EMBO/sd-character-level-ner huggingface dataset:
Fig.Figure 5(A-H) 3D views of 232 x 232 μm2 fields of view of the AT (A, G) and endothelial layer (B, H) from infected LoCs reconstituted without macrophages at 1 (A, B) and 3 dpi (G, H). Orf1ab antisense RNA, S RNA, ACE2 mRNA, and nuclear staining with DAPI are false-colored pink, amber, spring green, and electric indigo, respectively. (C, D) Zooms corresponding to the region in (A) highlighted with white and yellow boxes, respectively. (C) An example of infection and intracellular replication in a cell with no detectable ACE2 expression (white arrow) as well as nuclear localization of viral RNA (white arrowhead). (D) An example of an uninfected cell with ACE2 mRNA expression (yellow arrow). (E, F) Zooms corresponding to the regions in (B) representing increased nucleic acid staining (hyperplasic) and normal levels of nucleic acid staining highlighted with white and yellow boxes, respectively. (E) Examples of infection of hyperplasic endothelial cells both with and without ACE2 expression (F) Examples of endothelial cell infection with no ACE2 expression, nuclear localization of viral RNA in an infected endothelial cell is indicated (white arrow). (I-L) Quantification of viral antisense RNA (I, J) and viral genomic RNA (K, L) from pairs (n=2 biological replicates) of otherwise identical LoCs reconstituted without (I, K) and with macrophages (J, L) analyzed at 1 and 3 dpi. Plots show the number of spots normalized by the total number of cells in 4-6 field of view (technical replicates) detected using RNAscope and confocal imaging using identical imaging conditions for all chips. Data information: Bars represent the mean value, the solid line represents the median, and error bars represent the standard deviation. P-values are calculated using a Kruskal-Wallis One-Way ANOVA Test, * represents p≤0.05, ** represents p≤0.01, ** represents p≤0.001. (M) Quantification of ACE2 expression per cell in ATs and endothelial cells from 4-6 fields of view (technical replicates) in a pair of LoCs reconstituted without macrophages (n=2 biological replicates) analyzed at 1 and 3 dpi, respectively. Data information: Bars represent the mean value, the solid line represents the median, and error bars represent the standard deviation. P-values are calculated using a Kruskal-Wallis One-Way ANOVA Test, * represents p≤0.05, ** represents p≤0.01, ** represents p≤0.001.
Figure 1(A) Structure of the PP2A-B56γ-hSgo1 complex (adapted from Xu et al, PDB: 3FGA). The hSgo1 coiled-coil homodimer interacts with both PP2A catalytic and B56 regulatory subunits. The model shows a LxxIxE peptide bound to B56 at its conserved binding pocket. (B) YFP pull down from cells stably expressing YFP (control) or YFP-B56α enriches the entire PP2A-B56α holoenzyme on the beads. PP2A-A, scaffold subunit; PP2A-C, PP2A catalytic subunit. (C) Coomassie-stained SDS-PAGE of the purified hSgo1 full length (FL) and hSgo11-155. (D) Competition assay with the purified hSgo1 proteins shown in (C). Binding of YFP-B56α to indicated proteins was determined. Representative of 3 independent experiments. (E) Peptide competition assay with a WT LxxIxE peptide or a mutated variant that does not bind B56 (LxxAxA). Binding of YFP-B56α to indicated proteins was determined and quantified by LiCor. (F) A PP2A-B56γ-BubR1516-715 complex was reconstituted (blue box) and peak fractions pooled and incubated with hSgo11-155. Following incubation this complex was analyzed by size-exclusion chromatography and fractions analyzed by SDS-PAGE (black box). (G) ﻿YFP-B56α pull down from cells stably expressing the indicated LxxIxE binding pocket variants of B56α and subsequent immunoblotting of indicated proteins. Representative of 4 independent experiments.
Figure 6Predicted kinases are indicated on top of each panel for which one of their marker substrate phosphorylations was assayed. UACC257 cells were serum starved and stimulated with PBS or EDN at 100 nM for 60 min (5 min for CaMKII). Ten μg protein from RIPA lysates were analysed by Western blot. Kinase marker substrate phosphorylations and corresponding loading controls are shown. For each kinase, two independent replicates were performed and are shown. Each gel also had control lanes with increasing concentration of kinase inhibitors to support phospho band identity
PGE2-mediated MEF2 activation is Gi/o-βγ dependent.Figure 2 A Illustration of the target points of the used pharmacological and protein-based inhibitors  B-D (B, D) NRVMs were infected with the 3xMEF2-Luciferase reporter and with recombinant adenoviruses encoding EGFP (AdGFP), RGS16, RGS2, p63ΔN, RGS-LSCII, or Gαt, as indicated. The cells were serum starved for 20 hours and stimulated with DMSO or 1 μM PGE2 for 24 hours. (C) NRVMs were infected with the 3xMEF2-Luciferase reporter and pretreated with the adenylyl cyclase inhibitor SQ22536 (100 μM) for 20 minutes or the Gi/o-protein inhibitor pertussis toxin (PTX, 100 ng/ml) for 20 hours in serum-starved conditions and treated with DMSO or 1 μM PGE2 for 24 hours. Data information: In (B-D), n=3, independent experiments, * represents significant interaction between the two treatments (P<0.05, Two-Way ANOVA, RGS16, P<0.0001; RGS2, P=0.1725; p63ΔN, P=0.0666; RGS-LSCII, P=0.0818, SQ22536, P=0,385; PTX, P<0.0001; Gαt, P<0,0001), values are mean±s.e.m. The exact n and P values can be also found in the Source Data excel file for Figure 2
Figure 6 A. Rif1 and Rif1-∆C association with coding sequences of highly transcribed genes. Left panel shows association with the housekeeping gene FBA1, middle panel association with the mating pheromone-induced gene FIG1, and right panel association with RNR1, which is expressed in S phase and induced further by hydroxyurea. Bottom two plots (dark grey) show results obtained at these loci using an 'Untagged' control strain  B. Rif1-associated genes tend to be highly transcribed. Genes showing occupancy by Rif1 or Rif1-∆C594 that extends across 90% of their coding sequences were selected, and the transcription levels of these genes plotted compared to the transcription levels of all genes (shown at left). Number of genes analysed are 18 (Rif1, G1), 68 (Rif1, HU), 42 (Rif1-∆C594, G1), and 64 (Rif-1∆C594, HU). Boxes show the range of 25th to 75th percentiles, with horizontal lines within the boxes representing 50th percentiles. Whiskers represent 95% confidence intervals. Outliers are presented as open circles. *** indicates that p-value obtained by Students' t-test was below 0.001  C. Plot comparing chromosome-wide association of Rif1 and Rif1-∆C594 with R loop-prone sites. Plot shows ChIP profiles of Rif1 and Rif1-∆C594 across entire chromosome VI. R loop-prone sites are marked in green ('DNA:RNA hybrid' track in green), as previously assessed by S1-DRIP-Seq analysis. Also shown are positions of predicted G4-forming sequences ("Predicted G4" track in orange), and positions of replication origins (ARS) and centromere (CEN6)
Changes in epithelial-associated fibroblasts in postmenopausal breast tissue.Figure 3A Microenvironment t-SNE map separated by menopausal status and colored by expression (red=high expression, grey=undetectable) of selected fibroblast markers (upper panel). The dotted lines indicate the pericyte subsets. Bottom panel shows percentage of cells expressing the markers for the cell clusters B Co-immunofluorescence staining of pre- versus post-menopausal tissue for E-cadherin (cyan), PDGFRβ (yellow) and F-actin (pink). DAPI is shown in grey. The arrowheads in (v) depict fibroblasts in direct contact with the myoepithelial layer. For 2D and 3D confocal imaging: n=13 premenopausal and n=10 postmenopausal specimens. Scale bars: wholemount and optical sections:100 μm (panels i-iv); enlargements, 30 μm (panel v). C t-SNE plot of the integrated scRNA-seq profiles of fibroblasts from pre-and post-menopausal tissue Cell colors correspond to tissue specimens. D Same t-SNE plot as in (C) showing 5 distinct clusters (clusters 3 and 5 were specific to one patient). E KEGG pathways enriched in cluster 1 versus 2 from (D) above (Fisher's exact test). F Heat map of same cells as (D) showing expression of the top 20 marker genes in each cluster. Color bars at the top of the plot show cluster membership (colors as in (D)) and pre- or post-menopausal status (blue and yellow, respectively).
Loss of PHB2 leads to changes in mitochondrial morphology.Figure 6A Immunofluorescence staining of mPhb2 shRNA and control podocytes with a Tom20 antibody revealed a disturbed mitochondrial network in Phb2-deficient podocytes (scale bar: 10 μm).B Quantification of tubular and fragmented mitochondrial morphology mPhb2 shRNA versus control podocytes seen in (A) (mitochondrial network of n = 100 individual cells of mPhb2 shRNA podocytes and n = 108 individual cells of scrambled shRNA podocytes was assessed).C Morphometric image analysis revealed an increased ratio of mitochondrial count versus mitochondrial area in Phb2-deficient podocytes compared to control podocytes (n = 75 cell patches for scrambled shRNA, n = 58 cell patches for mPhb2 shRNA).D Morphometric image analysis showed a decrease in branch numbers in Phb2-deficient podocytes compared to control podocytes (n = 75 cell patches for scrambled shRNA, n = 58 cell patches for mPhb2 shRNA).E Morphometric image analysis detected a decrease in mitochondrial size in Phb2-deficient podocytes compared to control podocytes (n = 75 cell patches for scrambled shRNA, n = 58 cell patches for mPhb2 shRNA). F Measurements of mitochondrial respiration with complex II substrates (succinate and glycerol-3-phosphate) did not show significant differences between Phb2-deficient and control podocytes (n = 6). G Quantification of mtDNA as a measure for mitochondrial mass showed similar levels of mtDNA in Phb2-deficient podocytes compared to control podocytes (n = 3). H FACS analysis of MitoTracker- and MitoSOX-stained Phb2-deficient podocytes and control podocytes.I Quantification of mean fluorescence intensity of MitoTracker and MitoSOX by FACS analysis revealed the same mitochondrial mass and the same ROS levels in Phb2-deficient podocytes compared to control podocytes (n = 3, P = 0.7782 for MitoTracker, P = 0.1088 for MitoSOX).
Direct stacking of SAS-6 rings (A-D) Computationally assembled CrSAS-6[6HR] single (A, C) or double ring in register (ribbon diagram shown in different shades for clarity) (B, D) fitted into the 3D maps of the 36 % T.Figure 5Computationally assembled CrSAS-6[6HR] single or double ring in register (ribbon diagram shown in different shades for clarity) fitted into the 3D maps of the 36 % T. mirabilis class (A, B) Dashed box shows longitudinal section through hub element (left), double chevron viewing point of longitudinal external views (right). Extra unaccounted densities in single ring fitting are indicated by arrowheads Note that each elongated hub density can accommodate two tightly stacked CrSAS-6[6HR] rings Computationally assembled CrSAS-6[6HR] single or double ring in register (ribbon diagram shown in different shades for clarity) fitted into the 3D maps of the 45 % T. agilis class (C, D). Dashed box shows longitudinal section through hub element (left), double chevron viewing point of longitudinal external views (right). Extra unaccounted densities in single ring fitting are indicated by arrowheads Note that each elongated hub density can accommodate two tightly stacked CrSAS-6[6HR] rings , and that only one ring can fit into the thin hub element in the 45% T. agilis class (C, D). Indicated distances stem from measurements on the fitted models. (E) Purified CrSAS-6[NL] protein self-organized into stacks and analyzed by cryo-ET; one longitudinal and one transverse view are highlighted by a dashed rectangle and a dashed circle, respectively. (F, G) 2D views through STA of in vitro self-assembled CrSAS-6[NL] proteins. Transverse sections at the indicated heights through one assembly unit (F), as well as longitudinal view (G) of the area delineated by a dashed box in (F); white arrowhead denotes position of line scan along the vertical axis at the level of the hub, with corresponding pixel intensities in arbitrary units (AU). The average distance between two hub elements is 4.5 ± 0.3 nm (N=7; dashed lines). (H) Longitudinal view of in vitro self-assembled CrSAS-6[NL] proteins STA 3D map. Note densities bridging successive hubs vertically (arrows). (I) Two CrSAS-6[HR] single rings fitted into the 3D map of in vitro self-assembled CrSAS-6[NL] proteins. Dashed box indicates longitudinal section through hub element (left), double chevron indicates viewing point of longitudinal external view (right). Note that the 3D map can accommodate rings stacked 4.4 nm apart (measured on fitted ring models), and that vertical densities linking hubs are partially occupied.
FLNA editing in cardiac patients from GTEX donors.Figure 1(A) ADAR2 is most strongly expressed in vascular tissues. Graph showing ADAR2 gene expression data derived from GTEx for few representative human tissues. Note the highest expression of ADAR2 in the tibial artery followed by colon and esophagus. Boxes represent the 25th and the 75th percentile with median represented by the black line in the box. The whiskers depict the minimum and the maximum value. (B) FLNA gene expression in few representative human tissues from >500 different donors using GTEx data. Tibial artery, colon and esophagus show highest gene expression similar to ADAR2 expression. Boxes represent the 25th and the 75th percentile with median represented by the black line in the box. The whiskers depict the minimum and the maximum value. (C) Bar graph shows FLNA RNA editing (%) among few representative human tissues. Note very high editing levels in the arterial system (red bars). (D) Scatter plot shows the log fold change in editing levels of several candidates between ventricles of healthy donors and heart samples of cardiomyopathy patients. FLNA marked in the graph is one significant discriminator. Y axis is the -log 10 of the p value for the difference between healthy and sick. The colors (red vs green) reflect the threshold cutoffs randomly assigned to distinguish the sites, which demonstrate large, highly significant differences between the two groups. Fold change cutoff of 0.9 was used. (E) Scatter plot shows the FLNA RNA editing in control and diseased human tibial arteries and aorta. At least 4 control and 7 diseased human donors were used in each case. P value <0.05 measured by t test was considered significant.
Figure 8Proposed model of how palmitoylation of Golga7b regulates plasma membrane localisation of DHHC5
Metaphase targeting mutants of M18BP1-1 do not support robust CENP-A assembly A) Representative immunofluorescence images showing incorporation of exogenous Myc-CENP-A at centromeres.Figure 6A) Representative immunofluorescence images showing incorporation of exogenous Myc-CENP-A at centromeres. Sperm nuclei were incubated in M18BP1-depleted interphase Xenopus egg extracts complemented with the indicated M18BP1-1 protein. Extracts were supplemented with RNA encoding Myc-CENP-A to track new CENP-A assembly and in vitro translated HJURP. Scale bar, 10 μm. Insets are magnified 3X. B) Quantification of Myc-CENP-A loading at Xenopus sperm centromeres in (A). Values are normalized to the centromere signals in mock-depleted extract. Dashed lines indicate the Myc-CENP-A assembly signal observed upon M18BP1 depletion (bottom) and Flag-M18BP1-1WT add-back (top) as points of reference for mutant rescue. Graph shows the mean ± SEM of four independent experiments. C) Interphase Mis18 complex formation is unaffected by M18BP1-1 mutations that prevent binding to CENP-C. Extract depleted of endogenous M18BP1 was supplemented with Myc-Mis18α, Myc-Mis18β, and Flag-M18BP1-1. M18BP1-1 species added to each reaction and cell cycle state of the extract is indicated at the top. Co-immunoprecipitation of Myc-Mis18α/β was assessed by α-Myc immunoblot following Flag precipitation. D) HJURP association with M18BP1-1 is unaffected by mutations that prevent CENP-C binding. Extract depleted of endogenous M18BP1 was supplemented with Flag-M18BP1-1. M18BP1-1 species added to each reaction and cell cycle state of the extract are indicated at the top. Coimmunoprecipitation of Flag-M18BP1-1 was assessed by α-Flag immunoblot following HJURP precipitation.
Model.Figure 8Through the synthesis/secretion of soluble chemoprotective factors including IL-6, CAFs induce pancreatic cancer cell chemoresistance. High protein synthesis rate in CAFs is dependent on the robust intrinsic activation of the mTOR pathway resulting in inhibition of the protein translation inhibitor 4E-BP1. Secreted IL-6 participates in an autocrine feed-forward loop in the intrinsic activation of mTOR and subsequent high protein synthesis.CAFs express the somatostatin receptor sst1. Upon activation with the novel SOM230 somatostatin analogue (Pasireotide® Novartis) presenting a high affinity for sst1, the mTOR pathway is inhibited, resulting in reduced synthesis/secretion of chemoprotective factors including IL-6 through inhibition of mRNA translation. As consequences, the autocrine feed-forward IL-6/mTOR/protein synthesis/IL-6 loop is abrogated, and pancreatic cancer cells are re-sensitized to the apoptotic action of chemotherapies.
Fig.Figure 2(A) Gastrocnemius weight normalized to tibial length in C26-tumor bearing mice on day 12 post C26-injection (n=6-7). Data information: For all data, n represents the number of biological replicates. Statistical significance was calculated by unpaired, two-tailed Student's T-test. Data are mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. (B) TFR1 mRNA levels normalized to 18s (n=6-7) Data information: For all data, n represents the number of biological replicates. Statistical significance was calculated by unpaired, two-tailed Student's T-test. Data are mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. (C) TFR1 protein expression and densitometric quantification in mouse gastrocnemius (n=5). Data information: For all data, n represents the number of biological replicates. Statistical significance was calculated by unpaired, two-tailed Student's T-test. Data are mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. (D) Cytosolic aconitase activity in mouse quadriceps (n=4) measured following subcellular fractionation (n=4). Data information: For all data, n represents the number of biological replicates. Statistical significance was calculated by unpaired, two-tailed Student's T-test. Data are mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. (E) Representative Western blot of IRP2 in mouse gastrocnemius and densitometric quantification (n=5). Data information: For all data, n represents the number of biological replicates. Statistical significance was calculated by unpaired, two-tailed Student's T-test. Data are mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. (F) Binding of IRE-BPs to the ferritin IRE. The biotin labeled IRE probe was incubated with 6 μg of cytosolic gastrocnemius extracts from mice, in native or reducing conditions (with EDTA 0.5 mM and DTT 1 mM) (n=3). Data information: For all data, n represents the number of biological replicates. Statistical significance was calculated by unpaired, two-tailed Student's T-test. Data are mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. (G) Representative protein carbonylation blot and densitometric quantification in mouse quadriceps (n=6-7). Data information: For all data, n represents the number of biological replicates. Statistical significance was calculated by unpaired, two-tailed Student's T-test. Data are mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. (H) Representative Western blot of ferritin Heavy and Light chains in mouse gastrocnemius and densitometric quantification (n=4). Data information: For all data, n represents the number of biological replicates. Statistical significance was calculated by unpaired, two-tailed Student's T-test. Data are mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. (I-J) ICP-MS quantification of total (I) and protein-bound (J) iron in mouse quadriceps (n=4-5). Data information: For all data, n represents the number of biological replicates. Statistical significance was calculated by unpaired, two-tailed Student's T-test. Data are mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.
Overview of all identified vemurafenib resistance mechanisms in patient and PDX samples.Figure 8Overview of all identified vemurafenib resistance mechanisms in patient and PDX samplesResistance mechanisms discovered in each of the lesions have been identified: a) in patients samples by WES (BRAFV600E amplification in M032R1, R2 and R5; MEK1T55delinsRT in M032R4 and M032) and immunoblotting (of alternative form of BRAF in M032R3); and b) in PDX by IHC and qPCR (BRAFV600E amplification in M032R2 and R5) and PCR analysis (MEK1T55delinsRT in M032R1 and R4). Gray arrows indicate that the lesion was at the back side. ampl., amplification; alt., alternative.
TIP30 inhibits protein synthesis in cardiomyocytes.Figure 7(A) Western blot analysis of isolated neonatal rat cardiomyocytes (NRCM) after adenoviral transduction either with control adenovirus (Con) or Ad.TIP30 (TIP30) followed by stimulation with phenylephrine (PE, for 3 hours) and puromycin incorporation (for 30 min). (B) Quantification of the Western blot shown in (A) (N=3 samples/group). (C) Quantification of cell surface area of isolated neonatal mouse cardiomyocyte of Tip30 Het and WT mice treated with endothelin-1 (ET-1) and Narciclasine (Narci) or without stimulation as indicated (N=6 samples/group). (D) Schematic representation of Narciclasine study design. Quantification of HW/TL ratio (E), N=6 to 10 mice/group, in Tip30 Het or WT mice 2 weeks after TAC. Animals were treated with Narciclasine daily for 14 days after TAC as indicated. Quantification of , echocardiographic wall thickness (F), N=6 to 10 mice/group, in Tip30 Het or WT mice 2 weeks after TAC. Animals were treated with Narciclasine daily for 14 days after TAC as indicated. Quantification of cardiomyocyte area (G), N=5 mice/group in Tip30 Het or WT mice 2 weeks after TAC. Animals were treated with Narciclasine daily for 14 days after TAC as indicated. Quantification of fractional area change (FAC, H, N=6 to 10 mice/group) in Tip30 Het or WT mice 2 weeks after TAC. Animals were treated with Narciclasine daily for 14 days after TAC as indicated. (I) Western blot analysis of puromycin incorporation in hearts of Tip30 Het and WT mice 3 days after TAC or sham surgery and daily Narciclasine (Narci) injection and their quantification (N=2 to 4 mice/group). Puromycin was injected 3 hours prior to sacrifice.