EMBO/sd-character-level-ner huggingface dataset:
Figure 1(A) IDH1 acetylation levels upon treatment with NAM or TSA. Flag-tagged IDH1 was ectopically expressed in HEK293T cells treated with NAM (5 mM) and/or TSA (0.5 mM) for the indicated time period. (B) Five putative lysine residues were mutated. Acetylation levels of Flag-bead-purified IDH1 were determined by Western blot analysis using a pan-anti-acetyllysine antibody. Relative IDH1 acetylation ratios were calculated after normalizing against Flag. (C) Mutation of IDH1 K224R and K224Q resulted in altered acetylation levels using Western Blot. (D) IDH1 catalytic activity of IDH1 K224 mutants in vitro. Wild-type IDH1 and K224R, K224Q mutants were expressed in HEK293 cells. Proteins were purified by immunoprecipitation (IP), IDH1 levels were normalized for protein, and activity assays were performed. Data information: All results were expressed as mean ± SD of three independent experiments (n ≥ 3 per experimental condition). For multiple comparisons in (D), a one-way ANOVA with Newman-Keuls post hoc test was used to test for statistical significance. (E-F) Steady-state kinetic analysis of IDH1 and variants in HEK293T cells. Comparison of IDH1 WT and K224R and K224Q showed that the K224 site was important in catalysis. Data information: All results were expressed as mean ± SD of three independent experiments (n ≥ 3 per experimental condition). For (E-F), statistical significance was determined using the two-way ANOVA followed by Tukey's post hoc test. *P<0.05 and **P<0.01. (G) IDH1 directly interacted with SIRT2 but not SIRT1. SIRT2 deacetylated IDH1 effectively. Flag tagged IDH1 and HA tagged SIRT1 or SIRT2 were co-transfected into cells. The acetylation level of Flag-bead-purified IDH1 and the protein association between IDH1 and SIRT1 or SIRT2 were determined by Western blot. (H) Co-immunoprecipitation showed endogenous proteins of IDH1 and SIRT2 interacted with each other. (I) SIRT2 interacted with IDH1 in vitro. GST pull-down assay with GST-fused SIRT2 and Flag-tagged IDH1 proteins. (J) Acetylation level of endogenous IDH1 K224 when treated with SIRT1 or SIRT2 by Western blot analysis. (K) IDH1 K224 deacetylation was dependent on SIRT2 catalytic activity. SIRT2 H187Y was a catalytically inactive mutant.
STAT1Y701F mutant reduces IFNβ-stimulated nuclear translocation of STAT2.Figure 4A Analysis of STAT2 nuclear translocation by immunofluorescence. Bone marrow derived macrophages (BMDMs) of wild type (WT), Stat1Y701F (YF), Stat1-/- (S1), Stat2-/- (S2) and IRF9-/- (IRF9) mice were seeded on cover slips and stimulated with 250 IU/mL of IFNβ for 30 min or 24 h. The cells were fixed and stained for STAT2 specific antibody followed by Alexafluor® 488 conjugated secondary antibody (green). Nuclei were stained with DAPI (blue). Studies are representative of more than three independent experiments. The scale bars represent 10 µm.B Quantitative evaluation of STAT2nuclear translocation. The intensity of STAT2-dependent immunofluorescence over DNA staining (DAPI) was quantified using ImageJ software in 20 cells from two independent experiments. Bars represent a mean with standard deviation (SD) and asterisks denote the level of statistical significance (***, p≤ 0.001); p-value was calculated using unpaired t-test.
Figure 4Nucleoplasmic YTHDC1 (A) and WTAP (B) intensities upon HSATIII knockdown. Control and HSATIII knockdown cells after thermal stress exposure (42°C for 2 h and recovery for 1 h at 37°C) were stained by HSATIII-FISH and IF using anti-YTHDC1 or anti-WTAP antibodies. nSB and whole nuclear areas were defined by binarized images of HSATIII and DAPI, respectively. Box plots represent the average intensities of YTHDC1 and WTAP in the non-nSB nucleoplasm area of each nucleus. The first and third quartiles are the ends of the box, the median is indicated with a vertical line in the box, and the minimum and maximum except for the outliers are the ends of the whiskers. The outliers are indicated with open circles. The mean is indicated with X. P-value (Mann-Whitney U-test) is shown above (n=40). (B, D) Correlation between the intensity of YTHDC1 (B) or WTAP (D) in non-nSB nucleoplasm and the nSB occupancy in individual nuclei. nSB occupancy is the ratio of the nSB area to the whole nuclear area of each nucleus in (A) and (C). Pearson coefficient of correlation (R) and p-value are shown above (n=40). Western blot validation of WTAP and YTHDC1 protein levels after HSATIII knockdown. HeLa cells were transfected with HSATIII ASO and control sense oligonucleotide, cultured for 16 h, and exposed to thermal stress (42°C for 2 h and recovery for 1 h at 37°C). Cell extracts were used for western blotting. GAPDH is a control. Quantification of E showing mean ± SD values (n=3). P-values (Student's t-test) are shown above. HSATIII dependent sequestration of nucleoplasmic YTHDC1 (G) and WTAP (H) at four time points during thermal stress and recovery. The experiments same as A and C were performed at the indicated timepoints. The mean is indicated with X. The first and third quartiles are the ends of the box, the median is indicated with a vertical line in the box, and the minimum and maximum except for the outliers are the ends of the whiskers. The outliers are indicated with open circles. P-values (Kruskal-Wallis test, followed by Dunn's multiple comparison test) are shown above (n=40).
Figure 6A. 4x106 LNCaP cells were injected into NSG mice (5 per group) followed one day later by transfer of human PBMC (107 i.p.) and application of bsAbs (10µg) at day 1 and 4 (IgGsc) and daily at day 1-6 (Fabsc), respectively. At day 21, mice were killed and tumor cells in the lung were quantified by flow cytometry after enzymatic digestion. Statistical analysis was performed using the unpaired t test. Error bars represent standard deviation of the mean value. B, C. 106 LNCaP cells were injected s.c. into the right flank of NSG mice (5 per group) and treatment with PBMC and bsAbs was started when tumors had reached a diameter of 5mm. BsAbs (2µg per dose) were applied at day 1, 8 and 15 (IgGsc) and daily at day 1-3, day 8-10 and day 15-17 (Fabsc), PBMC were applied at day 1,8 and 15. Mean values and standard deviations of five mice per group are indicated.
Figure 3(a) HUWE1 and MYC Western blot analysis of epithelial cell extractions from control and Huwe1 deficient intestines. Levels of MYC protein are significantly increased in normal intestines lacking HUWE1 (Mann Whitney, p = 0.04, n = 3). (b) QRT-PCR analysis of Myc expression in control and Huwe1 deficient intestines. (c) MYC IHC in tumours from Vil Apc and Vil Apc Huwe1hom mice. Scale bars = 50 µm. (d) MYC Western blot in protein extracts from Vil Apc and Vil Apc Huwe1hom tumours. Levels of MYC protein are significantly increased in tumours lacking HUWE1 (Mann Whitney, p = 0.04, n = 3). (e) QRT-PCR analysis of Myc expression in tumours from Vil Apc and Vil Apc Huwe1hom mice.
Establishment of patient-derived organoids from solid HGSOC deposits.Figure 1Summary of cancer patient data with TNM and FIGO classifications showing advanced stage of disease at the time of surgery. Graphic representation of the standard experimental procedure for tumor patient material. Samples were obtained at the time of primary debulking surgery from the high purity tumor deposits in peritoneum/omentum. In vitro niche dependency of HGSOC tumor cells. Phase-contrast pictures illustrate that isolated ovarian cancer cells rely on EGF supplementation for growth, while they do not grow at all in Wnt3a supplemented medium. Also, inhibition of BMP signaling through Noggin has strong negative effect on the initial growth. Scale bar: 500 µm. E - EGF, F - FGF10, N -Noggin, R - R-spondin1, B - Basic medium, P - Passage Cancer organoids express HGSOC markers Pax8 and EpCAM and have lost the cystic phenotype suggesting complete breakdown of epithelial polarity as seen on confocal images from two representative organoid lines. Scale bar: 20 µm. HE staining of organoids and respective tissue confirm high similarity in cellular structure and tissue organization. Scale bar: 100 µm. HGSOC organoids show differential response to carboplatin treatment, confirming patient-specific sensitivity of the cultures. Cell viability assay was performed after 5 days of treatment with different concentrations of carboplatin on mature organoids from three different donors. Data represent mean ±SD of technical triplicates.
| Endothelial cells of the zebrafish embryonic cerebral vessels develop "kugeln"; large spherical membrane protrusions.Figure 1MIP of cerebral vessels of 3dpf Tg(kdrl:HRAS-mCherry)s916 embryo (grey LUT; inverted). Higher magnification panel showing two kugeln (arrowheads) arising from the middle mesencephalic central artery (MMCtA). Diameter of kugeln at 3dpf (mean ± s.e.m. 10.13 ± 0.49; n=93 kugeln from 32 3dpf embryos; 3 experimental repeats). Number of kugeln per embryo in 3, 4 and 5dpf animals was not statistically significantly different (mean ± s.e.m. 3dpf 5.47 ± 1.09, 4dpf 7.47 ± 1.46, 5dpf 7.09 ± 1.55; p = 0.8571; 3dpf: 175 kugeln from 32 embryos; 4dpf: 239 kugeln from 32 embryos, 5dpf: 227 kugeln from 32 embryos; 4 experimental repeats; Kruskal-Wallis test). Kugeln (arrowheads) could be observed in 28dpf animals. MIP of cerebral vessels of 4dpf Tg(fli1aep:CAAX-eGFP) embryo (grey LUT; inverted). Higher magnification panel shows a kugel (arrowhead) protruding from the posterior mesencephalic central artery (PMCtA). Single Z-plane micrograph of cerebral vessels of a 3dpf Tg(gata1:dsRed), injected with a Tol2-fli1a:myr-Cherry construct showing two kugeln (arrowheads) in the higher magnification panel protruding from the MMCtA. Locations and proportion of kugeln on cerebral vessels (colour coded by vessel; total n=107 kugeln from 34 4dpf embryos; 3 experimental repeats). AMCtA - anterior mesencephalic central artery, BCA - basal communicating artery, CaDI - caudal division of internal carotid artery, CMV - communicating vessel, CtA - central arteries, MMCtA - middle mesencephalic central artery, MtA - metencephalic artery, PCS - posterior communicating segment, PMBC - posterior midbrain channel, PMCtA - posterior mesencephalic central artery, PrA - prosencephalic artery. Location of kugeln by vessel and laterality (107 kugeln from 34 4dpf embryos; 3 experimental repeats comparison of left vs right p 0.3592; Mann-Whitney U test).
The molecular signature of KPM cells is enriched in human mesothelioma.Figure 9A Unsupervised hierarchical clustering shows distinctive gene expression of KPM versus PMC. B Volcano plot showing some top KPM versus PMC differentially expressed genes. C KPM and PMC expression of classic mesothelioma markers (top) and top KPM versus PMC overexpressed genes (bottom). D Gene set enrichment analysis, including enrichment score and nominal probability value of the 150 gene-signature specifically over-represented in human mesothelioma compared with other thoracic malignancies derived from 113 patients (GSE42977) within the transcriptome of KPM cells versus PMC shows significant enrichment of the human mesothelioma signature in KPM cells.
Figure 7A, Schematic representation of the experimental design. Zeitgeber time 0 (ZT0) corresponding to lights ON. B, Immunostaining for GFP and HA in Ctrl and Fxr1 mouse brain slices. Arrows indicate presence and arrowheads indicate an absence of GFP tagged Fxr1 granules. C, Quantification of colocalization of GFP and HA labeled neurons (GFP+HA+ 71.3%±3.7, n=3, GFP-Fxr1+HA+ 79.7%±2.8, n=3). D, Enrichment of differentially expressed transcripts from Ctrl/S vs Ctrl/SD comparison in GO:BP. Top 10 clusters with the most number of enriched pathways (nodes) are shown. E, SynGO enrichment for synaptic localization and function of differentially expressed transcripts from Ctrl/S vs Ctrl/SD comparison. F, Venn diagram showing overlap of differentially expressed transcripts between Ctrl/S vs Ctrl/SD and Ctrl/SD vs Fxr1 overexpression (Fxr1)/SD comparisons. G, Heat map showing transcripts that have bidirectional expression changes between Ctrl/S, Ctrl/SD, and Fxr1/SD conditions. H, Enrichment of commonly affected transcripts between Ctrl/S vs Ctrl/SD and Ctrl/SD vs Fxr1/SD comparisons in GO:BP. I, SynGO enrichment for synaptic localization and function of commonly affected transcripts between Ctrl/S vs Ctrl/SD and Ctrl/SD vs Fxr1over/SD comparisons.
A wild-type Tex11 knockin allele at the autosomal Tex19.Figure 2Schematic diagram of Tex11 gene structure, Tex19.1 gene structure, targeting vector and the final Tex11 knockin allele containing the wild-type Tex11 ORF. Western blot analysis of TEX11 in the testes of 3-month-old mice with different Tex11 gene dosages. ACTB serves as a loading control. Testis weight of 3-month-old males. Sperm count of 3-month-old males. Histological analysis of testes from adult wild-type, Tex11 KI/KO, and Tex11−/Y males. RS, round spermatids; ES, elongating spermatids; Pa, pachytene spermatocytes. Scale bar, 50 μm. The Tex11 knockin allele rescues chromosomal synapsis defects in Tex11−/Y spermatocytes in 3-month-old males. Chromosomal synapsis defects were assessed by SYCP1 and SYCP2 immunostaining of spread nuclei from 100 pachytene spermatocytes per male; for each genotype, three males were analyzed. Abbreviations for Tex11 genotypes: −/Y, Tex11 knockout (Yang et al, 2008); KI;−/Y, Tex11 knockin and knockout; +/Y, wild type; KI;+/Y, Tex11 knockin plus wild-type Tex11.
Carboxy-terminal region of the pilin involved in aggregation(A) Pilus structure (PDB: 5KUA) showing a cluster of charged amino acids surrounding the protruding K140 involved in aggregation.Figure 3Pilus structure (PDB: 5KUA) showing a cluster of charged amino acids surrounding the protruding K140 involved in aggregation. N138 is in light blue, Q122 and K140 in orange and E99, K103, K144 and H149 in dark blue. Piliation per bacterium as measured by flow cytometry using the 20D9 monoclonal antibody and the F10 nanobody. Visualization of type IV pili by immunofluorescence either using the 20D9 antibody or F10 nanobody as indicated (magenta). In the case of the K140Q mutant labeled with the 20D9 antibody the background was made visible to convincingly show absence of pilus labeling. Scale bar: 2 µm. Aggregation expressed as a function of piliation per bacterium measured with 20D9 antibody except for pilEQ122E and pilEK140Q for which the F10 nanobody value is reported. Each dot represents values for a PilE point mutant relative to the SB strain. Hypo-aggregative mutants are highlighted in orange while hyper-aggregative mutants are in light blue. solid lines indicate the 95% confidence interval. Quantification of PilX present in purified pili preparations sheared from the bacterial surface using western blot. PilX levels were normalized using PilE as a reference and normalized to pilESB values. Mean ratio ± SEM is indicated. N=3 independent experiments. Paired t-test.
Figure 8(A) Representative micro-CT images of trabecular bone from the femoral metaphysis of Cdc20f/f with negative control lentivirus, Sp7-Cre;Cdc20f/f with negative control lentivirus, and Sp7-Cre;Cdc20f/f with p65sh lentivirus. Scale bar, 500 μm. (B) Histomorphometric analyses of Cdc20f/f with negative control lentivirus, Sp7-Cre;Cdc20f/f with negative control lentivirus, and Sp7-Cre;Cdc20f/f with p65sh lentivirus. (n=6) (C, D) The ALP staining (C) and ALP quantification (D) of BMSCs from Cdc20f/f mice and Sp7-Cre;Cdc20f/f mice after 7 days osteogenic differentiation treated with negative control or p65si. (n=5) The ALP staining (E) of BMSCs from Cdc20f/f and Sp7-Cre;Cdc20f/f mice after 7 days osteogenic differentiation treated with BAY 11-7082 (NF-κB pathway inhibitor). (n=5) ALP quantification (F) of BMSCs from Cdc20f/f and Sp7-Cre;Cdc20f/f mice after 7 days osteogenic differentiation treated with BAY 11-7082 (NF-κB pathway inhibitor). (n=5) (G) The expression of RUNX2 in BMSCs from Cdc20f/f mice and Sp7-Cre;Cdc20f/f mice after 7 days osteogenic differentiation treated with negative control or p65si by western blot analyses.
Fig.Figure 1(A) Western blot analysis of NDUFS3 protein levels in homogenates from quadriceps muscle of 15-days old (upper panel) and 1-month old animals (lower panel). (B) Body weight comparison over time of Ndufs3 smKO male mice (filled blue squares; n=14) and age matched wild-type (WTFlx; n=15) males (empty blue squares). Ndufs3 smKO females (filled red circles) showed similar results (n=10 for smKO, and n=12 for WTFlx group). P values were calculated by Student's t test; (C) Representative image of a smKO male Ndfu3s3-smKO mouse and a WTFlx littermate of 6-month old showing decreased body weight. (D) Survival curve of Ndufs3 smKO female mice (red line) and males (blue line). P values were calculated using Log-rank (Mantel-Cox) Test; p=0.0010 for males (n=19), and p=0.0004 for females (n=10). (E) Nocturnal ambulatory activity of Ndufs3 smKO and aged matched wild-type littermates from 1 and 2 months old male mice. P values were calculated by Student's t test. P values were calculated by Student's t test to determine the level of statistical difference. ; *p<0.05, **p<0.01 and ***p<0.0001. (F) The number of falls in the treadmill of WTFlx and Ndufs3 smKO male mice, starting at 2 months (n=8). Bars represent means ± standard error (SE). P values were calculated by Student's t test to determine the level of statistical difference. n.s.-not significant; *p<0.05, **p<0.01 and ***p<0.0001. (G) Rotarod performed by Ndufs3 smKO and wild-type male mice. (n=8). Bars represent means ± standard error (SE). P values were calculated by Student's t test to determine the level of statistical difference. n.s.-not significant; *p<0.05, **p<0.01 and ***p<0.0001.
Fibrosis in Sox9fl/fl;AlbCre+/- mice.Figure 4A-C Representative images shown for olive oil treated (n=5) or chronic CCl4 (n=5) induced fibrosis or following sham operation (n=6 Sox9fl/fl;AlbCre-; n=5 Sox9fl/fl;AlbCre+) or BDL (n=5 Sox9fl/fl;AlbCre-; n=8 Sox9fl/fl;AlbCre+) induced fibrosis. SOX9 immunohistochemistry (brown; A), in situ hybridization for Sox9 (brown) and α-Sma (red) and collagen deposition by PSR staining (red; B) in control (Sox9fl/fl;AlbCre-) or Sox9-null (Sox9fl/fl;AlbCre+) mice following fibrosis. Higher magnified image of SOX9 localization in discrete cells within the scar is shown for CCl4 and BDL in the Sox9fl/fl;AlbCre+ mice (A).  A-C Representative images shown for olive oil treated (n=5) or chronic CCl4 (n=5) induced fibrosis or following sham operation (n=6 Sox9fl/fl;AlbCre-; n=5 Sox9fl/fl;AlbCre+) or BDL (n=5 Sox9fl/fl;AlbCre-; n=8 Sox9fl/fl;AlbCre+) induced fibrosis. SOX9 immunohistochemistry (brown; A), in situ hybridization for Sox9 (brown) and α-Sma (red) and collagen deposition by PSR staining (red; B) in control (Sox9fl/fl;AlbCre-) or Sox9-null (Sox9fl/fl;AlbCre+) mice following fibrosis. Higher magnified image of SOX9 localization in discrete cells within the scar is shown for CCl4 and BDL in the Sox9fl/fl;AlbCre+ mice (A). A-C Representative images shown for olive oil treated (n=5) or chronic CCl4 (n=5) induced fibrosis or following sham operation (n=6 Sox9fl/fl;AlbCre-; n=5 Sox9fl/fl;AlbCre+) or BDL (n=5 Sox9fl/fl;AlbCre-; n=8 Sox9fl/fl;AlbCre+) induced fibrosis. SOX9 immunohistochemistry (brown; A), in situ hybridization for Sox9 (brown) and α-Sma (red) and collagen deposition by PSR staining (red; B) in control (Sox9fl/fl;AlbCre-) or Sox9-null (Sox9fl/fl;AlbCre+) mice following fibrosis. Higher magnified image of SOX9 localization in discrete cells within the scar is shown for CCl4 and BDL in the Sox9fl/fl;AlbCre+ mice (A). D, E. Quantification of surface area covered by the PSR staining in (C). D, E. Quantification of surface area covered by the PSR staining in (C). F Expression of SOX9 protein by immunohistochemistry in quiescent (Q) and activated (A) HSCs extracted from Sox9fl/fl;AlbCre+ mice. G Individual fluorescent channels showing localization and expression of α-SMA (left panel), SOX9 (middle panel) and composite image for α-SMA (green) and SOX9 (red) in right panel in activated Sox9fl/fl;AlbCre+ HSCs (genotyping shown in Appendix Figure S9). Two-tailed unpaired t-test was used for statistical analysis. Data in bar charts show means ± s.e.m. Size bars = 100 μm (A), 10 μm (B) 200 μm (C), 25 μm (G).
Figure 1A Scatterplot representing differences in ssGSEA immune signatures in TERThigh versus TERTlow tumours (n = 4,632 tumours in each group) across TCGA. -log10(padj) for enrichment of ssGSEA immune signatures in TERThigh versus TERTlow tumours is shown on the y-axis. Signatures more highly represented in TERThigh tumours are shown on the right, while those in TERTlow tumours are shown on the left. B Heat map for ssGSEA of immune signatures in TERThigh and TERTlow tumours (n = 4,632 tumours in each group) across TCGA. Th1, Th2, Th17: T helper 1, 2, 17 cells, Tcm: central memory T cells, Tem: effector memory T cells, Tgd: gamma delta T cells, TFH: follicular helper T cells, Treg: regulatory T cells, MDSC: myeloid-derived suppressor cells, DC: dendritic cells, aDC: activated DCs, iDC: immature DCs, pDC: plasmacytoid DCs, NK cells: natural killer cells. C Heat map of interferon-related genes in U2OS cells with TERT-WT or TERT-K626A ectopic expression. D-F RT-qPCR analysis of interferon-related genes (D), western blot analysis of TERT, TBK1, pTBK1, IRF3, pIRF3, and GAPDH levels (E), and ELISA of IFNβ and CXCL10 in culture supernatant (F) in U2OS and HeLa cells ectopically expressing TERT-WT or TERT-K626A. G-I RT-qPCR analysis of interferon-related genes (G), western blot analysis of TERT, TBK1, pTBK1, IRF3, pIRF3, and GAPDH levels (H), and ELISA analysis of CXCL10 in culture supernatant (I) in HeLa and HCT116 cells with TERT knockdown.
A working model.Figure 8 Figure 8: A working model. In wild type, ddaC neurons establish minus-end-out MT orientation which is likely required for dendrite pruning. In msps or tacc mutant neurons, minus-end-out MT orientation in dendrites is disrupted, which likely results in impaired dendrite pruning. MT depolymerization, which is induced by both MDA treatment and katanin overexpression, also causes mixed MT orientation in dendrites as well as dendrite pruning defects. This study supports a link between minus-end-out MT orientation and dendrite pruning. Figure EV legends
Figure 5Trajectories of the different tau biomarkers in preclinical AD using a local weighted regression method (Loess curve). Changes on biomarkers levels in CSF are represented as z-scores using Aβ PET in centiloid scale (CL) as a proxy of disease progression. Abnormal biomarker levels were determined as two standard deviations (SD) above the mean. Incipient Aβ pathology positivity was determined as Aβ PET higher than CL 12 (Mila-Aloma et al., 2020). All samples were run in singlicates.