EMBO/sd-character-level-ner huggingface dataset:
Transverse sections from muscle biopsies from subject 3.figf2Transverse sections from muscle biopsies from subject 3.1 (top) and a normal control (bottom) were stained with monoclonal antibody against NBR1 or polyclonal antibody to MURF2 and counterstained with antibody to the M-band titin, titin M8. Accumulation of NBR1 in puncta (autophagosomes, arrowheads) and of MURF2, as well as fiber inhomogeneity with marked fiber atrophy, characterizes Vici syndrome muscle. Numerous fibers of very small cross-sectional area (arrows) with high content of MURF2 and NBR1 puncta are frequently seen. Scale bar, 10 μm.
Figure 7Brcc3+/+ and Brcc3−/− BMDMs were left untreated (Con), treated with LPS alone (LPS) or pretreated with LPS and then stimulated with ATP, nigericin, MSU, Alum, poly (dA:dT) or flagellin. CBA analysis of IL-1β secretion in the culture supernatants. Immunoblot analysis of cleaved caspase-1 and IL-1β in culture supernatants (SN) of LPS-primed Brcc3+/+ and Brcc3−/− BMDMs treated with ATP, nigericin, MSU, flagellin, and poly (dA: dT). Immunostaining of endogenous ASC specks in LPS-primed Brcc3+/+ and Brcc3-/- BMDMs left untreated or treated with nigericin or poly (dA:dT). ASC speck positive cells were counted and analyzed as described in Fig. 1I. UD, undetectable. LPS-primed Brcc3+/+ and Brcc3−/− BMDMs were left unstimulated or stimulated with nigericin. Immunoblot analysis of NLRP3 ubiquitination (detected by mouse anti-ubiquitin antibody) in cell lysates immunoprecipitated with rabbit anti-NLRP3 antibody. Survival of Brcc3+/+ and Brcc3−/− mice subjected to LPS (15 mg/kg). n=20 per group. ELISA of serum IL-1β from Brcc3+/+ and Brcc3−/− mice 3 h after intraperitoneal injection of LPS (15mg/kg) or vehicle (PBS). n = 6 for PBS groups and n = 12 for LPS groups. Brcc3+/+ and Brcc3−/− mice were intraperitoneally injected with MSU (1 mg per mouse) or vehicle (PBS) for 6 h. ELISA of IL-1β and TNF-α in the peritoneal lavage fluid (G) Brcc3+/+ and Brcc3−/− mice were intraperitoneally injected with MSU (1 mg per mouse) or vehicle (PBS) for 6 h. flow cytometric analysis of peritoneal cell exudates (H). n = 6 for PBS groups and n = 12 for MSU groups.
Actionable cell cycle dysregulation in crizotinib-resistant cells.Figure 2A) GSEA enrichment analysis using the KEGG gene set identifiers. Shown are the significantly dysregulated pathways (P < 0.05). B) Cell cycle enrichment plot from A). C) Western blot analysis of H3122 parental and isogenic drug-resistant cell lines for the indicated proteins. D) Western blot analysis of Ste-1 parental and isogenic Alectinib resistant cell lines for the indicated proteins. E) Proliferation assay of CrizR1 cells treated with the indicated concentrations of alvocidib ± 1uM crizotinib for 72h. P=0.2 (n=4). F) As above, in a proliferation assay, H3122 and CrizR1 cells were treated in parallel with DMSO or 1uM crizotinib as drug control. H3122 vs DMSO P <0.0001, CrizR1 vs DMSO P=0.1 (n=4). Proliferation assay of CrizR1, CrizR4, CrizR5, CeritR isogenic drug-resistant cell lines, treated with the indicated concentrations of alvocidib(G) for 72h. P >0.05 was calculated for IC50 shift, as indicated in the methods. H) Proliferation assay of AlecR isogenic drug-resistant cell lines, treated with the indicated concentrations of dinaciclib (H) for 72h. P >0.05 was calculated for IC50 shift, as indicated in the methods.
Figure 3nanoCAGE sequencing was applied to determine transcription start sites in shERα BM67 cells. The number of detected transcription start sites (peaks) and RefSeq transcripts when sampling increasing number of RNAseq reads are indicated to evaluate the complexity of nanoCAGE RNAseq libraries. Boxplots for offset and non-offset mRNAs comparing 5' UTR weighted lengths (log2 scale), GC content (%), free energy (log10 scale, kcal/mole). In boxplots, the solid horizontal middle line is the median; the lower and upper box limits correspond to the first and third quartiles; the whiskers extend from the box limits to the most extreme value but not further than +/- 1.5 x the inter-quartile range (IQR) from the box limits. The notches extend to 1.58 x the IQR / √n where n is the number of data points. Data beyond the end of the whiskers are plotted as points. Wilcoxon-Mann-Whitney tests (two-sided) were used to assess differences between translationally offset and non-offset mRNAs. Number of transcripts harboring at least one (dark gray) or no (light gray) uORF in a strong Kozak context were visualized as a bar chart and differences were tested using one-sided Fisher's exact tests. A global Bonferroni adjustment was applied on p-values. Analysis was performed on nanoCAGE data from 3 replicates of shERα BM67 cells; the numbers of transcripts in each group are indicated in the figure.
mediated arginine methylation of p14ARF.Figure 6HeLa cells were transfected with the indicated siRNAs (two control/non-targeting siRNAs and two PRMT1-specific siRNAs) and irradiated at 150 J/cm2 UVC or not irradiated. After 24 hours, cell cycle distribution was analyzed by flow cytometry using propidium iodide (PI) DNA staining for a representative experiment ((A), Appendix Fig. S8). The subG1 fraction of the siControl_4 and siPRMT1_4 condition was quantified for three independent experiments in (B) (mean ± SD, **: p-value ≤ 0.005 using Welch´s t-test). PRMT1 depletion and p14ARF methylation of the samples (from (A)) were monitored by immunoblotting using the indicated antibodies (C). The p14ARF and the methylated p14ARF signals were densitometrically quantified and normalized to the respective β-TUBULIN signal, as specified by the numbers below the blots, with siControl_4 condition set to 1. D: CRISPR/Cas9 control (CTR_1 and CTR_2) or PRMT1-deleted (KO_1 and KO_2) HeLa cell lines were irradiated at 150 J/cm2 UVC. After 0, 2, and 4 hours, cell lysates were analyzed by immunoblotting using the indicated antibodies. The percentage of PARP cleavage was densitometrically quantified and is specified below the blot. E: CRISPR/Cas9 control (CTR_1 and CTR_2) or PRMT1-deleted (KO_1 and KO_2) HeLa cell lines were irradiated at 150 J/cm2 UVC. After 4 hours, the apoptotic cell fraction was analyzed by flow cytometry using FITC-labeled AnnexinV and propidium iodide (PI). The increase in the apoptotic cell fraction upon UVC irradiation was quantified for three independent experiments (mean ± SD, **: p-value ≤ 0.005 and *: p-value ≤ 0.05 using Welch´s t-test). F: HeLa CRISPR/Cas9 PRMT1 deleted (KO_1) cells were transfected with Flag-tagged mutant RK or RF p14ARF-containing plasmid and subsequently irradiated at 150 J/cm2 UVC. After 4 hours, the apoptotic cell fraction was quantified by flow cytometry using FITC-labeled AnnexinV and propidium iodide (PI) for seven independent experiments (mean ± SD, *: p-value ≤ 0.05 using the paired t-test). G: GST alone, GST-tagged wild type (wt) and mutant (RF) p14ARF proteins were coupled to Glutathione beads and incubated with baculoviral expressed, purified Flag-tagged p32. Pulldown reactions and input of p32 protein were resolved by SDS-PAGE and analyzed by immunoblotting using α-Flag as well as α-p14ARF antibodies. Short and long exposure times are specified. Staining results derive from the same blot and exposure times with white lines indicating where tracks were cut. H: CRISPR/Cas9 control (CTR_1 and CTR_2) or PRMT1-deleted (KO_1 and KO_2) HeLa cell lines were irradiated at 150 J/cm2 UVC or not irradiated. After 4 hours, immunoprecipitations (IP) of endogenous p14ARF were performed from cell lysates using α-p14ARF antibody or IgG as negative control. IP reactions and input lysates were analyzed by immunoblotting using the indicated antibodies. Short and long exposure times are specified.
CCN1 restricts NSC pool size and niche capacity in the adult V-SVZ.Figure 2V-SVZ whole-mount staining showing reduced CCN1 expression in Ccn1cKO mice 1 week after TAM treatment. Scale bar: 10 μm. V-SVZ whole-mounts stained for γ-tubulin (cyan) and GFAP (red). Arrowheads point to B1 cells in the pinwheel center. Scale bars: 500 μm (left), 50 μm (middle), 10 μm (right). Densities (cells/mm2) of B1 cells in the adult V-SVZ. n=4, ***p=0.0005. V-SVZ whole-mounts stained for γ-tubulin (cyan), GFAP (red) and VCAM1 (white). Arrowheads point to VCAM1+ B1 cells in NSC units. Scale bars: 50 μm (left), 10 μm (right). Densities (cells/mm2) of VCAM1+ B1 cells in the adult V-SVZ. n=4, **p=0.002. Densities (units/mm2) of NSC units in the adult V-SVZ. n=4, **p=0.0022. Distribution of NSC units with different numbers of B1 cells. n=4, ****p<0.0001 (1 B1 cell/unit), **p=0.0025 (2 B1 cells/unit), **p=0.0063 (3 B1 cells/unit), *p=0.0385 (4 B1 cells/unit).
Figure 2A. Representative PET images of a sagittal view of the thoracic region of fasted lean mice treated with either MP-10 (30mg/kg) (n = 3) or vehicle (n = 4) prior to receiving [18F]-FDG. Interscapular brown adipose tissue (BAT) in each image is highlighted by the crosshairs. The mean standardized uptake value (SUV) of [18F]-FDG in BAT after both treatments was calculated for each group. ** P = 0.0013 using unpaired 2-tailed Student's t test. B. The percentage (%) change in core body temperature and associated area under the curve (AUC) of mice treated with MP-10 (30mg/kg) (n = 4) or vehicle (n=4) prior to a cold challenge study for 4 hours at 8oC. * P = 0.0108 (1st hour), * P = 0.0218 (2nd hour), * P = 0.0121 (3rd hour), * P = 0.0168 (4th hour) and * P = 0.0264 (AUC) using unpaired 2-tailed Student's t tests. C. RT-qPCR analysis of relative mRNA expression of Pde10a in BAT, peri-ovarian visceral white adipose tissue (VAT) and inguinal subcutaneous white adipose tissue (SAT) of lean mice (n = 5). ** P = 0.0063 (SAT vs VAT), ** P = 0.0011 (SAT vs BAT) and ** P = 0.0037 (VAT vs BAT) using unpaired 2-tailed Student's t test. D Cyclic GMP (cGMP) and cyclic AMP (cGMP) concentrations normalized to protein concentration in SAT, VAT and BAT 30 minutes after treatment of lean mice with MP-10 (30 mg/kg) (n=6) or vehicle control (n = 6). ** P = 0.0085 (cGMP in VAT), ** P = 0.0011 (cGMP in BAT) and * P = 0.04 (cAMP in VAT) using unpaired 2-tailed Student's t tests. E RT-qPCR analysis of relative mRNA expression of thermogenic genes in SAT, VAT and BAT after treatment of lean mice treated with MP-10 (30 mg/kg) (n=7) or vehicle control (n = 6). **** P < 0.0001 (Pgc1alpha in VAT), ** P = 0.0012 (Ucp1 in VAT), ** P = 0.0071 (Pgc1alpha in BAT) and * P = 0.0486 (Ucp1 in BAT) using unpaired 2-tailed Student's t tests.
Figure 5(A) UMAP visualisation of the B cell/B-ALL compartment. From left to right panel, cells are coloured according to: unsupervised clustering (Louvain - resolution 0.6), experimental condition and marker-based custom cell-type classification, unequivocally identifying leukemic cells by the presence of the tTA transcript. (B, C) Violin plots show the distribution of IFNγ (B) and MHC-II gene (C) signatures for each experimental condition, within each custom cell-type. Wilcox Rank Sum Test for IFNγ signature, condition IFNγ d12 vs. CTR d12: B-ALL p= 2.68x10-19, B cells p=0.5, plasma cells p= 0.78; Wilcox Rank Sum Test for MHC-II signature, condition IFNγ d12 vs. CTRL d12: B-ALL p= 4.26x10-46, B cells p=1x10-9, plasma cells p= 0.32. (D) Reduction of OFP expression in B-ALL cells in the IFNγ treated versus CTRL (mean). (E) Viability of OFP+ B-ALL cells at sorting before undergoing single-cell RNA sequencing processing (mean, CTRL = 4 mice, IFNγ = 6 mice; * p = 0.0333, Mann-Whitney test). (F) Expression of Mki67 transcripts in B-ALL cells from CTRL d12 and IFNγ d12 conditions, extracted from the scRNAseq dataset (**** p ≤ 0.0001, Welch's test). (G) IFNγ d12 B-ALL cells were split according to MHC II gene signature values and compared for gene expression profile in order to identify significantly enriched terms (within the hallmarks gene set MsigDB collection) by using the GSEA approach (H). On the left, hallmarks terms downregulated (negative NES - blue labels) in the MHCII high leukemia cell population are shown, whereas the right shows upregulated hallmarks terms (positive NES - red labels) in the MHC II high leukemia cell population, as compared to the MHC II low counterpart. (I) Loss of IFNγ response over time in B-ALL cells from the IFNγ group, potentially facilitated by reduced expression of Ifngr1, Ifngr2, Jak1, Stat1 and Irf1 (data extracted from the scRNAseq dataset statistical analysis by Two-Way ANOVA).
Extensive growth potential of recessive dystrophic epidermolysis bullosa (RDEB) epidermal keratinocytes.Figure 2Keratinocytes were isolated from the skin of a 4-year-old patient with severe-generalised RDEB linked to homozygous insertion-deletion in COL7A1 (Hilal et al, 1993). Cultured RDEB cells (blue line) were serially passaged for more than 4 months, displaying a growth potential similar to non-diseased control cells (YF29) isolated from the foreskin of a newborn (black line). To calculate the percentage of growing colonies, 100 to 1,000 cells were plated into indicator dishes at each passage. Cells were grown for 12 days, fixed and stained with rhodamine B. Colonies were scored as growing or aborted (Barrandon Green, 1987). Clonal analysis demonstrated the presence of stem cells (holoclones) in a passage VII RDEB culture (95% of growing colonies).
Figure 5(A-B) CYLD and A20 inhibit CARD14sh-induced NF-kB activation. HEK293T cells were transfected with NF-kB reporter plasmid and FLAG-tagged CARD14sh (30ng), with or without the indicated concentrations of CYLD (A) or A20 (B). Luciferase activity in cell lysates was measured 24 h later. Closed arrowheads indicate full length A20 and CYLD; arrows indicate cleaved A20 and CYLD. Data are representative of three independent experiments. (C) A20 and ABIN-1 inhibit CARD14-induced NF-kB activation. HEK293T cells were transfected with NF-kB reporter plasmid, A20 (3 ng) or ABIN1 (15ng) and the indicated concentrations of FLAG-tagged CARD14sh. Luciferase activity in cell lysates was measured 24 h later. Values are the mean of triplicates ± s.e. Protein expression of transfected plasmids was verified by western blot and is shown in the accompanying panels. (D) A20 inhibits CARD14-induced NF-kB activation through its ZnF domains. HEK293T cells were transfected with NF-kB reporter plasmid, FLAG-CARD14sh (20ng) and wild-type (WT) or mutant A20 expression plasmids (3ng) as indicated. Luciferase activity in cell lysates was measured after 24 h. CARD14 and A20 expression was measured by immunoblotting (bottom panel). Data are representative of two independent experiments.
CDC7 is required for efficient fork restart and togher with MRE11 regulates fork speed.Figure 4A. MCF10A cells were either mock or treated with 10 μM XL413 and labelled with IdU (red) for 30 minutes at which point 4 mM HU was added for 2 hours. HU was then washed off and cells labelled with CldU (green) in the continued presence or absence of XL413. A set of representative DNA fibres from each condition is shown and the percentage of IdU (red) only tracts is plotted. At least 200 replication forks were analysed for each condition. Error bars represent the SEM from three biological repeats and statistical significance was assessed by the student t test (** p˂0.01 B. U2OS cells were either mock or treated with 10 μM XL413, 50 μM Mirin or both, and labelled with IdU (red) for 30 minutes at which point 4 mM HU was added for 2 hours. HU was then washed off and cells labelled with CldU (green) in the continued presence or absence of XL413 and/or Mirin. A set of representative DNA fibres from each condition is shown and the percentage of IdU (red) only tracts is plotted. At least 200 replication forks were analysed for each condition. Error bars represent the SEM from four biological repeats and statistical significance was assessed by the student t test (*p<0.05, ** p˂0.01 C. U2OS cells were either mock or pretreated for the indicated times with 10 μM XL413, 50 μM Mirin or both, and labelled with CldU (red). Then 50 nM CPT was added and cells labelled with of IdU (green) for further 30 minutes. A set of representative DNA fibres from each condition is shown and the IdU/CIdU tract length ratio is plotted. The box extends from the 25th to the 75th percentile with the line in the box representing the median. Whiskers indicate the 10-90 percentiles with data outside this range for individual outliers being plotted as dots. At least 100 replication forks were analyzed for each condition. P values were calculated using one way ANOVA Kruskal-Wallis test and Dunn's multiple comparison post test (ns, not significant, **** p<0.0001) and are related to the experiment shown. Similar results were obtained in a second independent experiment. D. U2OS cells were transfected with control (siLuc) or MRE11 targeting siRNA (siMRE11). After 48 hours cells were either mock or pretreated with 10 μM XL413 and labelled with CldU (red) for 30 minutes. Where indicated 50 nM CPT was added and then cells were labelled with IdU (green). A set of representative DNA fibres from each condition is shown and the IdU/CIdU tract length ratio is plotted. The box extends from the 25th to the 75th percentile with the line in the box representing the median. Whiskers indicate the 10-90 percentileswith data outside this range for individual outliers being plotted as dots. At least 100 replication forks were analyzed for each condition. P values were calculated using one way ANOVA Kruskal-Wallis test and Dunn's multiple comparison post test (ns, not significant, **** p<0.0001) and are related to the experiment shown. Similar results were obtained in a second independent experiment.
Detection of protein elution via protein-centric analysis.Figure 3Peptide-level SEC chromatograms are grouped by UniprotKB identifier to detect co-elution signals indicative of protein elution ranges/peaks. Based on external size calibration of the apparent analyte molecular weight per SEC fraction, signals can be attributed to likely assembled or monomeric state (also see Appendix Figure S4). For TCPE, three distinct elution signals, numbered 1-3, are detected, two in the assembled and one in the monomer elution range. Global statistics of protein signal attribution to assembled or monomeric state. The majority of proteins (66 %) and protein mass (55 %) appear in assembled state in SEC-SWATH-MS. Proteins are observed eluting in 1-6 distinct peaks and with a wide range of apparent vs. monomeric molecular weight ratios (distributions, left panels). The molecular weight ratios of the three peaks detected for TCPE (displayed in A) are indicated. Many of the proteins eluting in a single peak (top panel and bar) appear assembled. Proteins eluting multiple times (27 % of the proteins) do so preferentially in the assembled range, suggesting frequent participation in multiple differently sized macromolecular assemblies (lower panels and pie chart). For a list of all detected protein peaks, see Dataset EV3. Proteins observed in multiple assembled peaks (n = 659) are enriched in components of the proteasome and other known large complex assemblies.
Figure 7Figure 7. CHCHD10 mutant fibroblasts are less sensitive to apoptotic cell death compared to control fibroblasts. A. Control (C1, C2) and patient (P1, P2) fibroblasts were treated with 1 μM of staurosporine (STS) for 16 h. Cell death was determined by flow cytometry using Annexin V/DAPI staining. Three independent experiments were performed per condition with two points analyzed per experiment. Differences between the control and patient fibroblasts were analyzed by Student's t-test (two-sided): significant (*: p=0.0116) or extremely significant (***: p=0.0001). B-C. Fibroblasts were treated with 1 μM STS for 4, 6 or 16 h (O/N), and caspase activation was determined either by western blot (B) assessing the cleavage of PARP and the caspase 3 (actin was used as a loading control) B-C. Fibroblasts were treated with 1 μM STS for 4, 6 or 16 h (O/N), and caspase activation was determined either by western blot (B) assessing the cleavage of PARP and the caspase 3 (actin was used as a loading control) or by DEVDase activity measurement (C). Two (B) or three (C) independent experiments were performed per condition. Differences between the control and patient fibroblasts were analyzed by Student's t-test (two-sided): extremely significant (***: p=0.0001). D-E. Mitochondrial membrane potential (ΔΨM) was measured by flow cytometry in control and patient fibroblasts after treatment of 1μM STS for 24 h (E) and quantified in D from two independent experiments. Differences between the control and patient fibroblasts were analyzed by Student's t-test (two-sided): significant (*:0.05>p>0.01). Patient 1 (P1) versus control (C2): *: p=0.0414 (24h). Patient 2 (P2) versus control (C2): *: p=0.0461 (24h). D-E. Mitochondrial membrane potential (ΔΨM) was measured by flow cytometry in control and patient fibroblasts after treatment of 1μM STS for 24 h (E) and quantified in D from two independent experiments. Differences between the control and patient fibroblasts were analyzed by Student's t-test (two-sided): significant (*:0.05>p>0.01). Patient 1 (P1) versus control (C2): *: p=0.0414 (24h). Patient 2 (P2) versus control (C2): *: p=0.0461 (24h).
Trf1 downregulation in K-RasG12V-transformed lung cells leads to a decreased tumor growth and decreased metastatic potential in allograft and xenograft models.Figure 5A-C The latency (A), volume (B), and weight (C) of subcutaneous tumors generated by control and Trf1-downregulated K-RasG12V-transformed lung cells in athymic mice.D Representative images of the subcutaneous tumors. E Trf1 expression levels measured by qPCR in the injected cell line and in the generated subcutaneous tumors. F-H Number of Ki67-positive (F), number of γH2AX-positive (G), and number of active caspase-3-positive (H) cells per field in the subcutaneous tumors. I Representative images of aberrant giant nuclei and anaphase bridges in the Trf1-downregulated subcutaneous tumors compared to the normal nuclei of control tumors. J TRF1 immunofluorescence shows the downregulation of Trf1 in lung tumors of the mice intravenously injected with control and Trf1-downregulated cells. K Tumor area measured in the lungs of the mice intravenously injected with control and Trf1-downregulated cells.L Representative images of the lungs colonized by control and Trf1-downregulated cells, respectively. M-O Number of Ki67-positive (M), number of γH2AX-positive (N), and number of active caspase-3-positive (O) cells per field in the lung tumors. P Trf1 expression levels measured by qPCR in the A549 cell line infected either with sh-scrambled or sh-Trf1. Q Growth of A549-derived tumors.