EMBO/sd-character-level-ner huggingface dataset:
| RNR inhibitor and ara-C synergy is dependent upon functional SAMHD1 in cancer cell models.Figure 1Schematic detailing proposed interplay between RNR and SAMHD1. Immunoblot of lysates prepared from the indicated SAMHD1-proficient (+/+), deficient (-/-) and rescue (WT, H233A) cell line pairs with the indicated antibodies. Representative of 2 independent experiments. Proliferation inhibition analysis of ara-C and RNRi combination treatment in SAMHD1+/+ or -/- THP-1 cells. Error bars indicate s.e.m. of two (HU and dF-dC) or three (3-AP) independent experiments, each performed in duplicate. Ara-C EC50 values plotted as a function of RNRi concentration in SAMHD1+/+, -/- and rescue (WT, H233A) THP-1 cell line pairs. EC50 values in the absence of RNRi are indicated by the black and red dotted line. Error bars indicate s.e.m. of two (HU and dF-dC) or three (3-AP) independent experiments, each performed in duplicate. Drug synergy plots for ara-C and the indicated RNRi in SAMHD1+/+, -/- and rescue (WT, H233A) cell line pairs. Each data point indicates an average delta score from a single dose-response matrix experiment performed in duplicate. Zero, >0, or <0 corresponds to additive, synergy, or antagonism, respectively, whilst >5 indicates strong synergy. The horizontal line and the error bars indicate the mean and s.d., respectively, statistical significance was determined using a two-tailed unpaired t-test: ns, not significant, P ≥ 0.05; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. Spearman correlation of relative SAMHD1 protein abundance and synergy delta scores for ara-C versus HU or dF-dC in a panel (n = 9) of haematological cancer cell lines. Error bars indicate s.e.m. Each data point corresponds to SAMHD1 protein levels determined by immunoblot analysis (n = 4 for each cell line, representative blot shown in Appendix Fig S2) and an average delta score from repeated dose-response matrix experiments each performed in triplicate: THP-1, n =4; HuT-78, n = 2; HL-60/iva, n = 1; KBM-7, n = 2 (HU) and 3 (dF-dC); K562, n = 3 (HU) and 4 (dF-dC); CCRF-CEM, n =3 (HU) and 4 (dF-dC); MV-4-11, n = 2 (HU) and 3 (dF-dC); Jurkat, n = 2 (HU) and 3 (dF-dC); MOLT-4, n = 2 (HU) an 3 (dF-dC). Immunoblot analysis of lysates prepared from SAMHD1+/+ or -/- THP-1 cells treated for 24 h with ara-C and HU, as indicated. Representative of 3 independent experiments.
Quantitative analysis of Pom1 gradient reveals two systems’ properties buffering against high variability of Tea4 at cell poles.Figure 1The standard diffusive gradient (top) is exponential and its decay length λ (the distance at which the concentration is reduced to some fraction of its amplitude) is independent from the amplitude. Variations in amplitudes are therefore not buffered and translate into large variations in positional information L. By contrast, a gradient that reduces its decay length at high amplitudes (bottom) buffers variations in amplitude and conveys more robust positional information (L1≈L2). Pom1-tdTomato (red) and Tea4-GFP (green) profiles were quantified along the cortex of the same Schizosaccharomyces pombe cells. Curves are averages from 385 individual profiles. Smoothed Pom1 gradients averaged in batches of 5% (n=19 or 20) from lowest to highest Tea4 amplitude. Amplitudes at the pole show a high variability. Corresponding smoothed and averaged Tea4 profiles. The variability at the pole is even higher than for Pom1. Pom1 gradients decay length decreases with the amplitude at the pole. Each dot corresponds to one average profile as in (C). The trans-phosphorylation model predicts a slope of -1/2 in log-log space (red line), close to the observed linear regression of -0.52 (black line) and well within the two SE confidence interval (shaded area). Each data point is weighted by its inverse variance of the mean in the linear regression. Pom1 amplitude increases with Tea4 concentration at the pole. Linear regression (black line) in the log-log scale shows a slope smaller than one indicative of a sub-linear relationship between Pom1 and Tea4 at the pole (see also Fig. 2B) and thus buffering of Tea4 fluctuations. Each dot corresponds to one average Tea4 and corresponding Pom1 profiles as in (C-D). The trans-phosphorylation model predicts the slope to be 2/3 (red line), close to the observed 0.63 (black line) and within the two SE confidence interval (shaded area). Each data point is weighted by its inverse variance of the mean in the linear regression.
Model of HOTAIR activity.Figure 6In epithelial cells the LSD1-interacting domain of HOTAIR perturbs LSD1 function at enhancers and promoters resulting in alteration of the transcriptional response and change in expression of genes involved in control of cell migration.
Fig.Figure 6(A) Solution NMR structure of cytosolic N-terminal domain of YqgP (YqgPNTD, amino acids 1-177), showing ensemble of 30 structures with lowest energy, and the structure with minimum energy displayed as cartoon. (B) Electrostatic surface of YqgPNTD structure reveals highly negatively charged areas. Left view is oriented as the left view in panel (A). (C) Chemical shift perturbation values and relative peak intensities for backbone resonances (15N and 1H) of YqgPNTD calculated for complexes of 400 μM YqgPNTD with 10 μM (light grey, light orange) or 40 μM MnCl2 (dark grey, dark orange bars). Only residues 20-100 of YqgP are displayed for simplicity. Residues identified as potential manganese-binding region based on their chemical shifts and intensity changes upon NMR titrations are depicted in orange. The X sign marks residues whose chemical shifts were not calculated due to the lack of resonance signal. (D) Details of 2D 15N/1H HSQC spectra of free (green) and Mn-titrated YqgPNTD (10 μM MnCl2 in red and 40 μM MnCl2 in blue) for the residues marked in orange in panel (C). As a reference, residues marked in black do not display spectral changes upon titration, e.g. peak intensity does not change for S88N-H upon Mn titration (overlay of green, blue and red contours), but peak intensity is severely depleted for D29N-H upon Mn titration (absence of red contours). Full titration data are available in Dataset S3. (E Detection of steady-state conversions of endogenous MgtE by YqgP variants bearing single-point mutations in putative Mn-binding region (BS184; 187; 196-203, Table EV2), cultivated in modified M9 minimal medium supplemented by low (1 μM) or high (100 μM) MnCl2. Black arrow marks full-length MgtE, and red arrow marks its N-terminal cleavage product by YqgP. F) quantification of steady-state conversions of endogenous MgtE by YqgP variants bearing single-point mutations in putative Mn-binding region (BS184; 187; 196-203, Table EV2), cultivated in modified M9 minimal medium supplemented by low (1 μM) or high (100 μM) MnCl2. (G) Overview of residues affected by Mn2+ binding to YqgPNTD mapped onto its solution NMR structure. The view is oriented as the left view in panels (A and B).
Fig.Figure 2Mice were intranasally given 1011 vg of AAV-vector in 50 µl volume three times in three-week intervals. 20 µg of Cal/7/9 WIV per 50 µl was given i.n. two times. Blood samples were taken at indicated time points (red drops). After influenza challenge, mice were monitored for survival and weight loss for two weeks before necropsy. Total IgG ELISA titers expressed as area under the curve (AUC) against homologous Cal/7/9 virus of pre-immune and pre-challenge sera of individual animals of the indicated vaccine groups (AAV-HA, -cHA, -GFP, WIV n = 18, AAV-NP n = 11). Background reactivity of pooled AAV-GFP pre-challenge serum is shown as dashed line. ELISAs were performed in technical duplicates. IgA ELISA titers in post-challenge lung homogenates against Cal/7/9 of individual mice of the indicated vaccine groups (AAV-HA, -cHA, -GFP, -NP, WIV n = 11). of the Cal/7/9 (blue symbols) and PR8 low dose (red symbols) challenge groups. ELISAs were performed in technical duplicates. D to G Total IgG ELISA titers expressed as log10 of the mean AUC against indicated viruses in pre-challenge sera in AAV-HA (D, n = 18), AAV-cHA (E, n = 18), AAV-NP (F, n = 11) or WIV (G, n = 18) immunized animals (grey area). Reactivity of sera of AAV-GFP immunized animals is indicated as white area in the center of each web diagram. ELISAs were performed in technical duplicates.
Figure 1A) Neutrophils and lymphocytes count. B) Levels of creatine kinase (CK) and creatinine. C) Levels of procalcitonin (PCT) and C-reactive protein (CRP). D) Levels of erythrocyte sedimentation rate (ESR) and ferritin. E) Levels of D-dimer and lactate dehydrogenase (LDH). F) Proportion of total CD3+ and CD4+ T cells. G) Proportion of CD8+ T cells and CD4+/CD8+ T cell ratio. H) Proportion of B and natural killer (NK) cells. cytokine profile of COVID-19 patients for IL-2, IL-4 (I) cytokine profile of COVID-19 patients for , IL-6, IL-10 (J) and TNF-α and IFN-γ (K).
Fig.Figure 1p53+/+ and p53-/- C57BL/6N male mice were treated as in A. Data are means ± SD (n=6 to 8). B Representative liver photos of mice livers. C Representative changes in liver weight. D H&E (hematoxylin-eosin) staining (left) and oil red O staining (right) of livers. Scale bar, 50μm. E Serum cholesterol concentrations of each group. Liver cholesterol concentrations (F) of each group were examined. Liver triglyceride levels (G) of each group were examined. H and I Cholesterol levels in p53+/+ and p53-/- HepG2 cells (H) or HCT116 cells (I) were examined. Protein expression was shown by western blotting (bottom panel). J Cholesterol concentrations of p53+/+ and p53-/- HCT116 cells were determined by FilipinⅢ staining. K Heat map analysis of 17 sterol biosynthesis genes and 3 other SREBP2 target genes from RNA-seq data using mice livers of p53+/+ and p53-/- mice fed with normal or HFD diet. Expression levels were normalized to the mean level of each gene among all samples and compared to p53+/+ normal diet mice. Color scale indicates the expression fold change of target gene. L Enrichment of cholesterol biosynthesis genes in the liver of normal diet (left) and HFD diet (right). FDR: false discovery rate; NES: normalized enrichment score.
Fig.Figure 4(A) Magnesium intake in 34-week-old untreated and treated LmnaG609G/+ mice (n=16). (B) Plasma magnesium concentration in 34-week-old untreated and treated LmnaG609G/+ mice (n=16). (C) Calcium content of aortas obtained from 34-week-old wild-type mice and untreated and treated LmnaG609G/+ mice (n=20). (D) Body masses of 34-week-old untreated and treated LmnaG609G/+ mice. (E) Kaplan-Meier graph for untreated and treated LmnaG609G/+ mice (n=16). (F) Representative photographs of 40-wk-old wild-type, untreated and treated LmnaG609G/+ mice.
a) Domain organization of FKBP38, Omp25 and the chimeric proteins Omp25/FKBP38 and FKBP38/Omp25, as well as a summary of their localization and ability to undergo translocation during mitophagy as determined in (b).f4(a) Domain organization of FKBP38, Omp25 and the chimeric proteins Omp25/FKBP38 and FKBP38/Omp25, as well as a summary of their localization and ability to undergo translocation during mitophagy as determined in (b). (b) MEFs stably expressing FLAG-Parkin and EGFP-tagged forms of FKBP38, Omp25, Omp25/FKBP38 or FKBP38/Omp25 were treated with CCCP (30 μM) for 4 h, fixed, permeabilized and subjected to immunofluorescence analysis with antibodies to cytochrome c and to FLAG (rabbit polyclonal). The fluorescence of EGFP was also monitored directly. Scale bar, 10 μm. (c) MEFs stably expressing mCherry-tagged Parkin and EGFP-tagged forms of FKBP38, Omp25, Omp25/FKBP38 or FKBP38/Omp25 were incubated in the absence or presence of CCCP (30 μM) for 24 h and then subjected to immunoblot analysis.
Figure 3A knockout cell line of MITRAC15 (MITRAC15-/-) was created in HEK293T cells by using CRISPR/Cas9 technology. Alignment of respective mutated region compared to wild type shows premature STOP codons (*) in MITRAC15-/-. Mitochondria from wild type and MITRAC15-/- cells were isolated and subjected to steady-state protein analyses by western blotting. Mitochondria from wild type and MITRAC15-/- cells were isolated and analyzed by BN-PAGE. Triton X-100 (left site) or digitonin (right site) were used for solubilization. The amount of complex I was quantified utilizing the NDUFS1 signal and normalized against SDHA levels (error bars indicate SEM from three biological replicates). Complex I and citrate synthase activity were measured in WT and MITRAC15-/- cells (error bars indicate SEM from three biological replicates; *p < 0,05; unpaired t-test). Pulse-chase [35S]methionine radiolabeling of mitochondrial translation products in wild type and MITRAC15-/- cells. Samples were analyzed by SDS-PAGE and digital autoradiography. Signal intensity of ND2 was normalized to the ATP6 signal and the relative ND2 stability was calculated by relative chase ND2 signal/relative pulse ND2 signal (error bars indicate SEM from three biological replicates; *p < 0,05; unpaired t-test). Pulse radiolabeling after re-expressing MITRAC15FLAG in MITRAC15-/- cells. Samples were analyzed by SDS-PAGE and digital autoradiography. Relative pulse signal intensity of ND2 was normalized to ATP6 (error bars indicate SEM from three biological replicates; *p < 0,05; **p < 0.01; unpaired t-test).
Figure 1A, Schematic diagrams of full-length (FL), N-terminus (NT) and C-terminus CCT domain of TOC1 used in this manuscript. The 5 phosphorylated residues identified from our mass spectrometry were progressively substituted by alanine, and their positions are displayed on top of TOC1FL. B, Mutations in S175 and 5X attenuate PRR5's enhancement on TOC1 phosphorylation. Wild type and phosphosite mutants of TOC1NT were transiently expressed with or without PRR5 in N. benthamiana leaves, total proteins were extracted 3 days after infiltration. C, Phosphorylation of TOC1 is eliminated or largely reduced in 5X and S175A. Total protein extracts (ZT13) from 10-day-old seedlings grown under 12h/12h light/dark cycles were subject to heat (30ºC 20min), pyrophosphatase (PP) and NaF/Na3VO4 (phosphatase inhibitors) treatments. TOC1-GFP at different phosphorylation states were separated by 8% SDS-PAGE (acrylamide:bisacrylamide 150:1) and subsequently detected by α-GFP.
TGIF1 inhibits Twist1 transcriptional activity A PL45 stably expressing both Dox-inducible TGIF1 and Tam-inducible Twist1 (Twist1ER) were treated with Tam and/or Dox for 48 hr and analyzed for Twist1 expression by immunoblotting or qRT-PCR (n=6).Figure 4PL45 stably expressing both Dox-inducible TGIF1 and Tam-inducible Twist1 (Twist1ER) were treated with Tam and/or Dox for 48 hr and analyzed for Twist1 expression by immunoblotting or qRT-PCR (n=6). MIAPaCa-2 cells were transfected with wild-type or mutated (Twist1 binding site) Twist1 luciferase reporters (TGTLuc) together with increasing amounts of Twist1 expression vector in the presence of empty vector (EV) or TGIF1 expression vector. Luciferase activity was measured 48 hr following transfection and normalized on the basis of co-transfected Renilla luciferase (n=6). Total lysates from pancreas of control, KC or KTC mice were pooled and analyzed by immunoblotting using antibodies to E-Cadherin, Vimentin, p16Ink4A and β-Actin (n=6). Expression of Cdh1, Vimentin, and p16Ink4A in pancreas from control, KC or KTC mice (same as in C) was analyzed by qRT-PCR (n=6). FFPE pancreatic sections from control, KC or KTC mice were stained with H&E or immunostained with antibodies to E-Cadherin, Vimentin or p16Ink4a and revealed by IF or IHC. Representative pictures at 20x are shown (n=30). Scale bars, 200 μM.
Figure 6At steady state, nuage and piP-body accommodate piRNA factors both stably localized at the condensate or transiently docked to it. Siwi, while does not accumulate in piP-body, transiently enters piP-body and interacts with BmSpnE and BmQin. An active slicer is required for Siwi to segregate from piP-body and go back to nuage, where BmVasa uses its ATPase to remodel RNP interaction and modulates the ping-pong cycle. Some Siwi-interacting factors like BmMael and BmArmi can enter multiple compartments where Siwi is present. When Siwi-D670A is expressed, the slicer mutant is expelled from nuage by RNP remodeling activity of BmVasa and is trapped in piP-body while co-aggregating with piP-body factors, as well as those freely shuttling between compartments. A hypothetic fidelity determining factor could be similarly trapped in piP-body by the mutant, resulting in the up-regulation of non-TE piRNAs. This production could be done in nuage, where native piRNA biosynthesis machineries may still function. Of note, the length distribution and the presence of 2′-O-methyl modification in non-TE piRNAs suggest that these piRNAs are likely to be processed on the outer mitochondrial membrane and in the cytoplasm. It is also possible that non-TE piRNA is directly generated from piP-body, where a battery of piRNA biosynthesis factors abnormally accumulated.
Figure 1(a) Transverse slice of live E10.5 Venus::HES5 homozygous knock-in mouse showing the ventral HES5 domain in spinal cord ex vivo (left panel); Draq5 live nuclear stain with nuclear segmentation overlay (right panel); scale bar 30 μm. (b) Venus::HES5 nuclear signal corresponding to tissue in (a) obtained by applying nuclear segmentation onto Venus channel. (c) Pseudo-color look-up table applied to mean nuclear Venus::HES5 intensity (Materials and Methods) corresponding to segmented image in (b). Black line surrounding representative microclusters. (d) Dimensions of microclusters in cell numbers with high and similar levels of HES5 in apical-basal axis (left panel) and dorso-ventral axis (right panel) at E9.5 (10 microclusters, 3 slices, 3 exps), E10.5 (10 microclusters, 9 slices, 3 exps) and E11.5 (10 microclusters, 3 slices, 3 exps). NS - no significant difference in one-way ANOVA p=0.46 (A-B), p=0.38 (D-V). Bars show mean ±SD. (e) Pearson correlation coefficient observed in segmented E10.5 homozygous Venus::HES5 spinal cord ex-vivo slices showing correlation between mean nuclear Venus::HES5 intensity in any cell compared to up to 8 nearest neighbours (see Materials and Methods); dots indicate average per slice; bars indicate mean and standard deviation of 5 slices from 3 experiments (dataset is different from (d). (f) Pearson correlation coefficient of mean nuclear Venus::HES5 intensity in relationship to distance; red dots indicate average Venus::HES5 correlation per slice of 12 slices from 3 experiments with corresponding red line indicating polynomial fit (order 2); gray dots with black line indicate correlations and polynomial fit from 5 randomizations of intensities analysed in the same way (see Materials and Methods). (g) Transverse slice of live E10.5 Venus::HES5 homozygous knock-in mouse showing the ventral HES5 domain in spinal cord ex vivo. Scale bar 30 μm, D- dorsal, V- ventral. (h) Pseudo-color look-up table applied to mean nuclear Venus::HES5 intensity of (g); centre of intensity shown with *. (i) Pseudo-color look-up table applied to mean nuclear Venus::HES5 intensity in (h) (only 1 side of ventricle) after radial gradient removal (see Materials and Methods). (j) Pearsons correlation coefficient of mean nuclear Venus::HES5 intensity with distance after subtraction of radial gradient in Venus::HES5 intensity; red dots represent average in each of 12 slices from 3 experiments.
Zebrafish paqr3b regulates GABAergic neuron development A-B.Figure 5A-B. GABAergic neuron defects in wild-type fish treated with ER/Golgi traffic inhibitor BFA (n=9; ****p<0.0001; ns, not significant; One-way ANOVA). C-D. Overexpression of paqr3b mRNA improve the number of GABAergic neurons in 3dpf chd7 mutant fish (n=7 for chd7-/- and n=11 for chd7-/- + paqr3b mRNA; ****p<0.0001; **p<0.05; One-way ANOVA). Of note, the rescue experiment was performed at 3 dpf given the transient nature of mRNA. E. ERK activation by phosphorylation in chd7-/- mutants was restored to normal levels upon overexpression of paqr3b mRNA shown by Western blot quantification (N=4; **p<0.05; One-way ANOVA, N is the number of experimental repeats.
Figure 5Characterization of the nanoscale organization of GABAARs and GlyRs at mixed inhibitory synapses. (A) Synaptic receptor cluster rendered from detections of both GABAARs (labelled with Alexa 647) and GlyRs (labelled with Cy3B). (B) Detections of GABAARs (red dots) and GlyRs (blue dots) overlaid with binary synaptic mask (white). Characterization of the nanoscale organization of GABAARs and GlyRs at mixed inhibitory synapses. (C-E) Detections of GABAARs, but not GlyRs were strongly decreased at mixed synapses after 4-AP treatment (KS test, p < 0.0001 in C and E, p < 0.001 in D). Scale bar: 200 nm. Sub-synaptic characterization of GABAAR and GlyR domains. (F) Watershed segmentation of sub-synaptic domains (SSDs). Each SSD is shown in different colors. (G) Detections of GABAARs (red dots) and GlyRs (blue dots) overlaid with SSDs (white). Sub-synaptic characterization of GABAAR and GlyR domains. (H-J) Detections of GABAARs but not GlyRs at the same SSDs were reduced after 4-AP treatment (KS, p < 0.0001 in H and J, p < 0.01 in I). Number of synapses: n = 439 in TTX, n = 531 in 4-AP condition, from three independent experiments.
Figure 5A, Immunoblots of H358 and A549 cells treated for 24h with vehicle (DMSO), AZD 4547 (5 µM) and BI2536(5 nM), alone or in combination. B, A549 cells stably expressing mCHerry-GFP-LC3B were exposed to vehicle (DMSO), AZD4547 (5 µM) and BI2536 (5 nM), alone or in combination for 24h. Cell were then fixed and processed for immunofluorescence, scale bar=10 µm. C, A549 and H358 cells expressing mCHerry-GFP-LC3B were treated with vehicle (DMSO), AZD 4547 (5 µM) and BI2536 (5 nM), alone or in combination for 24h before flow cytometry-based analysis for mCherry and GFP (upper). Shown underneath is quantification of the data. **P<0.01 and ***P<0.001 by two-way ANOVA with Tukey's multiple comparisons test. Data are shown as mean ± SD (error bar) of three independent experiments (n=3). D, Apoptotic assay of A549 and H358 cells treated with vehicle (DMSO), AZD 4547 (5 µM) and BI2536 (5 nM) for 48h in the presence or absence of HCQ (10 µM). Data are presented as mean ± SD (n=3). *P<0.05 and ns P＞0.05 by two-way ANOVA with Tukey's multiple comparisons test. E, Flow cytometry-based ROS measure in H358, A549 and MIA PaCa-2 cells treated with vehicle (DMSO), AZD4547 (5 µM) and BI2536 (5 nM), alone or the combination, in the presence or absence of HCQ (10 µM) for 24h. Quantification of relative ROS levels was shown to the right. Data are presented as mean ± SD (n=3). *P<0.05, **P<0.01, ****P<0.0001 and P＞0.05 (ns) by two-way ANOVA with Tukey's multiple comparisons test. F, Immunoblots of H358 cells transfected with ATG5-specific or control siRNAs for 48h and before treated for 24h with vehicle (DMSO) or combined AZD 4547 (5 µM)/BI2536 (5 nM).
Functional VCP is required for autophagic protein degradation.Figure 4.(A) Lysates from siRNA-treated U20S cells (scramble control [Scr] or VCP siRNA KD) after autophagic induction via nutrient deprivation for 0, 2, or 4 h and immunoblotting for LC3 and α-tubulin. LC3II degrades over time in control but not in KD cells. One of two experiments is shown. (B) Lysates from U20S or tetracycline-inducible VCP-WT, -EQ, -RH, or -AE cells after autophagic induction via nutrient deprivation for 0, 2, 4, or 6 h and immunoblotting for LC3 and α-tubulin. LC3II degrades in control and VCP-WT but degrades less efficiently in mutant (EQ, RH, or AE)-expressing cells. (C) Graphical representation of densitometric evaluation of LC3II and α-tubulin at each time point from three independent experiments.