EMBO/sd-character-level-ner huggingface dataset:
Figure 1A. Representative photomicrographs of mouse liver sections following a two-thirds PH of Ki67 positive hepatocytes (brown) visualized by IHC. Images are representative of 3 independent animals per time point. B. Western blot detection of p-MST1, MST1, p-LATS1, LATS1 from proteins isolated from homogenised liver tissue at several time points. TBP was used as a loading control. Results of a single experiment with n = 3 animals per group. Each band is from an independent animal. Protein levels were normalized to TBP and mean value of the ratio of p-MST to total MST and p-LATS to total LATS is plotted. C. Nuclear enriched protein fractions obtained from sham-operated livers and liver 48 h post PH were analyzed by Western blot for YAP expression. Sp1 was used as a loading control. Image is representative of a single experiment with n = 3 animals per group. D. Photomicrograph of YAP1 expression in sham and regenerating liver 48 h post PH visualized by immunoflourescence microscopy. Images are representative of 3 independent animals per condition. E. Livers from sham and 48 h following PH were digested to isolate the hepatocyte population. Protein extracts were analyzed by Western blot for YAP and TAZ expression. β-actin was used as a loading control. Results of a single experiment with n = 2 animals per group. Each band is representative of an independent animal. F. RT-PCR analysis of RNA isolated from mouse liver for Birc5, FoxM1B and cyclin B1. Data are presented as log2 fold change and were calculated using non-operated mouse liver tissue as a control. Unpaired, two-tailed Student t-test was used to determine the significance between the log2 values of livers 6, 24 and 48 h post PH compared to the sham operated controls. Log2 values are plotted on a linear scale as mean ± SD.
Figure 2(A) Classification of the wing size-associated RNAs at FDR < 20% based on association types. RNAs that exclusively belong to the association type 1 (without overlaps with association type 2) have a type-3 association with wing size. (B) Association of RNAs with the whole wing size variation that encompasses both sexes. Volcano plot of p-values against the slope of the fitted lines. The horizontal lines indicate 10 and 20 % FDR thresholds. (C) Association of RNAs with between-line wing size variation (absolute/ relative CS, adjusted for sex). (D) GO enrichment analyses on the wing size-associated RNAs (at FDR < 20%). Biological processes associated with wing size at FDR < 0.1 are shown. (E) Association of canonical growth regulator transcripts with wing size (absCS and relCS). P-values from the tests for association type 2 (ANCOVA) and type 3 (ANOVA on sex) are plotted. The dashed and dotted lines indicate 10% and 20% FDR thresholds, respectively. Note that canonical growth genes mostly exhibit type-3 association. (F) Sexually dimorphic expression pattern of canonical growth genes. Plots of RNA levels confirm type-3 association with wing size. (G) Exceptional growth genes that are associated with between-line size variation (association type 2).
Figure 5Filter trap binding assays show that ISR proteins are enhanced by all stressors. Significant increases following cellular stress treatments are seen in the ratio of phospho-eif2α to total eif2α as well as phospho-PERK and ATF4 levels compared to untreated controls. Changes in ISR proteins are normalized to GAPDH and shown relative to non-stressed or DMSO-only treated cells (CTRL) depending on the vehicle compounds were dissolved in. n = 4. Fluorescent intensity measurements show the levels of two ISR-related proteins, PERK and relative phospho-eif2α (phospho-eif2α / total eif2α), are increased following different excitotoxic stress treatments in cortical neurons. Representative images indicate fluorescent intensity differences for phospho-eif2α and phospho-PERK levels 24 h post treatment with different excitotoxic stressor paradigms. Blocking of AMPA and NMDA receptors using known antagonists significantly reduces ISR-related protein levels, which parallels reduced DPR levels (Figure 3B). Changes in ISR proteins are relative to non-stressed cells (CTRL).
IFN signaling is decreased in bone-metastatic PCa (A-C) Compositional analysis of RNA-seq data (FDR < 0.Figure 7Compositional analysis of RNA-seq data (FDR < 0.05; GLM by edgeR) from bone metastases (n = 9) and primary tumors (n = 12) revealed (A) immune cells and (B) tumor cells were relative (% of total) across all samples, with a mean tumor purity >85% as determined by ARMET. Median shown. Upper and low box hinges denote first and third quartiles. Upper and lower whiskers mark the values ± 1.5IQR from the hinge. Data points beyond the whiskers are shown individually. p values not significant by Wilcoxon rank sum test (R). Hallmark gene set responses associated with bone metastasis by camera analysis shows (C) enrichment of type I (IFN-α) IFN responses in suppressed genes (barcode plot). (D) Heatmap of type I IFN DE IRGs (normalized log2CPM; counts per million) significantly suppressed in bone metastases (n = 9) compared to primary (n = 12) and benign (n = 3) tumors, grouped by type I IFN specific, and type II and III IFN co-regulation status. Scale truncated at ± 5 relative log2CPM. GLM by edgeR. (E) IHC for IRF9 expression in primary prostate tumors and bone metastases with matched samples indicated. IRF9 indicated by brown (DAB) staining. Scale bar represents 100 μm. Full-face slides shown in Appendix Fig S7C. (F) Heatmap of HLA genes (normalized log2CPM) suppressed (p <0.05; FDR <0.05) in bone metastases (n = 9), primary tumors (n = 12) and benign tumors (n = 3). Scale truncated at ±5 relative log2CPM. GLM by edgeR. (G) CIBERSORT analysis of absolute leukocyte proportions (arbitrary units, AU) in mRNA samples of bone metastases (n = 9), primary tumors (n = 12) and benign tumors (n = 3). p values by Mann-Whitney U. (H) Kaplan Meier Curve of human prostate (n = 499 from 498 TCGA samples) biochemical recurrence based on primary tumor alterations in a core 8-IRG signature (Appendix Fig S7D). Patient groups stratified by mRNA z-Score (RNA-seq V2 RSEM) ± 1.5. Hazard ratio 1.674 (95% confidence interval (CI) 2.707 to 1.035); *p = 0.03 by log-rank test.
AMPK phosphorylates Ser121 to inactivate HSF1.Figure 3A In HEK293T cells that stably express an shRNA targeting the 3′ UTR of human HSF1, HSF1WT or HSF1S121A was expressed. Following treatment with 10 mM metformin overnight, the levels of HSF1 phosphorylation at Ser121 were detected by immunoblotting using a specific phospho-HSF1 Ser121 antibody (A8041, Assay Biotechnology). B Primary Ampkα1fl/fl; Ampkα2fl/fl MEFs were transduced with adenoviral GFP or Cre. Following treatment with 10 mM metformin overnight or treatment with 10 μM A-769662 for 3 h, HSF1 Ser121 phosphorylation was examined by immunoblotting. C HSF1WT or HSF1S121A was co-expressed with either GFP or GST-AMPKαCA in HSF1-deficient HEK293T cells. HSF1 Ser121 phosphorylation was examined by immunoblotting. D AMPK complexes were immunoprecipitated from HEK293T cells treated with and without 10 mM metformin overnight. Aliquots of AMPK complexes were incubated with 400 ng purified recombinant His-tagged HSF1 proteins with and without 100 μM AMP or 20 μM compound C (CC). HSF1 Ser121 phosphorylation was detected by immunoblotting. E, F HSF1 activities were measured by the dual reporter system in HSF1-deficient HEK293T cells. Either FLAG-HSF1WT or FLAG-HSF1S121A was co-expressed with LacZ orAMPKα1CA (E). E, F HSF1 activities were measured by the dual reporter system in HSF1-deficient HEK293T cells. Either FLAG-HSF1WT or FLAG-HSF1S121A was co-expressed with LacZ or AMPKα1CA (E). Following expression of HSF1WT or HSF1S121A, cells were treated with and without 10 mM metformin overnight (F) (mean ± SD, n = 5, one-way ANOVA, n.s., not significant, *P < 0.05, **P < 0.01, ***P < 0.001). G, H FLAG-HSF1WT or FLAG-HSF1S121A plasmids were co-transfected with LacZ or AMPKα1CA plasmids into HSF1-deficient HEK293T cells for 3 days (G). Following transfection with FLAG-HSF1WT or FLAG-HSF1S121A plasmids for 3 days, HSF1-deficient HEK293T cells were treated with and without 10 mM metformin overnight (H). Nuclear proteins were extracted to measure HSF1-DNA binding by the ELISA-based DNA binding assay using anti-FLAG antibodies (mean ± SD, n = 3, one-way ANOVA, n.s., not significant, *P < 0.05, **P < 0.01, ***P < 0.001). I, J Following transfections and metformin treatment as described in (G) and (H), cytoplasmic and nuclear proteins were extracted for immunoblotting using anti-FLAG antibodies.
Figure 1A, Schematic illustration of the nuclear envelope, depicting the link between the lamina, SUN proteins, giant nesprins and actin. B, Mouse embryonic fibroblasts genetically modified using CRISPR to introduce a GFP sequence immediately after the start codon of Syne2 display fluorescence at the nuclear envelope. C, Brightfield and fluorescence images of a cell migrating through a 2 µm constriction. Arrows indicate areas of accumulation of nesprin-2 at the front of the nucleus as it is deformed through the constriction. D, Schematic illustrating the 5 time points studied in the analysis: Before nucleus deformation ("Before"), once the front of the nucleus reaches the middle of the constriction ("Front@mid"), once the middle of the nucleus reaches the middle of the constriction ("Middle@middle"), a timepoint halfway between Front@mid and Middle@middle ("between"), and late time point after the deformation ("After"). E, Brightfield and fluorescence images of a cell migrating along a 15 µm control channel. F, Quantification of the intensity of the fluorescent signal around the perimeter of the nucleus in a 2 µm constriction and a 15 µm control channel. The average intensity at the front and the back is normalized to the intensity at the sides, demonstrating an increase in the signal at the front of the nucleus as it is deformed and a reduction after deformation. Error bars correspond to the standard deviation. Constriction: n=17 cells over 4 biological replicates, 15 µm channels: n=20 cells over 3 biological replicates. A Wilcoxon signed-rank test is used to compare matched samples, stars (*P < 0.05, **P < 0.01) denote comparisons to the measurement at the side, and pounds (#P < 0.05, ##P < 0.01) denote a comparison to the "before" time point. G, GFP-nesprin-2 cells deformed by the pressure gradient in a micropipette aspiration device. Note that the GFP signal does not increase at the tip but increases at sides of the nucleus.
Figure 7Schematic overview of the different phenotypes observed in the retina, optic nerve, thalamus and visual cortex of Nr2f1 Heterozygous (HET) and Knock-out (KO) mice. Some of the defects characterized in the mouse model (green boxes) are reminiscent of the major features reported in BBSOA patients (light blue boxes). Nr2f1 haploinsufficiency causes a shift of NR/OS molecular domains (in blue), leading to aberrant positioning of the optic stalk (OS) and ultimately optic disc (OD) malformations at early stages. Pale and abnormal ODs have also been reported in human patients. Optic nerve (ON) atrophy, the most characteristic feature of BBSOA patients, is most probably caused by: (i) delayed RGC differentiation (red cells) at early embryonic stages and increased apoptosis at perinatal stages; (ii) affected oligodendrocyte migration and maturation (green cells) leading to ON fiber demyelination and reduced volume; (iii) reduced axonal innervation and connectivity to the atrophic thalamic dLGN and V1 neocortex; (iv) reactive gliosis consistent with astrocyte inflammation and (v) possible associated mitochondrial dysfunctions. All these defects identified in Nr2f1 mutants contribute to form a reduced and hypomyelinated ON and ultimately affect visual axonal conduction velocity. While the myelination defect could be rescued by Miconazole treatment (blue box), the high proportion of astrocyte precursors and reactive astrocytes (yellow cells) leading to gliosis could not be rescued and might lead to ON degeneration at adult stages. The majority of these defects are already present in HET but further worsened in KO mice, suggesting a gene dosage effect during development of the visual system (red gradient).
Figure 2A. Concentration of the various indicated isotypes in the serum of 6-8 week old unimmunized WT and Shld2−/− mice. Values are mean concentration ± SD of 4 biological replicates; ** P ≤ 0.01, unpaired two-tailed t-test. B. B cells were purified from spleens from WT and Shld2−/− mice, and stimulated to undergo CSR to the various indicated isotypes using different stimulation cocktails [33]. Cells were then analyzed by flow cytometry for expression of the various indicated isotypes and the percent expression of each isotype are reported. Values are mean frequency ± SD of 4 biological replicates; **** P ≤ 0.0001, unpaired two-tailed t-test. C. WT, 53bp1−/−, and Shld2−/− mice were immunized with NP-CGG and the serum was withdrawn 2 weeks post immunization and serial dilutions were subjected to ELISA analysis for NP-specific antibodies of the indicated isotypes. Values are mean absorbance ± SD of 4 biological replicates. D. WT, 53bp1−/−, and Shld2−/− mice were immunized with NP-CGG, spleens were isolated and anti-IgG secreting cells were enumerated by the ELISPOT assay. Values are mean frequency ± SD of 4 biological replicates, except for Shld2-/- (n=3); * P ≤ 0.05, ** P ≤ 0.01, unpaired two-tailed t test.
Inhibition of cell fusion by ACE2-Ig.Figure 3.Inhibition of cell fusion by ACE2-Ig.Potent inhibition of cell fusion mediated by the SARS-CoV (left) or 2019 nCoV (right) spike (S) glycoprotein. Cells expressing diffferent S glycoprotein were incubated with indicated fusion proteins and mixed with ACE2-expressing cells. The activity of the reporter gene, β-gal, was measured as a correlate of fusion. The curves represent the best fit to the experimental data and were used for calculation of the IC50. Bars indicate the S.E.
Figure 4A. Mitochondria visualized by MitoTracker (Red) when wild-type GASZ or GASZ with MLS mutation was expressed in HeLa cells. Scale bar: 15 m. B. EM on 293T cells with empty vector control (Ctrl), wild-type GASZ, or GASZ with F to P mutation in MLS. Scale bar: 1 m. N: nucleus. C. Averaged percentage of HeLa cells (determined from 100 cells in each experiment) with different types of mitochondria determined by MitoTracker (Red) staining when transfected with GASZ, its SAM/ZIP deletion mutant, or GASZ with MLS mutation. D. HeLa cells transfected with COX4-DsRed, mito-PAGFP, and empty vector control, GASZ or GASZ with MLS mutation, followed by photoactivation of PAGFP. Representative images of PAGFP and DsRed were taken at 0, 10, and 20 min post photoactivation. Scale Bar: 10 m. Insets show images with higher magnification. The averaged exponential decay constant (±s.e.m.) of normalized PAGFP fluorescence within the designated region (white circle) at photoactivation area over a period of 10 min was shown as a bar graph below. n>20 cells for each group. E. Averaged percentage of C18-4 cells (determined from 100 cells in experiment) with different types of mitochondria determined by MitoTracker (Red) staining when transfected with Su9-GFP or GFP-GASZ. F. Real-time PCR analyses of mtDNA copy numbers on testes at different time points postnatal, relative to a single copy gene, PECAM, encoded by nuclear DNA. n=5 per genotype. G. Histochemistry of respiratory enzymes, COX, NADH, and SDH on testical sections from mice 10 days postnatal. Scale bar: 100 m.
Figure 2A) Representative image of M. florum cells observed by transmission electronic microscopy (TEM). Scale: 100 nm. B) Representative image of PicoGreen (DNA) and mCLING (cellular membrane) double stained M. florum cells observed by stimulated emission depletion (STED) microscopy. Scale: 1 µm. C) Frequency distribution of M. florum average cell diameter measured by TEM and STED respectively. The average cell diameter was obtained by averaging the minor and major axis values measured for each cell. A Gaussian curve fit is indicated for each method, and the calculated correlation coefficients are shown. Bins: 50 nm. D) Boxplots showing the median and interquartile range of the average cell diameter calculated from 389 and 169 individual cells analyzed by TEM and STED, respectively. Whiskers indicate the 10-90 percentile range. E) Picture of M. florum cells analyzed by discontinuous density gradient centrifugation in Percoll. The approximative density of each Percoll layer is indicated (g/ml) and colored in blue if trypan blue was added to the layer. The position of the cell pellet is marked. F) M. florum biomass quantification. The mass of each macromolecular constituent is shown as well as its relative fraction in the quantified cellular dry mass. Bars represent the mean and standard deviation values obtained from three independent biological replicates (dry mass) or four technical replicates (proteins, RNA, lipids, DNA, carbs). The "Other" category bar represents the residual mass obtained by the subtraction of all quantified macromolecule masses from the total dry mass value. G) Graph showing the relation between the M. florum cell diameter (d) and its cell mass (CM) according to cell mass Equations 4-7 (see Materials and Methods). For each equation, the mean cell mass (CMmean) is indicated by a colored line, and the range of probable values (CMmin - CMmax) is shown by a light-colored shading. The mean values of the average cell diameter measured by TEM and STED (see panel D) are indicated by black dashed lines. The portion of the graph where all the CMmean curves converge is enlarged and devoid of colored shadings for representation purposes. CMmean interception points encompassing all other interception points are encircled, and their corresponding x and y coordinates are indicated by fuchsia dashed lines (most probable cell diameter and most probable cell mass ranges).
POT1 mutations are tolerated in hematopoietic stem and differentiated cells in vitro A) Editing efficiency of hCD34+ cells at the AAVS1 control locus or in exon 9 of POT1 using sgRNA-1.Figure 4A) Editing efficiency of hCD34+ cells at the AAVS1 control locus or in exon 9 of POT1 using sgRNA-1. Shown are the average results of the three different nucleofection experiments of CD34+ cells. Error bars represent the standard deviation. B) Plot showing frequencies of different alleles determined by NGS two days after nucleofection. The vertical dashed line shows the site of CAS9 cut and the base pairs noted in red show insertion. A dash inside a gray box indicates a deleted base pair. C) Schematic of the differentiation protocol for edited CD34+ cells into erythroid cells. After editing, hCD34+ cells were cultured for 7 days in IMDM media containing indicated chemicals and cytokines. Next, culture medium was replaced with IMDM containing same cytokines except for hydrocortisone and IL-3 for another 4 days. The differentiated cells were cultured for 3 days more days with in IMDM supplemented with 5% human AB serum, heparin, insulin, transferrin and EPO before genomic DNA was extracted for genotyping. D) Quantification of edited or unedited colonies isolated in the in vitro differentiation described in (C) WT: wild-type, MT: mutant. E) Representative sanger sequencing chromatogram of four hCD34+ cell-derived heterozygous POT1-edited colonies shown in (C) and (D). WT: wild-type, MT: mutant. The red line indicates the CAS9 cleavage site, 3nt upstream of the PAM site.
Figure 2(A) Flow cytometric analysis of dissociated sensory neurons plotted according to their level of endogenous Ret-eGFP expression and IB4 binding (also indicated in Figure EV1). There are 5 well-defined subsets, 2 of which do not bind to IB4 but display a differential level of eGFP intensity (termed Ret-eGFPLo:IB4Neg, A, and Ret-eGFPHi:IB4Neg ,B, respectively). The other 3 subsets bind IB4 and display a range of eGFP intensities, termed Ret-eGFPLo:IB4Lo, C, Ret-eGFPHi:IB4Lo, D, and Ret-eGFPHi:IB4Hi, E. Percent of total events are indicated in each box. (B) No correlation between the median cell size of different Ret+ subsets and eGFP intensity or IB4 binding. The graph shows the forward scatter values plotted against the normalized number of cells, displayed as the percent of Max. (C−D) Variations in endogenous eGFP intensity are clearly visible with fluorescent microscopy. (C) Cultured neurons and (D) DRG section from Avil-Cre::Ret+/eGFPmice displaying native eGFPfluorescence and stained with IB4. Different levels of eGFP intensity (high -indicated by arrows and low -indicated by arrowheads) are detected across IB4+ and IB4- cells. Scale bar, 50 μm.
Fig.Figure 4A-B. Representative images (A) and quantification (B) of CD31, TUNEL and DAPI staining in small intestine sections of Casp8WT and Casp8ECko mice at an early disease stage (red dotted lines: epithelium; white dotted lines: endothelium) in (n= 5 WT, 5 ECko, multiple t-tests with Holm-Sidak method). Scale bars: 100µm. Data information: All data is shown as mean ± SEM. ns: not significant. C. Schematic representation of the strategy of tamoxifen treatment and timepoint of analysis in Casp8WT/MLKLko or Casp8ECko/MLKLko mice. D-E. Graphs showing survival and relative weight of Casp8WT/MLKLko and Casp8ECko/MLKLko mice upon tamoxifen treatment. (D; n= 5-8 WT/ MLKLko, 5-8 ECko/MLKLko; Log-Rank test, E; n= 7 WT, 8 ECko; two tailed unpaired Student's t-test). F. Representative pictures of intestines of Casp8WT/MLKLko and Casp8ECko/MLKLko mice at 30 days after tamoxifen treatment (n= 8 WT, 8 ECko). G. Representative images of stainings for CD31, Lyve1 and DAPI in small intestinal wholemounts from Casp8WT/MLKLko and Casp8ECko/MLKLko mice at 30 days after tamoxifen treatment. Scale bars: 100µm. H-K. Quantification of vessel area (H, I) and lacteal length (J, K) at two different timepoints after tamoxifen treatment in Casp8WT/MLKLko and Casp8ECko/MLKLko mice (n= 4-6 WT/MLKLko, 5 ECko/MLKLko; two tailed unpaired Student's t-test). Data information: All data is shown as mean ± SEM. ns: not significant. L. Graph showing quantification of extravasated 70kDa Dextran from small intestine tissue of Casp8WT/MLKLko and Casp8ECko/MLKLko mice at 30 days after tamoxifen treatment (n= 7 WT/MLKLko, 6 ECko/MLKLko; two tailed unpaired Student's t-test). Data information: All data is shown as mean ± SEM. ns: not significant. M-N. Representative pictures (M) and quantification (N) of lymphatic vessel density in Peyer's Patches of Casp8WT/MLKLko and Casp8ECko/MLKLko mice at 140 days after tamoxifen treatment (n = 8 WT, 7 ECko Peyer's Patches are pooled from 4 mice per genotype; two-tailed unpaired students t-test). Scale bars: 200µm. Data information: All data is shown as mean ± SEM. ns: not significant. O. Representative pictures of H&E stainings of small intestine tissue of Casp8WT/MLKLko and Casp8ECko/MLKLko mice (n= 4-6 WT, 4-5 ECko). Scale bars: 100µm. P-Q. Quantification of intestinal pathology in Casp8WT/MLKLko and Casp8ECko/MLKLko mice at two different timepoints after tamoxifen treatment (enteropathy score; n=4-6 WT/MLKLko, 5 ECko/MLKLko; two tailed unpaired Student's t-test). Data information: All data is shown as mean ± SEM. ns: not significant.