EMBO/sd-character-level-ner huggingface dataset:
Figure 3Tumour diameter before and after doxycycline withdrawal in 4 K and 5 KM breast tumours with cMet amplification. 0 indicates when doxycycline was removed. Each colour represents one tumour. Blue and green squares indicate the timeframe between doxycycline withdrawal and the moment in which tumours resumed growth. Genome-wide log2-ratio plots of chromosome 6 of two primary tumour biopsies and their corresponding non-regressed tumour showing no amplification in the primary tumour (upper panels) and a small amplification in the non-regressed tumours (bottom panels, yellow arrow). Immunostaining of phospho-cMet in 3 biopsied primary tumours (PT) and their corresponding non-regressed tumours (KM5, KM7 and KM8, which are also shown in Fig 2C). Scale bar 100 µm. Representative two-dimensional scatter plots constructed with overlaid dPCR data of the reference (VIC) and cMet (FAM) from one tumour without cMet amplification and one with cMet amplified. Dots represent results of independent PCR reactions in the wells of a digital PCR chip. Reactions in the bottom left corner (yellow) are negative for both targets, while the ones in the top right corner (green) are double positives. Reactions in the top left (blue) and bottom right (red) corners are positive for cMet and the reference targets, respectively. Representative photographs of FISH staining with a probe for Met (red signal) and a probe for a reference gene EML4 (green signals) The upper panel is a negative example for cMet amplification containing 2 red and 2 green dots (white arrows). The lower panel shows an example of a tumour with cMet amplification (several red dots) and 2 green dots (yellow arrow). Scale bar 10 µm.
Figure 7During cellular stress, Nox4 at the MAM augments the level of Akt activation secondary to redox inhibition of PP2a. Activated Akt in turn phosphorylates InsP3R and leads to an inhibition of calcium flux from ER to mitochondria. Mitochondrial calcium is maintained at a low level, the mitochondrial membrane potential (ΔΨm) is preserved, and cells remain viable. When Nox4 is deficient (bottom panel), high PP2a activity dephosphorylates Akt and leads to a reduced level of InsP3R phosphorylation. This removes the restraint on calcium transfer from ER to mitochondria resulting in increased mitochondrial calcium levels, triggering of the mPT, loss of ΔΨm and eventual cell death.
Figure 5A, B. HEK293 cells treated for 2, 4 and 8 h with 100 μM H2O2. After treatment, cells were incubated with 5 μM 2',7'-H2DCF-DA (A) or MitoSox (B) to evaluate the production of H2O2 or mitochondrial superoxide, respectively. Values are shown as units of DCF or MitoSox fluorescence relative to untreated cells (arbitrarily set as 1) and represent the means ± SD of n ≥ 3 independent experiments. *, p<0.05; **, p<0.01; ***, p<0.001. C. HEK293 were transfected for 48 h with siRNA against ATM (siATM), GSNOR (siGSNOR), or control siRNA (scramble, siScr). Afterwards, they were treated for 8 h with 100 μM H2O2 and mitophagy was assessed by Western blot of different mitochondrial complex subunits [i.e., NDUFB8 (complex I), SDHB (complex II), MTCO2 (complex IV)]. Basal and phospho ATM, as well as GSNOR were used to check the efficiency of siRNA-mediated knock down. Vinculin was used as loading control. D. Densitometry of mitochondrial protein immunoreactive bands of panel C (normalized to Vinculin) is indicated as H2O2-treated versus untreated cells (CTR) and expressed as arbitrary units. Values shown represent the means ± SD of n = 3 independent experiments. *, p<0.05; **, p<0.01; ***, p<0.001; n.s., not significant. E. In the same experimental settings, mitophagy was also assessed by RT-qPCR relative quantitation of D-loop (selected as measure of mtDNA) normalized to genomic actin (gActin). Results shown are the means ± SD of n = 8 experiments ***p<0.001; n.s., not significant. F. U2OS cells were depleted of endogenous ATM by repeated transfections with shRNA and induced, by doxycycline incubation, to express ATMWT, ATM2RA, or ATMCL mutant. Where indicated, cells were further transfected with a GSNOR-coding vector and then treated for 4 h with 100 μM H2O2. Mitophagy was assessed by RT-qPCR relative quantitation of D-loop normalized to genomic actin (gActin). Results shown are the means ± SD of n = 6 experiments. *p<0.05; ***p<0.001. In the same experimental settings, mitophagy was also assessed at 8 h by fluorescence microscopy analyses upon incubation with chloroquine (CLQ) to enhance differences in mitophagy. Percentage of mitochondria merging with LC3-positive puncta calculated by Fiji analysis software using the open-source plugin ComDet v. 0.3.7. Values are expressed as % of mitochondria (TOM20+ particles) co-localizing with LC3/cell and graphed as boxes (25th-75th interquartile range) and whiskers (minimum to maximum showing all points), with central bands representing the median of n ≥ 7 different cells analyzed. *p<0.05; **p<0.01; ***p<0.001. H. In the same experimental settings, mitophagy was also assessed at 8 h by fluorescence microscopy analyses upon incubation with chloroquine (CLQ) to enhance differences in mitophagy. Anti-TOM20 (red) was used to visualize mitochondria; anti-LC3 (green) was used to identify autophagosomes.
Figure 1FOXA1 and FOXA2 expression in human PDAC areas with distinct differentiation grade. Immunohistochemistry analysis of FOXA1 (top) and FOXA2 (bottom) in human PDAC. Representative G1, G2 and G3 areas from one patient are shown. Red dotted lines indicate the borders of poorly differentiated (G3) tumor areas. Percent of FOXA1- and FOXA2-positive nuclei in PDAC cells from areas of different grade. Tissue microarrays were automatically acquired and quantified using QuPath. The number (n) of analyzed areas of each grade is indicated. Expression of FOXA1 and FOXA2 in low-grade and high-grade PDAC cell lines. Vinculin is shown as loading control.
Figure 7Schematic representation of the relative contributions of Sct1p and Ole1p to the SFA (dark green)/UFA (light green) ratio, and its consequences for lipid flux from the endomembrane system (E) into mitochondria (M) in WT (i), WT overexpressing SCT1 (SCT1-oe) (ii), and cells lacking a tether that overexpress SCT1 (SCT1-oe tetherΔ) (iii). In the latter, the flux of SFA into the pool of membrane lipids is further enhanced by the depicted feedback mechanism in response to partially obstructing (red cross) lipid entry into mitochondria. The insets show the tendency of a di-unsaturated lipid to efflux from the endomembrane system for each condition, depicted as the extent of displacement from the bilayer.
Figure 6Heatmap showing differentially expressed genes after cyclin F knockdown, and rescued by additional E2F7/8 depletion. Highlighted genes are all involved in DNA repair. Cells were arrested with nocodazole for 16 hours prior to harvesting to minimize bias from potential differences in cell cycle progression between the different conditions. KEGG pathway analysis of genes downregulated by cyclin F knockdown, and rescued by additional E2F/8 depletion. Bars represent -log P values, such that larger values mean stronger statistical significance. The cut-off P value 0.05 is shown as a red dotted line. qPCR showing the RNA expression of atypical E2F- target genes that are involved in DNA replication or DNA repair. HeLa cells were transfected for 48 hours with siRNAs as indicated. Cells were incubated with nocodazole for 16 hours before harvesting. Data represent averages ± SEM (n=3); *P<0.05 or **P<0.01 (Student's t-test). n.s.: not significant. Immunoblotting showing the protein levels of Chk1 and Rad51 in the indicated siRNA conditions. HeLa cells were treated with nocodazole for 16 hours prior to harvesting.
Figure 3(A) Basal oxygen consumption in Flp-In 293 HA-IKKε wt, Flp-In 293 HA-IKKε KD-m and Flp-In 293 HA-IKKε UbLD-m cells following treatment with doxycycline (Dox, 16 hours), measured using Oroboros high resolution respirometry. Data are normalised to non-treated control cells. (B) Average TMRM staining intensity in Flp-In 293 HA-IKKε wt, Flp-In 293 HA-IKKε KD-m and Flp-In 293 HA-IKKε UbLD-m expressing cells induced by doxycycline (Dox, 16 hours). Data are normalised to non-treated control cells. (C) Basal mitochondrial oxygen consumption rate (OCR) in a panel of IKBKE (IKKε)-silenced breast cancer cell lines, measured using Seahorse XF96e or XF24 analysis. (D) OCR in mitochondria isolated from Flp-In 293 HA-GFP or HA-IKKε cells treated with doxycycline (50 ng/ml, 16 hours), measured using Oroboros high resolution respirometry. (E) Principal component analysis of differentially phosphorylated substrates in three independent single cell clones of Flp-In 293 HA-GFP or Flp-In 293 HA-IKKε cells treated with doxycycline (100 ng/ml, 16 hours). The phosphoproteomes in the three clones were analysed by mass spectrometry as described in material and methods. (F) Representative western blot showing l­evel of IKKε in three independent single cell clones of Flp-In 293 HA-GFP or Flp-In 293 HA-IKKε cells following treatment with doxycycline (100 ng/ml, 16 hours). (G) Relative pyruvate dehydrogenase (PDH) activity in Flp-In 293 HA-GFP or Flp-In 293 HA-IKKε cells treated with doxycycline (50 ng/ml, 16 hours). (H) Average TMRM staining intensity in Flp-In 293 HA-GFP or Flp-In 293 HA-IKKε cells treated with doxycycline (Dox) and DCA (both for 16 hours). Data are normalised to non-treated Flp-In 293 HA-IKKε cells. (I) Basal OCR in Flp-In 293 HA-GFP or Flp-In 293 HA-IKKε cells treated with doxycycline (50 ng/ml) in combination with dichloroacetate (DCA) for 16 hours, measured using Oroboros high resolution respirometry. (J) Basal OCR in Flp-In 293 HA-GFP or Flp-In 293 HA-IKKε cells treated with doxycycline (50 ng/ml) in combination with pyruvate deprivation for 16 hours, measured using Oroboros high resolution respirometry.
DIS3 compensates repeat RNA degradation in the absence of EXOSC10 A, B, C.Figure 5A, B, C. Double knockdown of EXOSC10 and DIS3 significantly increased poly-GA and repeat RNA expression compared with single knockdown of EXOSC10 in cells expressing (G4C2) 80 repeats. D. Overexpression of DIS3 compensated EXOSC10 knockdown-induced repeat RNA accumulation. E, F. Catalytically dead mutant EXOSC10 rescued EXOSC10 knockdown-induced GA increase.
NYKO mice manifest locomotor impairment of hind limbs.Figure 2Mean body weight of male wild type (WT, n = 15), NYKO (n = 12) and heterozygous NYKO (het NYKO, n = 7) mice from 4-32 weeks. Unpaired t-test was used for the comparison of two groups, ordinary one-way ANOVA for the comparison of three groups. * B Body composition analysis by nuclear magnetic resonance (NMR) of 31-32 week old male WT (n = 12) and NYKO (n = 7) mice. Unpaired t-test was used for the comparison of two groups, ordinary one-way ANOVA for the comparison of three groups. *P ≤ 0.05, *** P ≤ 0.001, ****P ≤ 0.0001. Data are means ± SEM. C Whole-body energy expenditure per kg lean mass of 26 week old male WT (n = 5) and NYKO (n = 5) mice after disease onset. Unpaired t-test was used for the comparison of two groups, ordinary one-way ANOVA for the comparison of three groups. Walking beam test of 6-7 week old (WT, n = 10; NYKO, n = 10), 17-18 week old (WT, n = 8; NYKO, n = 9) and 31-32 week old mice (WT, n = 12; NYKO, n = 19; het NYKO, n = 9). Unpaired t-test was used for the comparison of two groups, ordinary one-way ANOVA for the comparison of three groups. *P ≤ 0.05, *** P ≤ 0.001, ****P ≤ 0.0001. Data are means ± SEM. Walking beam test of 6-7 week old (WT, n = 10; NYKO, n = 10), 17-18 week old (WT, n = 8; NYKO, n = 9) and 31-32 week old mice (WT, n = 12; NYKO, n = 19; het NYKO, n = 9). Hind limb slips per run are shown in E. Unpaired t-test was used for the comparison of two groups, ordinary one-way ANOVA for the comparison of three groups. *P ≤ 0.05, *** P ≤ 0.001, ****P ≤ 0.0001. Data are means ± SEM. F Representative images of 31-32 week old WT and NYKO mice showing aberrant positioning of hind limbs.
Figure 2A. Genome browser view of AC and DMZ accessibility profiles for ectoderm-expressed (tfap2a and grhl3) and organizer-expressed (gsc and chrd) marker genes. B. Boxplots showing differential gene expression (AC versus DMZ) and associated ATAC-seq signals (Two biological replicates). The central band within the boxplot represents the median and the whiskers show 1.5 times the range between the first and third quartile (IQR). C. Hierarchical clustering of AC and DMZ ATAC-seq data on H3K4me3-positive (top) and p300-positive (bottom) ATAC-seq peaks. D. Heat map showing accessibility signal (log1p of fold over background) at p300-positive ATAC-seq peaks surrounding pluripotency genes (ventx1/ventx2, pou5f3 and sox2). The row labeled 'random' shows accessibility signals at random genomic loci. E. Single-cell ATAC-seq UMAP projection of cells derived from gastrula stage embryos (stage 10½), colored by cluster. F. Genomic tracks showing aggregated accessibility of single-cell ATAC-seq clusters at the t (tbxt), tfap2a, gsc, ctcf, lhx1 and sox11 loci.
Figure 6(A) Venn diagrams summarize global changes in gene expression caused by the inactivation of Foxn1 or Aff4 and detected by RNA-seq in murine skin at P6. The upregulated or downregulated gene populations of each mutant - and the extents to which they overlap - are indicated. (B) Gradient bars (green) signify the ranking of downregulated genes by fold-change, with genes ordered from most downregulated (gene #1) to least downregulated. Fold-change was determined by RNA-seq. Separate rankings were performed for each mutant (Foxn1 null or Aff4 cko). Pie charts indicate whether genes of a given rank in one mutant were downregulated in the other mutant. Percentages indicate the overlap in the mutants' downregulated gene populations. (C-E) KRT86 (red) is shown by immunofluorescence in skin sections from wild-type (WT), Foxn1 null, or Aff4 cko mice at P9. DNA is stained blue. HS marks examples of hair shafts. Scale bar, 40 µm.
Figure 7A Rosette phenotype of 3-weeks-old Col-0, lrx3/4/5 and 35S::LRR4-cit. Data information: Scale bars: 1 cm B Representative images and quantification of vacuolar morphology of late meristematic cells of Col-0 and 35S::LRR4-cit. PI (green) and MDY-64 (yellow) staining depict cell wall and vacuolar membrane, respectively. Col-0 (n=36) and 35S::LRR4-cit (n=32-36) seedlings were treated with DMSO or 50 µM EGCG for 22 h on solid medium. Kruskal Wallis test followed by Dunn´s multiple comparison (b: p < 0.05, c: p < 0.001). Data information: 5 µm (B). Boxplots: Boxplots: Box limits represent 25th and 75th percentile, horizontal line represents median. Whiskers display min. to max. values. C Relative root length of Col-0 (n=11-13) and 35S::LRR4-cit (n=11-14) after 3 days of RALF1 treatment. Statistical analyses were performed for each concentration using Student´s t-test (***p < 0.001). Boxplots: Boxplots: Box limits represent 25th and 75th percentile, horizontal line represents median. Whiskers display min. to max. values. D Schematic model depicting the FER- and LRX-dependent cell wall sensing mechanism, which limits vacuolar expansion (WT) in late meristematic cells. In contrast, dysfunctional cell wall perception in lrx3 lrx4 lrx5, fer-4 and 35S::LRR4-cit triggers premature vacuolar expansion.
Deep mutational scanning of PilE(A) Representation of the overall work flow used in the study with the three selection schemes of pilE mutants and subsequent NGS based analysis.Figure 1Representation of the overall work flow used in the study with the three selection schemes of pilE mutants and subsequent NGS based analysis. Volcano plots of piliation, aggregation and adhesion mutation scores for all pilE mutations. Stop and synonymous mutations are highlighted in blue and orange respectively. Adhesion and aggregation mutation scores relative to piliation. The correlation appears as a red dotted line.
Figure 1Formation and hydrolysis of (A) 4→3 and (B) 3→3 crosslinks. The disaccharide-pentapeptide unit is assembled from N-acetylglucosamine (GlcNAc), N-acetylmuramic acid (MurNAc), and five amino acids including meso-diaminopimelic acid (DAP), which is linked via its L (S) center to the γ-carboxyl group of D-Glu. (C) Hydrolysis of the D-Ala4-D-Ala5 and DAP3-D-Ala4 peptide bonds by carboxypeptidases of D,D and L,D specificities, respectively.
Figure 1A. Body weight gain of male mice with the specified genotypes under normal (ND) or high-fat diet (HFD). B. Quantification of fat, free fluid and lean mass by nuclear magnetic resonance (NMR) of mice in HFD in panel A. C. Organ weight (percentage of total weight) of different fat depots, liver and quadriceps of Pkd2wt/wt and Pkd2ki/ki male mice after 22 weeks in HFD. D. Quantification of the average adipocyte size in SubWAT and EpiWAT of male mice of the specified genotypes in normal and high-fat diet. E. Representative pictures of H&E staining of SubWAT and EpiWAT of indicated male mice fed HFD. F. Triglyceride content in liver of Pkd2wt/wt and Pkd2ki/ki male mice in panel A. Relative to Pkd2wt/wt. G. Glucose tolerance test of the specified genotypes after 16 weeks in HFD. H. Insulin tolerance test of Pkd2wt/wt and Pkd2ki/ki male mice after 18 weeks in HFD. Triglycerides in circulation of specified mice after 18 weeks in HFD. free fatty acids (J) in circulation of specified mice after 18 weeks in HFD. Energy expenditure of mice after 20 weeks in HFD. energy intake (L) of mice after 20 weeks in HFD.
Btn2 triggers protein aggregation inside the nucleus A, B S.Figure 6A, B S. cerevisiae wt, hsp42Δ, btn2Δ, and hsp42Δ btn2Δ cells expressing GFP‐VHL (green, A) or GFP‐luciferase‐DM‐NLS (green, B) were grown at 30°C and shifted to 37°C for 90 min in the presence of MG132, and protein localizations were recorded. DNA was stained by DAPI (blue), and the nuclear envelope was stained by Nsp1immunofluorescence microscopy (red). Solubilities of GFP‐VHL and GFP‐luciferase‐DM‐NLS were determined after stress application by Western blot using GFP‐specific antibodies. Solubility of actin was determined as a control. T, total fraction; S, soluble fraction; P, pellet fraction. Scale bars, 2 μm. C S. cerevisiae wt, hsp42Δ, btn2Δ, and hsp42Δ btn2Δ cells expressing mCherry‐VHL were heat‐shocked to 38°C. Upon return to 30°C, protein synthesis was stopped by cycloheximide (CHX) addition and degradation of mCherry‐VHL was monitored by Western blot analysis. The stability of GFP‐luciferase‐DM‐NLS expressed in S. cerevisiae wt and btn2Δ cells was determined accordingly. Ccs1 or Zwf1 levels are given as loading controls. D S. cerevisiae hsp42Δ btn2Δ cells expressing either GFP or LuciDM‐GFP and Htb1‐mCherry were grown at 30°C and heat‐shocked to 37°C for 30 min. Line intensity plots of luciferase‐DM‐GFP and Htb1‐mCherry before and after heat shock are given. The ratio of nuclear and cytosolic luciferase‐DM‐GFP fluorescence intensity was determined at 30°C and 37°C (n > 25). Scale bars, 2 μm.
Figure 3(A, B) In vitro degradation of model proteins by purified S. cerevisiae proteasome. The model proteins (UbL-DHFR-95) consisted of an N-terminal UbL domain, followed by an E. coli DHFR domain and a 95 aminoacid tail derived from S. cerevisiae cytochrome b2. UbL-DHFR-95 proteins were degraded by proteasome (red solid circle) and can be stabilized by removing the UbL domain (no UbL, black triangles), by removing the 95-amino-acid tail (no tail, blue diamonds) or by proteasome inhibitor (MG132, green squares). (A) Degradation of model proteins containing the UbL domain of S. cerevisiae Rad23. (A, B) In vitro degradation of model proteins by purified S. cerevisiae proteasome. The model proteins (UbL-DHFR-95) consisted of an N-terminal UbL domain, followed by an E. coli DHFR domain and a 95 aminoacid tail derived from S. cerevisiae cytochrome b2. UbL-DHFR-95 proteins were degraded by proteasome (red solid circle) and can be stabilized by removing the UbL domain (no UbL, black triangles), by removing the 95-amino-acid tail (no tail, blue diamonds) or by proteasome inhibitor (MG132, green squares). (A) Degradation of model proteins containing the UbL domain of S. cerevisiae Rad23. (B) Degradation of model proteins containing other UbL domains or ubiquitin. The graphs plot the amount of substrate estimated by electronic autoradiography in SDS-PAGE gel bands over time as normalized to the initial substrate amount as described in Methods. Data points represent mean values determined from at least three repeat experiments; error bars indicate s.e.m.
a) Top: LC3 immunostaining of 18Q-htt and 111Q-htt MEFs maintained in the presence (+) or absence (−) of serum and inhibitors of lysosomal proteolysis (inhib+).figf2(a) Top: LC3 immunostaining of 18Q-htt and 111Q-htt MEFs maintained in the presence (+) or absence (-) of serum and inhibitors of lysosomal proteolysis (inhib+). Bottom: mean number of LC3+ vesicles per cell in cells maintained in the presence (left) or absence (right) of serum. n = 4. (b) Top: LC3 immunoblot of the same cells after serum (S) removal or thapsigargin (TG) treatment. PI, protease inhibitors. Bottom: LC3-II levels and LC3-II flux (calculated as a multiple of LC3-II value in absence of PI; lateral numbers). n = 4. (c) LC3-II values and LC3-II flux in neuronal cultures from wild-type (WT) or HD94 mice (HD) grown on wild-type rat astrocyte monolayers, analyzed as in b. n = 4. (d) LC3 staining of lymphoblasts, from three unaffected controls (UC) and people with HD, maintained in the presence or absence of serum. Accession numbers are indicated above panels. Right: mean number of LC3+ vesicles. Extended study in Supplementary Figure 8b,c. (e) Immunoblots for LC3-II in UC and HD lymphoblasts treated or not treated with protease inhibitors. Right: LC3-II values and LC3-II flux. Extended study in Supplementary Figure 8a. Mean + s.d.; #P 0.05. Full-length blots in Supplementary Figure 20.
Figure 4A. F4L5.13 prevents BRB defects and restores vascular morphogenesis in Tspan12-/- mice. 10 mg/kg F4L5.13 or isotype control antibody was injected i.p. every 48h from P5 to P20. Multiple images acquired at 10x magnification were stitched together to create a high-resolution image of a representative part of the retinal flat mounts. Scale bar 500 µm. B. Optical sections of IB4-stained P20 retinas showing the mouse three-layered retinal vasculature (superficial, intermediate, and deep layers at 20x magnification) to document blood vessel formation or malformation. In Tspan12-/- mice, the intermediate capillary layer fails to properly develop and glomeruloid vascular malformations are present. The deep capillary layer is virtually absent. F4L5.13 fully restores intraretinal capillary development in Tspan12-/- mice. Scale bars: 100 µm. C. IB4-stained retinal cross sections show the restored retinal vasculature and the absence of misdirected angiogenesis after F4L5.13 treatment. Boxed areas are shown enlarged in the panels on the right. Scale bars: 500 µm. D. Quantification of the vascularized area in the outer plexiform layer. 10 mg/kg F4L5.13 or isotype control antibody was injected every 48h from P5 to P20. Data are presented as mean± SD, n=5 retinas (2-4 fields of view per retina) from 4-5 mice per group. Significance was calculated by one-way ANOVA with Bonferroni's multiple comparisons test. E E. F4L5.13 rescues barrier function defects in Tspan12-/- mice as shown by an increased expression of the tight junction component CLDN5 and decreased expression of the EC fenestration component PLVAP. IB4-Alexa647 was used to stain ECs. Scale bars: 100 µm.