red, upregulated. Data represent means of three independent experiments, each including the rosettes leaves of at least three plants grown in individual pots. Asterisks indicate significant differences from WT plants (Student's t test, p ≤ 0.05).
Figure 2A,C Double-immunolabeling of Sororin (green) and SYCP3 (red) on spread spermatocytes at (A) early zygotene (E. zyg.) and (C) late zygotene (L. zyg.).B,D Double-immunolabeling of SMC3 (pseudocoloured in cyan) and SYCP3 (red) on spread spermatocytes at (B) early zygotene and (D) late zygotene. E Double-immunolabeling of Sororin (green) and REC8-myc (pseudocoloured in purple) on a spread mid zygotene spermatocyte. The sex univalents (X,Y) are indicated. Scale bar: 10 µm.
Figure 1(A) The three top enriched DNA-binding motifs identified by HOMER known motif analysis on 200 bp regions centred on the ZEB1 peak summits. (B) Enrichment profiles of TEAD4 and AP-1 consensus motifs in 600 bp regions centred on the ZEB1 peak summits. The two grey dashed lines highlight the 200 bp regions around the ZEB1 peak summits which were used for HOMER known motif analysis (C) Overlap between ZEB1 and JUN DNA-binding sites. Sites are counted as overlapping when their peak summit positions are not more than 200 bp apart. (D) Enrichment profile of JUN peak summit positions in 1 kb regions centred on the ZEB1 peak summits. The two grey dashed lines highlight the region of +/- 200 bp around the ZEB1 peak summit positions where JUN peaks are counted as overlapping with ZEB1 peaks. (E) Heatmap representing the signal enrichment of JUN ChIP-seq reads in 2 kb regions centred on the ZEB1 peak summits. (F) Circle plot displaying the genome-wide distribution of ZEB1 (outer middle circle, blue), JUN (inner middle circle, green) and YAP (inner circle, red) peaks across all chromosomes (outer circle). Peak density is visualised as a heatmap with more intense colours indicating a higher number of peaks in a specific location. (G) ZEB1, JUN and YAP DNA-binding overlaps at 1,993 genomic sites in MDA-MB-231 cells. Triple overlap is assumed when both JUN and YAP peak summit positions are within 200 bp of a ZEB1 peak summit position.
RIPK3 K518R hypersensitize HT‑29 cells for TBZ-induced necroptosis A HT‑29 RIPK3 KO cells re-expressing Dox-inducible WT RIPK3 or the indicated RIPK3 mutants were incubated with 1 µg/ml Dox overnight.Figure 7A HT‑29 RIPK3 KO cells re-expressing Dox-inducible WT RIPK3 or the indicated RIPK3 mutants were incubated with 1 µg/ml Dox overnight. Cells were treated with 20 µM zVAD.fmk, 5 µM BV6 and 10 ng/ml TNFα for 4 h. Cell death was measured by analysis of PI-positive nuclei. B HT‑29 RIPK3 KO cells re-expressing Dox-inducible WT RIPK3 or the indicated RIPK3 mutants were incubated with 1 µg/ml Dox overnight, as indicated. Cells were treated with 20 µM zVAD.fmk, 5 µM BV6 and 10 ng/ml TNFα for 2 h and analyzed by Western blotting for RIPK3 and phosphorylated MLKL expression levels. β-Actin served as a loading control. C Schematic overview of the putative mechanistic roles of USP22 in necroptosis. Activation of TNFR1 by TNFα, upon caspase-8 inhibition by zVAD.fmk and cIAP1/2 inactivation by BV6, induces RIPK1/3 activation, necrosome formation and execution of necroptosis. In the absence of USP22 KO, TBZ-induced necroptosis is delayed, accompanied by increased RIPK3 phosphorylation and ubiquitination of RIPK3 at lysine 518. This USP22-mediated necroptotic signaling could be caused either by direct USP22-mediated (de)ubiquitination of RIPK3 and/or indirectly by, for example, regulating RIPK3 autophosphorylation or the activity of RIPK3-associated E3 ligases or kinases. Additionally, USP22-mediated retrograde effects on RIPK1 phosphorylation and RIPK3 oligomerization might be involved as well.
Beclin1 is required for HMGB1-mediated autophagy.Figure 6.(A and B) Knockdown of Beclin1 in MEFs by shRNA inhibits exogenous HMGB1-induced autophagy. Cells as indicated were stimulated with 1 µg/ml HMGB1 protein (rHMGB1) for 24 h, and LC3 expression was detected by Western blotting (A). (A and B) Knockdown of Beclin1 in MEFs by shRNA inhibits exogenous HMGB1-induced autophagy. Cells as indicated were stimulated with 1 µg/ml HMGB1 protein (rHMGB1) for 24 h, LC3 punctae formation (arrows) was assayed by immunofluorescence (B; n = 3; *, P < 0.05). (C and D) Knockdown of Beclin1 in MEFs by shRNA inhibits HBSS-induced autophagy with and without pUNO1-HMGB1 transfection. Cells as indicated were stimulated with Earle's balanced salt solution for 3 h, and LC3 expression was detected by Western blotting (C). (C and D) Knockdown of Beclin1 in MEFs by shRNA inhibits HBSS-induced autophagy with and without pUNO1-HMGB1 transfection. Cells as indicated were stimulated with Earle's balanced salt solution for 3 h, and LC3 punctae formation (arrows) was assayed by immunofluorescence (D; n = 3; *, P < 0.05). Data are means ± SEM.
Figure 6WCE and FLAG-tag NS3 IP assay immunoblots of 293T transfected with BTV-NS3-FLAG plasmids and increasing amounts of Ubiquitin-HA plasmids, and probed (IB) for ubiquitin (HA), BTV-NS3 (FLAG) or GAPDH expression. Immunofluorescence staining of Vero cells co-transfected with BTV-NS3 and ubiquitin and acquired with Airyscan super-resolution. Scale bar = 10 μm. Inset shows detailed area and arrowheads indicate NS3 and ubiquitin signal co-incidence (scale bar = 2 μm). Fluorescence intensity profile of cell section indicated by the white line (blue: DAPI, green: NS3; red: Ub). Arrowheads on image and arrows on intensity profile indicate matching fluorescence signal for NS3 and Ub. FLAG-tag IP assay of BTV-NS3 and WCE immunoblot of 293T co-transfected with BTV FLAG-NS3-, -NS3-K13R, -NS3-K15R, -NS3-K13/15R or -NS3-PPRY/AARH plasmid and Ubiquitin-HA plasmid and probed (IB) for ubiquitin (HA), BTV-NS3 (FLAG), STAT2 and GAPDH expression. WCE and HA-tag Ubiquitin IP assay immunoblots of 293T transfected with Ubiquitin-HA plasmids and increasing amounts of BTV-FLAG-NS3-K13/15R, and probed (IB) for ubiquitin (HA), BTV-NS3-K13/15R (FLAG) or GAPDH expression. ISRE-driven luciferase reporter assay in 293T cells transfected with empty expression plasmid (Control), BTV -NS2 (300 ng), -NS3 (50, 300 and 600 ng), or -NS3-K13/15R (50, 300 and 600 ng)-expressing plasmids. Mean % inductions of luciferase activity of three independent experiments are shown. FLAG-tag IP assay of BTV-NS3 and WCE immunoblot of 293T co-transfected FLAG-BTV-NS3 plasmid and HA-UB-WT or HA-UB lysine (K to R) mutants. IP were probed (IB) for ubiquitin (HA) and BTV-NS3 (FLAG) expression. WCE were probed for STAT1, STAT2, ubiquitin (HA), BTV-NS3 (FLAG), and GAPDH expression.
Figure 2(A). Cell growth assessment of ΔIndLon (upper plot) and ΔIndFtsH (lower plot) mutants grown under inducing (blue) or depleting conditions (red). Growth was monitored by measuring DNA and protein biomass over time. The average from two independent biological replicates is shown. (B). Protein aggregation in ΔIndLon (upper plot) and ΔIndFtsH (lower plot) mutants grown under inducing (blue) or depleting conditions (red, 48h and 72h of depletion for Lon and FtsH, respectively). Protein aggregates in Triton-X100 insoluble fractions of untreated or heat shock (15min at 45°C) treated cells were measured by thioflavin (ThT) staining. Bars represent the mean of three biological replicates (dots). Significance of comparisons was assessed by two-sided independent t-test (exact p-values are shown). (C). Assessment of cell membrane integrity of ΔIndLon (upper plot) and ΔIndFtsH (lower plot) mutants grown under inducing (blue) or depleting conditions (red, 72h of depletion for both, Lon and FtsH). Membrane integrity of untreated cells or after exposure during 30 min to 0.001% Triton-X100 was assessed by trypan blue exclusion staining. Bars represent the mean of three biological replicates (dots). Significance of comparisons was assessed by two-sided independent t-test (exact p-values are shown). ( (D). Role of Lon and FtsH under heat shock stress conditions. ΔIndLon and ΔIndFtsH mutants were grown under inducing (Lon+ or FtsH+) or depleting conditions (Lon+ or FtsH+, 60h of depletion for both, Lon and FtsH), and then exposed at 45°C during 0, 5, 10, 15 or 20 min. Then, growth after the heat treatment was monitored over time under inducing conditions by the 430/560 absorbance rate index that shows pH changes in the medium. The average from two independent biological replicates is shown for each condition.
Figure 4(A) Experimental scheme of pulse-labelling of DNA fibers in wild-type cells (shWRNIP1WT) or WRNIP1-deficient cells (shWRNIP1). Cells were labelled with IdU and exposed or not to 25 μM RAD51 inhibitor, then treated or not with 4 mM HU.(B) Representative IdU tract length distributions in shWRNIP1WT cells (left graph) or shWRNIP1 cells (right graph). Median tract lengths are reported in parentheses. See Tables S1 and S2 for details on the data sets and statistical test. Representative DNA fiber images are reported. Scale bars, 10 µm. (C) Scheme of DNA fiber tract analysis in shWRNIP1 cells. Cells were transfected with an empty vector or a plasmid expressing a wild-type human RAD51, and 48 h thereafter labelled with IdU and treated or not with 4 mM HU.(D) Representative IdU tract length distributions in shWRNIP1 cells or shWRNIP1 cells expressing exogenous wild-type RAD51 after HU exposure. Median tract lengths are given in parentheses. See Tables S1 and S2 for details on the data sets and statistical test. Representative DNA fiber images are given. Scale bars, 10 µm. Western blot shows the expression of the RAD51 protein in shWRNIP1 cells. The membrane was probed with an anti-RAD51. LAMIN B1 was used as a loading control.
24F4A induces rapid internalization of BDCA2 resulting in trafficking to LAMP1+ compartments and sustained IFNα inhibition.Figure 2A Whole blood was treated with the indicated concentrations of 24F4A or the IgG1 isotype control antibody for 0, 1, 3, 6, 9, and 24 h. FACS analysis was performed to determine BDCA2 levels on pDCs. Fluorescence minus one (FMO) represents background staining of BDCA2. Shown is a representative plot of three independent experiments. B Isolated pDCs were treated with 10 μg/ml 24F4A-AF647 at 4°C or at 37°C and analyzed at the indicated time points. Subcellular localization of BDCA2 (red) compared to LAMP1 (green) was assessed using confocal microscopy. Phalloidin was used to delineate the cell membrane. Yellow represents co-localization of BDCA2 and LAMP1. Shown is a representative image of four experiments conducted. C, D Whole blood was either treated with 24F4A for 1 h at 37°C (pre-incubation) or left untreated. PBMC were isolated from each condition. PBMC from the untreated whole blood were subsequently treated with 10 μg/ml 24F4A, and cells from all conditions were stimulated with CpG-A for 16 h. Plot represents mean of duplicate wells. Flow cytometry was used to measure BDCA2 levels (MFI). Shown is a representative donor of eight donors tested. E Whole blood was pre-treated for 0, 1, 3, 6, and 9 h with 10 μg/ml of 24F4 or the isotype control and then stimulated with CpG-A for additional 16 h. IFNα levels were measured by ELISA. Shown is a representative plot of two independent experiments.