EMBO/sd-character-level-ner huggingface dataset:
UPP1 induction deregulates pyrimidine metabolism and contributes to cell death A Heat map for pyrimidine pathway-related metabolites derived from a non-targeted metabolomics analysis, depicting relative levels in HCT116 cells treated as indicated for 18h (NEN (N) 1.Figure 4A Heat map for pyrimidine pathway-related metabolites derived from a non-targeted metabolomics analysis, depicting relative levels in HCT116 cells treated as indicated for 18h (NEN (N) 1.2 µM, Domperidone (Dom) 30 µM or in combination (Comb). B Relative UPP1 mRNA expression levels in HCT116 cells treated as indicated (NEN (N) 1.2 µM, Domperidone (Domp, D) 30 µM, Imipramine (Imi), Desipramine (Desi), Amitriptyline (Ami) each 30 µM, Clomipramine (Clomi) 20 µM), determined by qPCR. Data information: data (N=3) are presented as mean (SD) and were analyzed by one-way ANOVA with Tukey post-hoc test In (B), right panel, significance is indicated for the comparison of combinatorial treatments (NEN+TCAs) to controls. ** p< 0.01;*** p< 0.001; **** p < 0.0001. C Relative UPP1 mRNA expression levels in HCT116 cells either transfected with control (siCtrl) or combined TFE3- and MITF- targeting (siTFE3/siMITF) siRNAs and treated as indicated (NEN (N) 1.2 µM, Domperidone (D) 30 µM, ISRIB 1 µM), determined by qPCR. Data information: data (N=3) are presented as mean (SD) and were analyzed by two -way ANOVA with Tukey post-hoc test D Relative UPP1 mRNA expression in HCT116 cells transfected either with control (siCtrl) or UPP1-targeting (siUPP1) siRNAs and treated as indicated (NEN (N) 1.2 µM, Domperidone (D) 30 µM). Data information: data (N=3) are presented as mean (SD) and were analyzed by two -way ANOVA with Tukey post-hoc test E Ratio of toxicity and viability measurements of HCT116 spheroids (3D) and 2D cultures, treated as indicated (NEN 1.2 µM, Domperidone 30 µM). Cells were transfected either with control (siCtrl) or UPP1-targeting (siUPP1) siRNAs prior to spheroid formation or during cell seeding. Data information: data (N=3) are presented as mean (SD) and were analyzed by two -way ANOVA with Tukey post-hoc test F Immunoblot of HCT116 whole cell lysate using antibodies against the indicated proteins upon treatment of the cells as indicated for 16h (NEN (low) 0.6 µM or NEN (High) 1.2 µM, Domperidone (Domp) 30 µM) Cells were transfected either with control (siCtrl) or UPP1-targeting (siUPP1) siRNAs 48h prior to treatment. G Immunoblot of HCT116 whole cell lysates using antibodies against the indicated proteins upon treatment of the cells as indicated for 16h (NEN 1.2 µM, Domperidone (Domp), Imipramine (Imi), Amitriptyline (Ami) and Desipramine (Desi), each 30 µM, Clomipramine (Clomi) 20 µM). H Ratio of toxicity and viability of HCT116 spheroids (3D) treated as indicated (NEN 1.2 µM, Imipramine (Imi), Desipramine (Desi), Amitriptyline (Ami), each 30 µM). Cells were transfected with control (siCtrl) or UPP1-targeting (siUPP1) siRNAs 24h prior to spheroid formation. Data information: data (N=3) are presented as mean (SD) and were analyzed by two -way ANOVA with Tukey post-hoc test
kb MGE and 78-kb PAI derived from lineage III and II, respectively.Figure 4Two types of 127-kb tandem MGE detected in multi-drug resistant S. suis ST7 isolates in lineage III. The 127-kb MGE encodes eight antibiotic resistance genes. The integrative and conjugative elements (ICE, in orange) and prophage (in purple), both of which are flanked by att sites, are tandemly arranged in two possible orders. The antibiotic-resistance genes are indicated in red. The chromosomal conserved genes around MGE are indicated in black. Comparison of the novel 78-kb PAI reported in this study, with the 89-kb PAI reported in epidemic strains from two outbreaks in China. The key factors for virulence of epidemic strains are indicated in green, including SalKR, NisKR, a Type IV-Like Secretion System and a Tn916 element. The antibiotic-resistance genes are shown in red. Three regions in the 89-kb PAI are absent from the 78-kb PAI. No virulence-related genes were found within these regions.
Figure 5A. WB analysis of TALDO1 overexpression or knock down in two groups of CRC cells, which was quantified by gray analysis on the right (n = 3, biological replicates, mean±SEM, t test). Data information: * for P < 0.05; ** for P < 0.01. Control referred to the corresponding plasmid vector and si-NC was the abbreviation of commercial normal control of siRNA. B. Relative NADPH/NADP+ ratio (n = 3, biological replicates, mean±SEM, t test) C. GSH contents (n = 3, biological replicates, mean±SEM, t test) Data information: * for P < 0.05; ** for P < 0.01. Control referred to the corresponding plasmid vector and si-NC was the abbreviation of commercial normal control of siRNA. D. Cellular ROS was measured by flow cytometry and shown in both curve and bar charts (n = 3, biological replicates, mean±SEM, t test) Data information: * for P < 0.05; ** for P < 0.01. Control referred to the corresponding plasmid vector and si-NC was the abbreviation of commercial normal control of siRNA. E. MMP detection. Scale bar, 10 µm. F. IF detection of H2AFX for DNA damage by L-OHP. Scale bar, 10 µm. G. CCK-8 assay of the inhibition ratio by L-OHP (n = 3, biological replicates, mean±SEM, t test) with down- or up-regulation of TALDO1 in two groups of CRC cells. Data information: * for P < 0.05; ** for P < 0.01. Control referred to the corresponding plasmid vector and si-NC was the abbreviation of commercial normal control of siRNA.
Fig.Figure 112 month old Ptpn2fl/fl;p53+/- and Lck-Cre;Ptpn2fl/f;p53+/- mice were assessed for a) disease and tumour incidence. Lymphocyte subsets from 12 month old Ptpn2fl/fl;p53+/- and Lck-Cre;Ptpn2fl/f;p53+/- mice were analyzed by flow cytometry. Significance in (a) was determined using two-sided Fisher's exact test. AT-3-OVA breast cancer cells were injected into the fourth inguinal mammary fat pads of female Ptpn2fl/fl and Lck-Cre;Ptpn2fl/fl mice and (c) tumour growth monitored over 26 days. At day 26 (d26) the numbers of activated tumour infiltrating lymphocytes (TILs) were determined. The proportion of IFNγ+ versus IFNγ+TNF+ d26 TILs was determined by flow cytometry. d26 TILS were incubated with AT-3-OVA tumour cells isolated from tumour bearing C57BL/6 mice and the proportion of IFNγ+ T cells was determined.
NAF‐1 knockdown results in increased incidence and intensity of starvation‐induced Beclin 1‐dependent autophagy.Figure 4(A) Effect of NAF‐1 knockdown on starvation‐induced autophagy. H1299 cells infected with CTRL or NAF‐1 shRNA were starved for 4 h in EBSS with DMSO (vehicle) or Baf A1 (100 nM). Cell lysates were analysed by immunoblot. (B) Representative images of GFP‐LC3 staining in H1299 GFP‐LC3 cells transfected with LUC or NAF‐1 siRNA, untreated and starved. Scale bar represents 10 μm. (C) Quantification of autophagy is expressed as the percentage of GFP‐LC3‐expressing cells displaying punctate GFP‐LC3. A minimum of 100 cells per sample were counted; results represent the average±s.d. of three independent experiments. Cell lysates were analysed by immunoblot.
Uqcrh-/- mice displayed several essential characteristics of human CIII disorders A Representative picture of a WT and age- and sex-matched Uqcrh-/- mouse at 4 weeks of age.Figure 2A Representative picture of a WT and age- and sex-matched Uqcrh-/- mouse at 4 weeks of age. Scale expressed in centimetres. B Body weight of Uqcrh -/- mice and wild-type littermates at 1-2 weeks (n = 5 to 10), during the post-weaning period from 5-6 weeks of age (n = 3 to 7) and weekly from 6 to 10 weeks (n = 13 to 66), data are shown as mean ± SD. **** p < 0.0001, 2-Way ANOVA, from 7.5wk of age for Uqcrh-/- males and females versus sex-matched controls. Data are shown as single data points and median, or categorical data, males and females pooled, since genotype-related effects were comparable for both sexes from different cohorts. * p < 0.05, *** p <0.001, Wilcoxon rank-sum test. C, D Clinical chemistry plasma concentrations of parameters related to acid-base balance (Lactate and Ammonia) and metabolic state (Glucose) n = 15 Uqcrh-/- and 16 WT pups 1-2-week-old (C); n = 15 f, 15 m Uqcrh-/- and 15 f,15 m WT mice aged 8-9 weeks (D); sex not differentiated. Data shown as scatter dot plots with line at median. * p < 0.05, ** p < 0.01, **** p < 0.0001, Wilcoxon rank sum test. E, Measures related to behaviour and neurological function, data generated by the Open Field test (8 weeks old, E) n = 7f, 8m Uqcrh-/- and 8f, 9m WT animals. Prepulse inhibition of the acoustic startle as a parameter associated with sensorimotor gating (10 weeks, E right panel), n = 7 f/5 m Uqcrh-/- and 8 f/ 9 m WT mice. Sex is not differentiated. Data are shown as scatter dot plots with a line representing the median. *** p < 0.001, **** p < 0.0001, Wilcoxon rank-sum test. F Measures related to behaviour and neurological function, data generated by the SHIRPA (9 weeks old, F), n = 7f, 8m Uqcrh-/- and 8f, 9m WT animals. Prepulse inhibition of the acoustic startle as a parameter associated with sensorimotor gating (10 weeks, E right panel), n = 7 f/5 m Uqcrh-/- and 8 f/ 9 m WT mice. Sex is not differentiated. Data are shown as scatter dot plots with a line representing the median. *** p < 0.001, **** p < 0.0001, Wilcoxon rank-sum test. G Parameters related to heart function generated by Echocardiography (ECHO) and Electrocardiography (ECG), n = 15 f, 15 m Uqcrh-/- and 15 f, 15 m WT mice, 6 weeks old.
Figure 6Blastocyst (E4.5) and diapause embryos stained for Ronin, Sox2 and Sox17. Egg cylinder stage (E5.5) embryo stained for Ronin and Sox17. Nuclei are counterstained with DAPI. Transmission electron microscopy analysis of mitochondrial morphology in the epiblast of E4.5, diapause and E5.5 embryo. Mitochondria analysed, E4.5 (n mitochondria = 55), diapause (n mitochondria = 62), E5.5 (n mitochondria = 65).
Figure 10(A-C) Subdural EEG recordings from freely behaving mice at 4-9 months of age before (A top trace, B) and after (A bottom trace, C) injection of PTZ at a dose (30 mg/kg) that did not produce convulsions. (A) Representative EEG traces from an hTau-A152T(L1) mouse. Arrows indicate epileptic discharges. (B, C) Quantitation of spikes per hour at baseline (B) and of spikes per 20-min intervals after PTZ injection (C). n = 31 (B) or 23 (C) NTG; 13 (B) or 11 (C) CaMKII-tTA; 12 (B) or 10 (C) hTau-WT(L32); and 22 (B) or 21 (C) hTau-A152T(L1) mice. *P < 0.05, **P < 0.01, ***P < 0.001 by Welch's one-way ANOVA and two-tailed Welch's t test with Holm adjustment (B) or by two-way repeated-measures ANOVA and Tukey test (C). Values are means ± SEM.
Figure 3A-B. 3D7 (A) or parasites overexpressing EK-GFP (B) were used to prepare protein lysates followed by immunoprecipitation using anti-PfCDPK7 or anti-GFP antibody, respectively. Subsequently, protein lysate or indicated IP were subjected to Western blotting using anti-PMT (A), anti-GFP (B). PMT was found to co-IP with PfCDPK7 (A, lane 2) and EK-GFP with PfCDPK7 (B, lane 3). C. 3D7 and PfCDPK7-KO parasites at schizont stage (~40 h) were used for Western blotting using anti-PMT antisera. Actin served as a loading control. Right panel: Densitometry was performed for bands corresponding to PMT in Western blots and the measurements were normalized to Actin. Fold change in PMT was determined with respect to 3D7 (mean± SEM, n=4; biological replicates, **p<0.01, t-test).
DnaK compensates for the inferior gatekeeping potential of basic residues.Figure 6A) DnaK binding to cellulose-bound peptide array. Sequences of the aggregating cores are indicated in the top of the panel, the GKs flanking them in each instance are indicated above the blots. B) Schematic representation of folding-incompetent model protein. The β-Gal APR is flanked on either side by one of the charged GKs, and separated from the GFP moiety by a rigid linker. Additionally, the construct is C-terminally His-tagged. C) Representative Western blot of in vitro translation analysis of folding-incompetent model constructs. Bands show Total (T) and Soluble (S) fractions after centrifugation at 21,000 g for 30 mins. "+ Dnak" indicates addition of either DnaK mix - containing DnaK, DnaJ and GrpE. D) Bar plots showing mean solubility as determined through quantification of western blot in (C), whiskers indicate standard deviation (n=4). Statistically significant differences between the -DnaK and +DnaK conditions were determined through two-way ANOVA with Bonferroni's correction for multiple comparisons. P-values for significant differences are indicated. E) Representative western blot of the solubility analysis of GK variants upon overexpression in E. coli K12. Bands show Soluble (Sol) and Pellet fractions after centrifugation at 17,100 g for 15 mins. "-DnaK" indicates expression in E. coli K12 DnaK knockout line, "+ Dnak" indicates co-expression of pKJE7, encoding DnaK, DnaJ and GrpE. F) Barplots showing mean solubility as determined through quantification of western blot bands in (E), whiskers indicate standard deviation (n=7, except for the AA construct, where n=4). Statistical significance was determined through two-way ANOVA followed by Tukey's multiple comparisons test. P-values are indicated. G) Structured Illumination Microsopy (SIM) images of E. coli after three hours of expression of one of the GK variants. GFP fluorescence is shown in green. "ΔDnaK" indicates expression in E. coli K12 DnaK knockout line, "+ Dnak" indicates co-expression of pKJE7, encoding DnaK, DnaJ and GrpE.
Monomerisation by dilution enhances the AMPylation activity of wild-type FICD.Figure 3A Autoradiographs of in vitro reactions containing varying concentration of wild-type FICD protein and fixed concentrations of BiPT229A-V461F and [α-32P]-ATP as co-substrates, resolved by SDS-PAGE after the indicated incubation times. The proteins were visualised by Coomassie staining of the gel (bottom). The reactions shown on the right were performed in the presence of an excess of S-SFICDA252C-H363A-C421S (trap) to delay de-modification of BiP. Representative gels are shown, and similar results were observed in three independent experiments. B As in (A) but with 0.2 µM of the indicated FICD variant. The radioactive signals were detected by autoradiography, quantified, and normalised to the signal in lane 6. The mean radioactive signals ± SD from three independent experiments are given. The proteins were visualised by staining with Coomassie. C As in (A) but with dilutions of FICDC421S or covalently linked S-SFICDA252C-C421S. Reactions were preceded by a 16 h incubation of FICD in presence or absence of the reducing agent (DTT). Representative gels are shown of three independent experiments. D Forced dimerisation does not significantly alter deAMPylation rates. Time-dependent deAMPylation of fluorescent BiPV461F-AMPFAM by the indicated FICD proteins (at 7.5 µM) assayed by fluorescence polarisation A representative experiment (data points and fit curves) is shown E Representative autoradiograph of thin layer chromatography (TLC) plates revealing AMP produced from reactions containing [α-32P]-ATP and the indicated FICD enzymes in the presence of the co-substrate BiP. AMP signals were normalised to the total nucleotide signal in each sample and the graph below plots mean values ± SD from at least three independent experiments.
a,b) Multiple MBds associate with a PC3 cell (a) and a B-lymphoblast (b).figf1(a,b) Multiple MBds associate with a PC3 cell (a) and a B-lymphoblast (b). Insets: MBd labelling (a) and merged differential interference contrast microscopy image with MBd labelling to show cell boundaries (b). MKLP1, MBd marker (a,b; red); CD44, membrane (a; green); DAPI, DNA (a; blue). Scale bar, 5 μm (a) and 2 μm (b). (c,d) Three-dimensional reconstructions of polarized cells in a monolayer (c) and a HeLa cell (d) show intracellular MBds. (c) ZO-1, tight junction; MKLP1, MBds. Scale bar, 2 μm. Enlargement (bottom panel) of the box in the top panel shows five MBds (arrows). (d) Wheat-germ agglutinin, plasma membrane (red); MKLP1-GFP, MBds (green); DAPI, DNA (blue). Scale bar, 5 μm. (e) Electron microscopy of an MBd in a permeabilized MCF-7 cell showing immungold labelling with MKLP1 antibodies. Inset, lower magnification of the MBd (boxed) in the cell; nucleus, right. Scale bar, 200 nm. (f) Time-lapse images during extracellular trypsin treatment of HeLa cells show retention of most MBds (MKLP1-GFP, red). Two MBds (yellow arrows) are lost on treatment, indicating digestion and/or dissociation. Time (h:min) post trypsin. Scale bar, 5 μm. (g) Two-day co-cultures of HeLa cells expressing either MKLP1-GFP (MBd marker) or cytosolic RFP. Green MBds (arrows) associated with red cells (asterisk) indicate post-mitotic transfer of MBds between cells. Scale bar, 10 μm.
Figure 3Figure 3. Mmi1 promotes transcription termination of nam1 non-coding gene and prevents nam1 read-through transcription from repressing the downstream MAPKKK gene byr2. (A) Northern blots showing nam1 and byr2 RNA levels during the first 4 hours of sexual differentiation. Ribosomal RNAs (rRNAs) stained with ethidium bromide were used as loading controls. Black lines indicate probes used to detect nam1 and byr2 RNAs. Figure 3. Mmi1 promotes transcription termination of nam1 non-coding gene and prevents nam1 read-through transcription from repressing the downstream MAPKKK gene byr2. (B) ChIPs showing the occupancy of the elongating RNAPII (RNAPII-S2P) over nam1-byr2 locus, in WT and nam1-1 cells. RNAPII-S2P was immunoprecipitated with an antibody recognizing the heptameric repeats (present in the C-terminal domain of the polymerase) when it is phosphorylated on its serine 2. Black lines, genomic regions investigated. Average fold enrichment is shown with error bars that indicate mean average deviations (n=3) for (B, C, E and F). Figure 3. Mmi1 promotes transcription termination of nam1 non-coding gene and prevents nam1 read-through transcription from repressing the downstream MAPKKK gene byr2. (C) RT-qPCRs showing the accumulation of nam1 read-through transcripts (RT1-qPCR) and nam1 lncRNAs (RT2-qPCR) in cells with or without the transcription terminator Ttef inserted at the 3' end of nam1 (scheme). Black arrow, primer used for the strand-specific reverse transcription (RT); Black line, location of the region amplified by PCR. Average fold enrichment is shown with error bars that indicate mean average deviations (n=3) for (B, C, E and F). Figure 3. Mmi1 promotes transcription termination of nam1 non-coding gene and prevents nam1 read-through transcription from repressing the downstream MAPKKK gene byr2. (D) Microscopy images of, respectively, WT (h90), nam1-1 and nam1-1-Ttef cells, after induction of sexual differentiation for 24 hours. The percentage of sporulation and iodine vapor assays are shown at the bottom of the images. Scale bar, 10 µm. Figure 3. Mmi1 promotes transcription termination of nam1 non-coding gene and prevents nam1 read-through transcription from repressing the downstream MAPKKK gene byr2. (E) RT-qPCRs showing the accumulation of nam1 read-through transcripts in mmi1∆ and rrp6∆ cells. Black arrow and line as in (C). . Average fold enrichment is shown with error bars that indicate mean average deviations (n=3) for (B, C, E and F). Figure 3. Mmi1 promotes transcription termination of nam1 non-coding gene and prevents nam1 read-through transcription from repressing the downstream MAPKKK gene byr2. (F) ChIPs monitoring the enrichment of H3K9me2 over nam1-byr2 locus and mei4 gene in WT and rrp6∆ cells. Average fold enrichment is shown with error bars that indicate mean average deviations (n=3) for (B, C, E and F).
USP45 knockout cells are hypersensitive to DNA damage A Clonogenic survival assays were carried out in wild‐type (WT), USP45 knockout (KO) or USP45 KO cells re‐expressing wild‐type USP45 (RESWT) or catalytically inactive USP45 [Cys199Ala] (RESCA).Figure 5A Clonogenic survival assays were carried out in wild‐type (WT), USP45 knockout (KO) or USP45 KO cells re‐expressing wild‐type USP45 (RESWT) or catalytically inactive USP45 [Cys199Ala] (RESCA). The cells were treated with the indicated doses of mitomycin C (MMC) 16 h. Medium was changed and number of colonies quantified after 10 days. Each data point is the average of 3 experiments undertaken with 6 replicates ± SD. B Control immunoblot analysis demonstrating that in the USP45 KO U2OS rescue cell lines, similar levels of wild‐type and catalytically inactive USP45 [Cys199Ala] were expressed. C, D As above, except rescue experiments are undertaken using the non‐ERCC1‐binding USP45 [Asp25Ala, Glu26Ala] mutant. E, F As in (A), except that DNA damage is induced using UV‐C irradiation (E) or 16 h hydroxyurea treatment (F). Similar results were obtained in at two separate experiments.
Figure 2(A) ssDNA repair efficiency of cells expressing RAD18 from the GAL1 promoter as determined from the maximal and final percentages of cells with Rad52 foci during the time course (right panel). Cells were synchronized in G1 from galactose-containing medium and released into S phase in the presence of 0.033% MMS for 1 hour, treated with 2.5% sodium thiosulfate to inactivate the MMS, washed and released into fresh medium for different times. Cells were shifted to glucose-containing medium 1 hour after MMS inactivation. The percentage of cells with foci at each point was normalized to the highest value of the wild type, taken as 100 (left panel). See Figure EV1A for control cells released in galactose-containing medium during the entire time course. Data information: The mean and SEM of 4 independent experiments are shown. Statistically significant differences according to an unpaired two-tailed Student's t-test are shown, where one, two and three asterisks represent p-values <0.05, <0.01 and <0.001, respectively. (B) ssDNA repair efficiency of cells expressing MMS2 from the GAL1 promoter as determined from the maximal and final percentages of cells with Rad52 foci during the time course (right panel). Cells were synchronized in G1 from galactose-containing medium and maintained in galactose (a, c) or shifted to glucose (b) for 1 hour in G1; subsequently, cells were released into S phase in the presence of 0.033% MMS for 1 hour, treated with 2.5% sodium thiosulfate to inactivate the MMS, washed and released into fresh medium for different times. Cells were maintained either in galactose (a), glucose (b) or shifted to glucose 2 hour after MMS inactivation (c). The percentage of cells with foci at each point was normalized to the highest value in galactose, taken as 100 (left panel). Wild type and mms2∆ controls were analyzed in parallel Data information: The mean and SEM of independent experiments are shown. Statistically significant differences according to an unpaired two-tailed Student's t-test are shown, where one, two and three asterisks represent p-values <0.05, <0.01 and <0.001, respectively. (C-E) ssDNA repair efficiency of the indicated TLS mutants as determined from the maximal and final percentages of cells with Rad52 foci during the time course (D and E, right panel). Cells were synchronized in G1 and released into S phase in the presence of 0.033% MMS for 1 hour, treated with 2.5% sodium thiosulfate to inactivate the MMS, washed and released into fresh medium for different times. The percentage of cells with foci at each point was normalized to the highest value of the wild type, taken as 100 (C and E, left panel). Data information: The mean and SEM of and 3-12 12 (wild type), 6 (rev1∆, rev3∆, rad30∆, rev1∆ rad30∆, rev3∆ rad30∆), and 3 (rev1∆ rev3∆ and rev1∆ rev3∆ rad30∆) independent experiments are shown. Statistically significant differences according to an unpaired two-tailed Student's t-test are shown, where one, two and three asterisks represent p-values <0.05, <0.01 and <0.001, respectively.