EMBO/sd-character-level-ner huggingface dataset:
Figure 5Immunoblot analysis of parental HeLa cells or cells expressing UBR7-BirA*-HA confirms specific expression of UBR7-BirA*-HA. Representative images of parental cells or cells stably expressing UBR7 fused to BirA* ligase and HA tag (UBR7-BirA*-HA) after incubation with or without biotin. DNA is visualized via DAPI staining shown in cyan, immunofluorescence for UBR7-BirA*-HA is shown in green, and biotinylated proteins are shown in red. Scale bar = 5 μm. Quantification of log2 fold change and -log10 p-value of MS spectral counts from UBR7-BirA*-HA (heavy) compared to parental (light) cells. Positive log2 fold changes are presented. Full dataset displayed as inset bordered by dotted line. The two proteins most abundantly labeled by UBR7-BirA*-HA are UBR7 and NASP. Experiment was performed twice. Immunoblot analysis of anti-FLAG IP. Cells were fractionated 72 h post-transfection, and 3xFLAG constructs were immunoprecipitated from non-nucleosomal fraction and eluted with FLAG peptide. Immunoblot analysis of anti-FLAG IP. Immunoblot signals were normalized to anti-FLAG signal. Experiment was performed as in (D). Asterisk indicates non-specific bands in input. Quantification appears on the right. A two-tailed one sample t-test was used to calculate p-value (italics) associated with group mean difference from a hypothetical value of 1.0 (represented by dashed line). Experiment was performed twice. Immunoblot analysis for total and modified H3 and H4 in anti-FLAG IP. Experiment was performed as in (D). Dot plot error bars represent mean +/- standard deviation. Immunoblot signals were normalized to anti-H4 signal. A two-tailed one sample t-test was used to calculate p-value associated with group mean difference from a hypothetical value of 1.0. Experiment was performed twice. Representative images of HeLa cells transfected with sNASP-FL. DNA is visualized via DAPI staining shown in magenta and immunofluorescence for 3xFLAG is shown in green. Scale bar = 5 μm.   Quantification of the nuclear anti-FLAG immunofluorescence intensity in (G) demonstrating that sNASP-FL is highly susceptible to pre-extraction with Triton X-100. One way ANOVA was used to determine p-values (italics) associated with differences between treatments. Error bars represent mean +/- standard deviation. Experiment in (G-H) was performed twice.
Human Spectrin SPTAN1 and SPTBN1 are required for the YAP localisation response to cell density.Figure 8A Control Caco-2 epithelial cells plated a low density show strong nuclear YAP localisation.B Control Caco-2 epithelial cells plated at high density show relocalisation of YAP to the cytoplasm.C, D siRNA knock-down of SPTAN1 (C) or SPTBN1 (D) prevents relocalisation of YAP to the cytoplasm. E Quantification of YAP localisation in (B-D). F Western blotting reveals that SPTAN1 or SPTBN1 are required for normal levels of YAP phosphorylation, and of LATS1 phosphorylation. G siRNAs against SPTAN1 or SPTBN1 efficiently deplete their target proteins.
Figure 1(A) A diagram that outlines the steps of the genetic screen. ATXN1 reporter cell line produces RFP-conjugated ATXN1 and YFP internal control independently from a same transcript, and ATXN1 levels were monitored by measuring the ratio of red to yellow fluorescence intensity. 1) ATXN1 reporter cell line was transduced with lentivirus harboring shRNAs of 858 ubiquitination-related genes. 2)-3) The cells with the 5% lowest and 5% highest fluorescence ratio were sorted. 4) Illumina sequencing revealed enriched shRNAs in the two groups compared to the non-sorted group. 5) Identifying the genes targeted by these enriched shRNAs revealed 39 positive regulators including ZBTB7A and ZBTB7B, and 13 negative regulators of ATXN1 protein levels (B) Western blot analysis of Atxn1 levels in cerebellar granule neurons (CGNs) after knockdown of Zbtb7a and/or Zbtb7b (n = 3). Data information: Mean ± SD, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, one-way ANOVA. (C) Western blot analysis of Atxn1 levels in the cerebella of wild-type (n = 3), Zbtb7a+/- (n = 4), Zbtb7b+/- (n = 3), Zbtb7a+/-; Zbtb7b+/- (n = 4) mice. Data information: Mean ± SD, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, one-way ANOVA. (D) Western blot analysis of Atxn1 levels in CGNs after overexpressing Zbtb7a and/or Zbtb7b (n = 3). Data information: Mean ± SD, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, one-way ANOVA. (E) Genetic interaction of wild-type human ATXN1(30Q) with either ZBTB7A or ZBTB7B expressed in the Drosophila eyes. Co-expression of ATXN1(30Q) with ZBTB7A or ZBTB7B severely disrupted the external Drosophila eye structure and increased ATXN1 levels. Scale bar: 100 μm
Figure 1A. Ig sequence diversification in wild type and h3.3. Each line represents an IGVLR gene from a population cells that have expanded for 36 divisions after cloning. Blue bars = gene conversions; green lollipops = point mutations; green bars = ambiguous mutations (Sale et al, 2001).B. Quantification of sequence diversification in wild type and two h3.3 clones expressed as unique events (gene conversions or point mutations) per kb per generation. (Sequences analysed: WT n = 142, for h3.3 c20 n = 140, for h3.3 c32 n = 190). C. Quantification of endogenous AID mRNA in wild type and h3.3 cells. The graph represents the mean and SD of four independent RNA extractions of wild type and h3.3c20. p value calculated with the unpaired T test. D. Fluctuation analysis for the formation of sIg-loss variants in wild type (black circles) and h3.3 clones (black diamonds) and in two wild type clones (blue circles), two h3.3 clones (blue diamonds) expressing hAIDup (Wang et al, 2009) at 15 - 20 times the level of wild type AID. The final column shows h3.3 cells with hAIDup complemented with H3.3 (blue triangles). Clones were expanded for 36 cell divisions at which point the percentage of sIgM negative cells in each population was determined by flow cytometry. Error bars represent mean and 1 S.D. P values calculated with the Mann-Whitney test. E. Ig sequence diversification in wild type, h3.3 and complemented h3.3 cells overexpressing hAIDup. Key as in (A) plus * = deletion or duplication. F. Mutation rate as a function of hAIDup expression in wild type and h3.3. Filled symbols represent clones overexpressing hAIDup, open symbols clones with natural AID expression. Linear regression lines are shown. G. Spectrum of diversification in wild type and h3.3. GC = gene conversion; PM = point mutation; Amb = ambiguous; Other = deletions or duplications.
CRMP4 is mis localized in ALS motor units (A) Representative images of ALS patient or non-ALS human control intra-muscular nerves.Figure 2(A) Representative images of ALS patient or non-ALS human control intra-muscular nerves. Red: denotes NFH, Green: denotes CRMP4, White: denotes co-localization area using Imaris software. Scale bar: 20 μm. (B) Quantification of CRMP4 intensity levels in NFH positive intra-muscular distal nerves from 5 non-ALS controls and 4 sALS patients. We analyzed 40 terminal axons from the healthy samples (~8 axons per sample) and 36 terminal axons from sALS samples (~8 axons per sample). Data presented as mean ±SE. Student's t-test, *p = 0.0475. (C) Representative images of SOD1G93A/ChAT::tdTomato or WTChAT::tdTomato neuromuscular junctions at P90. White: denotes BTX, Red: denotes direct ChAT, Green: denotes CRMP4, Yellow: denotes Z projection of 3D Imaris co-localization of CRMP4 and ChAT. Scale bar: 10 µm. (D) Quantification of CRMP4 positive NMJs in gastrocnemius muscles from 3 WT or 3 SOD1G93A P90 mice. Total of 44 NMJ's in WT condition and 60 NMJ's in SOD1G93A condition. Student's t-test, n=3, Data presented as mean ±SE, *p = 0.0157. (E) Quantification of the percent of partially denervated NMJ's in the presence or absence of CRMP4 immunostaining in 3 different SOD1G93A mice. We counted 24 NMJ's in SOD1G93A CRMP4 negatives and 67 NMJ's in SOD1G93A CRMP4 positives. Student's t-test, n=3, Data presented as mean ±SE, *p = 0.0352. (F) Representative images of P90 SOD1G93A and WT sciatic nerves. Red: denotes NFH, Blue: denotes GFAP and green denotes CRMP4, Yellow: denotes the Z projection of 3D Imaris co-localization of CRMP4 and NFH. Scale bar: 5 μm. (G) Quantification of the co-localization area of CRMP4 with NFH in the sciatic nerve in 3 SOD1G93A mice compared to 3 WT mice using Imaris analysis. 14 WT sciatic nerve sections and 11 SOD1G93A sections were monitored. Data presented as mean ±SE. Student's t-test, n=3, ****p<0.0001. (H-I) Western blot analysis and quantification of 3 independent repeats of P90 SN tissues for CRMP4 expression levels (size of ~64 KDa) in SOD1G93A compared to WT. tERK was used as a loading control (size of ~44 KDa). Student's t-test, n=3, Data presented as mean ±SE, *p = 0.0215.
Figure 3A. Western Blot (WB) analysis of A549-ACE2 knock-out (KO) lines. Two different KO lines were generated using distinct CRISPR guide RNAs for both ADAM10 and ADAM17. A non-targeting control (NTC) guide RNA served as control. Shown are two replicates for each cell line. Mature (m) and immature (im) forms of ADAM10 and ADAM17 are indicated. ACE2 levels remained unaffected. α-tubulin served as loading control. *: remaining, low amount of immature ADAM10 and ADAM17. The double KO (dKO) was generated from guide 1 of each ADAM10 and ADAM17. B. Quantification of the abundance of mature ADAM10 and ADAM17 protein forms from (A). Data are normalized to loading control tubulin and to NTC (N = 3-4). The band of mADAM17 runs very close to three unspecific bands (marked with arrowheads) - one closely above and two closely below mADAM17 (A). Thus, quantification of remaining mADAM17 in the ADAM10 KO includes also these three unspecific bands. Two-sided independent Student's t-test with Benjamini-Hochberg FDR correction. Data information: Data are represented as means ± 95% CI from at least three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001. C. RT-qPCR quantification of viral RNA (SARS-CoV-2 N gene relative to RPLP0) upon infection of A549-ACE2 NTC or KO cells with SARS-CoV-2. Cells were treated with BB94 (10 µM) or DMSO for 6 h prior to infection and RNA was isolated at 24 hpi. Two-way ANOVA with Tukey's post-hoc multiple comparison test. Data are normalized to NTC DMSO (N = 3). Data information: Data are represented as means ± 95% CI from at least three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001. D-G. UMAP of 63,103 cells from BAL fluid derived from infected individuals (D) Individual cell types are indicated in color. (E) Normalized counts of SARS-CoV-2 reads, ADAM10, ADAM17, and TMPRSS2 gene expression levels. (F) Dot plot of ADAM10, ADAM17, and TMPRSS2 expression, grouped by disease severity. Expression levels are color-coded, the percentage of cells expressing the aforementioned genes is size coded. (G) Cells that are infected and additionally express ADAM17 (ADAM17+, red), ADAM10 (ADAM10+, blue), both ADAMs (ADAM10+ ADAM17+, purple), or TMPRSS2 (TMPRSS2+, orange) are indicated.
Fig.Figure 1(A) Protein levels of RPL factors in ribosomes purified from Hela RPS9SBP cells exposed to 10 µM of (PR) 20 for 16h, as identified by LC-MS/MS. (B) Protein levels of RPL factors in the input extracts used for ribosome purification from Hela RPS9SBP cells exposed to 10 µM of (PR) 20 for 16h, as identified by LC- MS/MS. ( (C) Representative polysome profiles obtained from HeLa cells untreated or treated with 10 µM of (PR)20 for 16h. The presence of halfmers is indicated (arrows). (D) Electron microscopy images from purified 40S and 60S ribosomal complexes (1 pmol each) assembled in vitro in the presence of MgCl2, and in the presence or absence of 5 pmol of (PR) 20. Assembled 80S particles are indicated (red arrows). Scale bar (white) represents 10 nm. (E) Quantification of 80S particles identified in (D) (n=1000) in non-assembly (1 mM MgCl2) or assembly (5 mM MgCl2) conditions. ( (F) In vitro translation of 100 ng of luciferase mRNA (quantified by Relative Luciferase Units (RLU)) in the presence of increasing doses of (PR) 20. (G) In vitro translation of 100 ng of luciferase mRNA in the presence or absence of 0.5 µM (PR) 20. Translation products were labeled with [35S]-Met/Cys and analyzed by SDS-PAGE and autorradiography. (H) In vitro translation of 100 ng of luciferase mRNA with different 5' UTR lengths in the presence (orange columns) or absence (grey column) of 0.5 µM (PR) 20 (n=3). Data represent mean values ± SD (*, P<0.05; t-test). (I) In vitro translation of 100 ng of luciferase mRNA in the presence or absence of 0.5 µM (PR) 20. In the right two columns, the mRNA was extracted from a translation reaction done in the presence of the DPR, and subsequently used in a new translation reaction performed in the absence of (PR) 20 (n=3). Data represent mean values ± SD.
β‐Catenin negatively regulates p62 expression but does not block autophagic flux.Figure 2(A) Protein expression of p62 increased following β‐catenin knockdown under normal and starved conditions in HT29 cells. (B) Comparison of HCT116 expression in isogenic HCT116β‐cateninWT/− and HCT116β‐catenin−/ΔS45 cells by western blotting showed decreased p62 expression in β‐catenin−/ΔS45 cells. (C) LC3 puncta number per cell during starvation and nutrient addback post starvation. Left panel: LC3 puncta number reduces in non‐targeting (NT) siRNA control with nutrient addback. Right panel: LC3 puncta number reduces in β‐catenin knockdown cells with nutrient addback (mean±s.e.m. of three independent treatments; n>200 cells in >20 fields of view per experiment). Western blotting for β‐catenin confirmed knockdown. (D) Western blotting of LC3 and p62 in HT29 cells following β‐catenin knockdown and starvation with lysosomal inhibitors. LC3‐II and p62 protein expression showed an increase with β‐catenin siRNA in the presence of autophagy flux inhibitors chloroquine (10 μM) or bafilomycin A1 (100 nM; applied for the final 30 min of the 8 h) compared to β‐catenin knockdown alone. Quantification by densitometry of the LC3‐II/β‐actin ratio (mean±s.e.m. of four independent experiments). See also Supplementary Figure S1A.Source data for this figure is available on the online supplementary information page.
Figure 3A. Zoom of the PfMyoA structure showing important amino acid interactions for stabilization of the rigor-like state that are thought to influence the kinetic properties of MyoA. The N-terminal extension is depicted in purple. It is located in proximity to Switch I (green) and the connectors Switch II (orange) and Relay (yellow). Phosphorylated serine 19 (SEP19) in the N-terminal extension interacts with lysine 764 (K764) in the converter and glutamic acid 6 (E6) in the N-terminal extension interacts with arginine 241 (R241) as well as phenylalanine 476 (F476) from switch I and II. Image taken from Robert-Paganin et al. (2019). The image was published under a Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/). B. Scheme of mutated MyoA versions that are expressed in clonal parasite lines at the endogenous myoA locus. Either the complete N-terminal extension is deleted (19 amino acids) or point mutations within amino acids that are located in the N-terminal extension are introduced. C. Blood stage growth rate of clonal parasite lines. A single parasite or 100 iRBCs were injected i.v. into a mouse and the growth rate was calculated from parasitemia at day 6-9. Black dots: individual mice; bars: mean. Statistical analysis: (C) One-way analysis of variance with Tukeys multiple comparison test. D. Proportion of motile ookinetes. Black dots: Individual movie; bars: mean. Data pooled from three independent biological replicates with 1 - 3 movies taken per experiment. Statistical analysis: (D) Kruskal-Wallis test with (D) Dunn's multiple comparisons test E. Speed of ookinetes of the different lines. Numbers of analysed parasites are shown above the graph. Data pooled from three independent experiments (biological replicates). Violin plots show median (line) and quartiles (dashed lines). F. Number of oocysts per infected mosquito. Numbers of analyzed mosquitoes are shown above the graph. Data pooled from at least three independent cage feeds originating from independently infected mice. Box‐and‐whisker plots depict the 25% quantile, median, 75% quantile, and nearest observations within 1.5 times the interquartile range (whiskers). Statistical analysis: (F) Kruskal-Wallis test with (F) Bonferroni's Multiple Comparison test.
Figure 1Mean ± SEM CB2 mRNA levels in cerebellar extracts of WT and ASM-KO mice (n = 5 mice per group). Western blot against CB2 and PSD-95 (used as loading control) and graph showing mean ± SEM CB2 protein levels in cerebellar extracts of WT and ASM-KO mice (n = 6 mice per group). Mean ± SEM CB1 mRNA levels in cerebellar extracts of WT and ASM-KO mice (**P = 0.0013, n = 6 mice per group, Student's t-test). Western blot against CB1 and GAPDH (used as loading control) and graph showing mean ± SEM CB1 protein levels in cerebellar extracts of WT and ASM-KO mice (n = 3 mice per group). Immunofluorescence images against CB1 and the following cellular markers: Calbindin for Purkinje cells (E), GFAP for astrocytes (F) and F4/80 for microglia (G) in the cerebellum of WT and ASM-KO mice. DAPI stains cell nuclei. Graphs to the left show mean ± SEM intensity associated to CB1 in the Purkinje cells (shown by white arrows, E), astrocytes (F) and microglia (G) expressed as percentage of the values obtained in WT mice. Graphs to the right show the Mander's coefficient that indicates degree of co-localization between CB1 and the cellular markers for Purkinje cells (E), astrocytes (F) and microglia (G) (E: **PFluorecence intensity = 0.0082, *PMander's Coefficient = 0.0158; F: *PMander's Coefficient = 0.0115); n = 5 mice per group, Student's t-test). Scale bar, 100 μm. H. Immunofluorescence images against CB1 and the Purkinje cell marker calbindin in the cerebellum of age-matched control and ASMD-affected children. Graph shows mean ± SEM intensity associated to CB1 in the Purkinje cells expressed as percentage of the values obtained in the control child (16 and 15 replicates in control and ASMD, respectively). Scale bar, 10 μm.
Figure 4(A HAP1 cells were fixed and stained with antibodies examining PI4P and PI4KB Data information: Nuclei were stained with DAPI (blue). Scale bars represent 10 μm. B) HAP1 cells were fixed and stained with antibodies examining Golgi morphology markers (B). (C) Western blot of GBF1, PI4KB and B-Actin levels in both wild-type and c10orf76 KO HAP1 cells. Data information: Nuclei were stained with DAPI (blue). Scale bars represent 10 μm. (D) Markers of Arf1 activation - the coatomer proteins COPIα/γ and βCOP act as a readout for GTP-bound Arf1, while the native coatomer was detected with the CM1 antibody. Data information: Nuclei were stained with DAPI (blue). Scale bars represent 10 μm.
Spatial distribution of the TME populations (A, B) tissue sections from patients p1 and p3 stained with HistoGene Staining Solution.Figure 6tissue sections from patients p1 and p3 stained with HistoGene Staining Solution. Laser captured zones are marked with dashed black line including: tumor core (tc), the border between the tumor and the fibrotic regions (ftb), tumor islets within the fibrotic regions (ti) and fibrotic zones (fz) in panel (A), and tumor border (tb) in panel (B). Small images in (A) are magnifications of the corresponding zones in the large image for the left. White dashed line in (B) marking the non-malignant liver (nml) border. Scale bars in all panels are 100µm. Spatial distribution of the different TME populations across the analyzed five zones. Values are the estimated proportions based on de-convolution of the transcriptome of each zone with the average transcriptome of the cell types (Methods). Y axes show the estimated fractions of the cells, empty circles are the medians over all repeats (n=8 for tb, 11 for tc, 15 for ftb, 14 for ti and 15 for fz)
Figure 4 (A) Left, circular dichroism (CD) analysis of purified wild-type Arm/Smc5-Nse2. Black line, wild-type Arm/Smc5-Nse2 without bound ligand (no 5kb ssDNA); dashed black line, denatured protein; blue line, ligand/Smc5-Nse2; numbers in graphs (dotted black lines), molar ratio of ligand/Smc5-Nse2. Right, conformational changes induced by 5kb circular ssDNA quantified by ligand titration until signal change in mean residue ellipticity (MRE) at 222 nm achieved saturation. Blue squares Arm/Smc5-Nse2 (wild-type); grey triangles Arm/Smc5-Nse2 (K333E/K344E); green circles Arm/Smc5-Nse2 (K764E); pink triangles Arm/Smc5-Nse2 (K333E/K344E/K764E); red diamonds Arm/Smc5-Nse2 (K743E/K745E)  (B) Left, tryptophan intrinsic fluorescence of wild-type Arm/Smc5-Nse2. Black line in each panel, native Arm/Smc5-Nse2 without bound DNA; blue line, 5kb circular ssDNA/Smc5-Nse2; pink line, 50b linear ssDNA/Smc5-Nse2; and green line, 25b linear ssDNA/Smc5-Nse2. Right, conformational changes of wild-type Arm/Smc5-Nse2 induced by different types of DNA quantified by changes in center of mass (CM, red shift) of fluorescence spectra upon titration of the ligand until signal change achieved saturation. Blue circles, 5kb circular ssDNA/Smc5-Nse2; pink diamonds and 50b linear ssDNA/Smc5-Nse2. Inset, titration curves of 5kb circular ssDNA/Smc5-Nse2 reactions containing 0 or 100mM NaCl (solid and hollow symbols). Below, table of the dissociation constants of the curves  (C) Degree of conformational changes in CM induced by 60b linear ssDNA. Blue bar, Arm/Smc5-Nse2 (wild-type); grey bar, Arm/Smc5-Nse2 (K333E/K344E); green bar, Arm/Smc5-Nse2 (K764E); pink bar, Arm/Smc5-Nse2 (K333E/K344E/K764E); red bar, Arm/Smc5-Nse2 (K743E/K745E). Reactions were performed at least in three different independent experiments. Data values are mean ± s.d. and n=3 technical replicates. Significance was measured by a two-tailed unpaired t-test relative to wild-type. ****P < 0.0001