EMBO/sd-character-level-ner huggingface dataset:
miR-155 post-transcriptionally represses Rheb expression by targeting its 3′UTR.Figure 8(A) Sequences of mouse and human miR-155 and their predicted interactions with conserved 7-mer 1A miR-155 seeds found within the Rheb 3′UTRs of different species are shown. The sequence of the Rheb 3′UTR seed mutant used for the reporter assays and the predicted disruption of the miR-155 interaction are also shown. B) RAW264.7 cells were co-transfected with control or miR-155 mimic and a wild-type (WT-Rheb) or mutated Rheb 3′UTR (mut-Rheb) luciferase reporter plasmid and assessed for luciferase activity at 24 h after transfection. Data are shown as the mean ± SEM of three independent experiments. ***, p<0.001; NS, not significant. (C-F) RAW264.7 cells were transfected with miR-155 mimic (C and E) or inhibitor (D and F) for 24 h and either left uninfected or infected with BCG. Protein expression levels of Rheb were detected by Western-blot. Values of Rheb/β-actin ratios are indicated below the representative blot (C and D) (C-F) RAW264.7 cells were transfected with miR-155 mimic (C and E) or inhibitor (D and F) for 24 h and either left uninfected or infected with BCG. Protein expression levels of Rheb were detected by Western-blot. Values of Rheb/β-actin ratios are indicated below the representative blot (C and D), and expression levels of Rheb mRNA were detected by RT-PCR (E and F).
a, Glycaemia drop in RagA+/+, RagAGTP/+ and RagAGTP/GTP and recovery in fasted RagA+/+ and RagAGTP/+ but not in RagAGTP/GTP neonates (+/+: n = 5, 18, 4, 5, 9, 8; G/+: n = 10, 26, 10, 13, 26, 21; G/G: n = 7, 20, 9, 10, 16, 11, for 0, 1, 2, 3, 6 and 10 h, respectively; mean ± s.f2a, Glycaemia drop in RagA+/+, RagAGTP/+ and RagAGTP/GTP and recovery in fasted RagA+/+ and RagAGTP/+ but not in RagAGTP/GTP neonates (+/+: n = 5, 18, 4, 5, 9, 8; G/+: n = 10, 26, 10, 13, 26, 21; G/G: n = 7, 20, 9, 10, 16, 11, for 0, 1, 2, 3, 6 and 10 h, respectively; mean ± s.e.m.). b, Rapamycin significantly increases glycaemia in RagAGTP/GTP fasted for 6 and 10 h (mean ± s.e.m.). NS, not significant. c, Extension of survival by glucose injections in fasted RagAGTP/GTP neonates (untreated: n = 10; glucose: n = 5). d, Normal expression of genes involved in glucose metabolism in neonatal liver (+/+: n = 2, G/+: n = 5; G/G: n = 4; mean ± s.e.m.). e, Left: representative electron microscopy images showing abundant glycogen content in RagA+/+ and RagAGTP/GTP hepatocytes before fasting (0 h, upper panels) and more pronounced glycogen depletion after 10 h of fasting (lower panels) in RagAGTP/GTP neonates. Right: quantification of hepatic glycogen content (+/+: n = 5, 3, 4, 4; G/+: n = 11, 7, 14, 15; G/G: n = 6, 4, 4, 6; for 0, 3, 6 and 10 h, respectively; mean ± s.e.m.; AU, arbitrary units). . f, Partial rescue of hepatic glycogen content in RagAGTP/GTP neonates fasted for 10 h and treated with rapamycin (rapa) (mean ± s.e.m.). g, Quantification of neonatal plasma amounts of branched-chain (BCAA) and essential amino acids at birth (left), after 10 h fasting (middle) and after 10 h fasting with rapamycin treatment (right) (n for +/+, G/+ and G/G, respectively: n = 4, 5 and 4 for 0 h; n = 4, 4 and 3 for 10 h; n = 2, 5 and 3 for rapamycin; mean ± s.d.). Values are expressed relative to RagA+/+ amounts at each time point. h, Extension of survival by injection of a combination of gluconeogenic amino acids (a.a.) in fasted RagAGTP/GTP neonates (untreated: n = 10; amino acids: n = 8). *P  0.05; **P  0.01; ns, P > 0.0
cfDNA fragmentation patterns enable classification of glioma patients from controls.Figure 6A: Schematic of the features extracted from the global cfDNA fragmentation patterns of urine samples. 10 features were calculated from the cfDNA fragments size (the proportion of fragments in specific size ranges: P30_60, P61_90, P91_120, P121_150, P151_180, P181_210, P211_240, P241_270, P271_300; and the amplitude of the 10bp oscillations: OSC_10bp). B: Workflow for the predictive analysis combining the urine fragment size features via LR, RF, SVM and GLMEN models. sWGS data from 40 urine samples from patients with gliomas and 53 urine samples from controls were split into 5 subsets for training/validation (80% of the samples) and testing (20% of the samples), according to a 5 fold cross-validation approach and 50 random iterations (see Methods). C: Principal component analysis comparing cancer (HGG and LGG) and control samples (healthy and other CNS diseases) using data from the urine fragmentation features. Red arrows indicate features tested during the predictive analysis. D: tSNE analysis comparing cancer and control samples using data from the same urine fragmentation features. E: ROC curves for binary classication of cancer and controls for each of the individual fragmentation features analysed. AUC values are added to the plots. F: AUC distribution for the unseen test-set (samples from patients with gliomas, 40; controls, 53) for four predictive models (LR, GLMEN, RF, SVM) trained and optimized following the scheme described in B and the Materials and Methods section. For each models are shown the AUC for the 50 iterations. Horizontal line within the bars represent median of the underlying population. Boxplot whiskers show 1.5 interquantile range of highest and lowest quartile. G: Accuracy were compared for the 4 classifiers and 50 iterations on the unseen test-set of baseline and follow-up samples (19 samples). Horizontal line within the bars represent median of the underlying population. Boxplot whiskers show 1.5 interquantile range of highest and lowest quartile.
Fluorescence images of the pag4 (strain STK4) mutant of P.Figure 10Fluorescence images of the pag4 (strain STK4) mutant of P. pastoris after (a) 0, (b) 3, or (c) 6 h of glucose adaptation. The vacuolar membrane proliferated extensively and surrounded peroxisomes during glucose adaptation.
a) Glycohydrolase enzyme activities (GCase, GBA2, β-galactosidase, β-hexosaminidase) were measured in enriched neurons from control, GBA-PD, isogenic gene-corrected control and GD iPSC-derived neurons at DIV65.f6(a) Glycohydrolase enzyme activities (GCase, GBA2, β-galactosidase, β-hexosaminidase) were measured in enriched neurons from control, GBA-PD, isogenic gene-corrected control and GD iPSC-derived neurons at DIV65. Enzyme activities are expressed as pmoles mg−1 h−1. Data are represented as mean+s.d.; experiments were independently repeated three times in triplicate. *P0.05; **P0.01; ***P0.001 compared with controls, one-way ANOVA. (b,c) Representative western blots of GCase protein levels in control, GBA-PD, corresponding gene-corrected and GD iPSC-derived neurons. Data are represented as mean+s.d.; experiments were independently repeated three times. *P0.05; **P0.01, one-way ANOVA and Student's t-test.
miR-132 and its targets p300 and BTAF1 control RP expression.Figure 3A. mRNA levels of indicated RP transcripts determined by qRTPCR in MEFs transfected with Non-targeting control (NTC) mimics (white) or miR-132-3p mimics (grey). * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001. B. p300 and BTAF1 protein levels in MEF transfected with NTC mimics or miR-132-3p mimics determined by Western blot. GAPDH was used as a loading control. Right panel indicates mean + SEM of 4 experiments. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001. C. mRNA levels of indicated RP transcripts determined by qRTPCR in MEFs transfected with NTC siRNA (white) or p300 siRNA (grey). * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001. D. mRNA levels of indicated RP transcripts determined by qRTPCR in MEFs transfected with NTC siRNA (white) or BTAF1 siRNA (grey). Data information: Statistical significance is determined by unpaired t-test E. mRNA levels of indicated RP transcripts determined by qRTPCR in MEFs transfected with NTC or miR-132-3p mimics and NTC siRNA or p300 or BTAF1 siRNAs for 48h. Levels are normalised to cells transfected with NTC siRNA and NTC mimic. Data information: Statistical significance is determined by unpaired t-test * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001. F. Puromycin incorporation (following 10-minute pulse and 50-minute chase) determined by western blot in MEFs transfected with NTC or miR-132-3p or miR-212-3p mimics. Data information: Statistical significance is determined by unpaired t-test
Figure 2A) Representative wild-type pachytene bivalents; pachytene-like Rec8−/−, zygotene-like Stag3 mutant and Smc1β−/− univalents. Nuclear spreads were immunostained for SYCP1, SYCP3 and ACA. Filled arrowheads indicate sites of LSAEs with detectable SYCP1. Bars, 1 μm. Bellow: graphs showing the percentages of sites of LSAEs with detectable SC assembly along sister-AEs of Stag3 mutant and Smc1β−/− univalents (n=120 and n=68, respectively). B) Representative zygotene-like Stag3 mutant univalents. Nuclear spreads were immunostained for SYCP3, ACA and: SYCE1, SYCE2 and TEX12. Filled arrowheads indicate SC assembly between sites of LSAEs. Bars, 1 μm C) Quantification of signal distribution within sites of LSAEs, in Stag3 mutant spermatocytes. Signal distribution measured for SYCP3 and: SYCP1, SYCE1, SYCE2 and TEX12. Empty arrowheads indicate signal intensity peaks that correspond to each of the two sister-chromatid axes, filled arrowheads indicate SYCP1 peaks.
PHD1 silencing impairs p53-mediated DNA repair by reducing p53 binding to the NER helicase XPB.Figure 7Western blot for pH2AX and vinculin (vinc) in p53wt/wt and p53−/− HCT116 cells after 24-h treatment with 300 μM 5-FU. Detection of pH2AX and vinc in p53S15A→p53−/− HCT116 cells silenced for PHD1 and treated with 300 μM 5-FU for 24 h. Detection of pH2AX and vinc in HCT116 silenced with a second silencing construct of PHD1 and treated for 24 h with 300 μM 5-FU. Western blot for pH2AX and vinc in HCT116 treated for 20 h with 200 nM SN-38 or 24 h with 20 μM oxaliplatin. Western blot for pH2AX and vinc in p53R248/− HCT116 cells silenced for PHD1 upon treatment with 300 μM 5-FU for 24 h. Detection by Western blot of p53 and XPB from whole cell extracts (WCE) or after immunoprecipitation of p53 from cells silenced for Scr control or siPHD1 and treated for 4 or 8 h with 300 μM 5-FU. Western blot for pH2AX and vinc in HCT116 silenced for PHD1 alone or in combination with silencing for XPB upon treatment with 300 μM 5-FU for 20 h. PHD1 hydroxylase activity allows the phosphorylation of p53 upon chemotherapy treatment in colorectal cancer cell lines. As a consequence, p53 can interact with the NER machinery and more specifically with XPB. This promotes DNA repair and results in the resistance toward the cytostatic effects of chemotherapy (left panel). On the other hand, when PHD1 is lacking, the p53-kinase p38α can no longer bind, and therefore, p53 phosphorylation upon chemotherapy treatment is prevented. In absence of Ser15 phosphorylation, p53 is no longer able to bind XPB, thereby blocking the DNA repair-promoting capacity of p53. This leads to increased DNA damage and thus enhanced apoptotic response to chemotherapy (right panel).
Figure 6In young flies the metabolic state and the variation in gene expression are well balanced. When flies reach midlife, metabolic increases in oxidative phosphorylation, citrate synthase and ATPCL activity result in higher acetyl-CoA levels. The higher acetyl-CoA result in increased histone acetylation followed by misregulation of the transcriptome. Attenuating the metabolism-histone acetylation axis by a reduction of ATPCL activity, or of the KAT Chameau, moderates age-associated transcriptomic changes, leading to a lifespan extension.
CLEC12A Associates with Bacteria Early after Entry into Host Cells and Restricts Intracellular Bacterial Replication.Figure 4(A) Gentamicin protection assay in HeLa cells showing fold replication of Salmonella in cells treated with the indicated siRNAs. Data are shown as mean ± SD; n = 6. Data are representative of four independent experiments. (B) Quantitation of CLEC12A-bacteria and LC3-bacteria colocalization at indicated time points. Data are shown as mean ± SD; n = 3 independent experiments. (C) Representative confocal micrographs of HeLa cells showing association of intracellular bacteria with GFP-CLEC12A at 20, 40, 60, and 80 min post-infection. The scale bars represent 10 μm.
Figure 5A. mRNA expression analysis of IgM and IgA was carried out by RT-PCR for Iglo subclones from WT and two independent 53bp1−/− CH12 (TA and T1) clones. The Iglo subclones derived from the 53bp1−/− CH12 TA clone are listed as D01-D18, while the Iglo subclones derived from the 53bp1−/− CH12 T1 clone are listed as F03-F24. Gels show the RT-PCR analysis for IgM cDNA (top), IgA cDNA (middle), and AID cDNA (bottom) as control. B. mRNA expression analysis as in A of Iglo subclones of two independent Shld2-/-/- (F2 and F3) clones, and IgA+ subclones from Shld2-/-/- clone F3. The Iglo subclones derived from the Shld2-/-/- CH12 F2 clone are listed as H03-H40, while the Iglo subclones derived from the Shld2-/-/-CH12 F3 clone are listed as J02-J109. C. Representative IgA cDNA sequence analysis from WT Iglo clones.
Figure 4(A) Transcription profiles of ΔIndLon mutants grown in inducing (+) or depleting (-) conditions across the cell division and the MPN525 (DnaB) operons. The respective estimated protein copy numbers based on MS data are shown below. (B) Time course experiments showing depletion levels of Lon over time and the corresponding accumulation of N-terminal FLAG-tagged derivatives of candidate substrates associated to cell division, including FtsA, FtsZ and DnaB. Protein levels of Lon and candidate substrates were assessed by immunoblot analysis using anti-Lon and anti-FLAG antibodies. LC, loading control. (C) In vivo degradation assays of FtsA, FtsZ and DnaB. ΔIndLon mutants expressing the different N-terminal FLAG-tagged derivatives were grown in depleting conditions for 36h. Then, Lon expression was transiently induced for 3h before translation was blocked with gentamicin (Gm). Samples were taken at the indicated time points after gentamicin treatment, and processed for immunoblot analysis using anti-FLAG antibodies. LC, loading control. As controls, non-induced cells were also treated with gentamicin and processed at the same time points. Immunoblots are representative of two independent experiments. (D) Estimated protein copy numbers based on MS data of components associated with the R-M system. (E and F) Similar to panel B (E) and C (F) but for a C-terminal FLAG-tagged derivative of MPN201 as a representative of an unstable HsdS subunit. (G) Estimated protein copy numbers based on MS data of FtsH candidate substrates. (H) Similar to panel B but for C-terminal FLAG-tagged derivatives of FtsH candidate substrates. Protein levels of FtsH and candidate substrates were assessed by immunoblot analysis using anti-FtsH and anti-FLAG antibodies. LC, loading control. (I) Similar to panel C, but for FtsH candidate substrates. In this case, C-terminal FLAG-tagged derivatives were grown in depleting conditions for 60h before transient expression of FtsH for 3h and gentamicin treatment.
KO of both UPF3 paralogs results in strongly impaired NMD A Western blot analysis of WT, UPF3B KO and UPF3A-UPF3B double KO cells (clones 1 and 2) (n=1).Figure 4A Western blot analysis of WT, UPF3B KO and UPF3A-UPF3B double KO cells (clones 1 and 2) (n=1). UPF3A and UPF3B protein levels were detected, Tubulin serves as control. The asterisk indicates unspecific bands. B Schematic depiction of the insertion/deletion in the UPF3A open reading frame resulting in the additional UPF3A KO in the UPF3B KO clone 90 generating UPF3 dKO clones. C Quantitative RT-PCR of the indicated samples with the indicated KDs. For RSRC2 and SRSF2 the ratio of NMD isoform to canonical isoform was calculated. ZFAS1 expression was normalized to C1orf43 reference. Data points and means are plotted as log2 fold change (log2FC, n=3). D Schematic overview of the globin reporter constructs and their functional elements. E Northern blot analysis of globin reporter and xrFrag. Ethidium bromide stained 28S and 18S rRNAs are shown as controls. Quantification results are shown as data points and mean (n=3).
Figure 5A and B. Time course analyses of mutant SOD1 levels at the MAM in SOD1G93Amousespinal cords (A) and brains (B). Immunoblots show levels of mutant SOD1 protein in the indicated fractions at various time points (left panels). Quantitative data at the right were plotted as mean ± SEM of three independent experiments. Arrows indicate a rapid reduction in SOD1 level at the MAM just following the onset of disease. C and D. Time course analyses of mutant SOD1 accumulation at the MAM in SOD1G85R (C) and SOD1G37R (D) mousespinal cords. Quantitative data from three independent experiments were plotted (bottom). The arrows in the bottom panel are the same as in A and B. E. Time course analyses of MAM-specific proteins at MAM and in the whole lysates of SOD1G93Amousespinal cords.
Figure 2(A) Structure of the reported PP2Aγ-B56-hSgo1 binding interface (top) and residues mutated in the B56α 5A mutant are shown (bottom). (B) IP of YFP-B56α from cells stably expressing the B56α WT, R222E, and 5A followed by immunoblotting of indicated proteins. Representative blots are shown (top). hSgo1 signals were normalized to YFP and plotted (bottom). Error bars represent SD (n=4). (C) Reciprocal IP of (B). YFP-hSgo1 expression construct was transfected into cells stably expressing FLAG-B56α WT, R222E and 5A, followed by YFP IP and immunoblotting of indicated proteins. Representative blots are shown (top). B56α signals were normalized to YFP and plotted (bottom). Error bars represent SD (n=4). (D) Representative images of chromosome spreads from the indicated conditions. Scale bar, 5μm. (E) Quantification of (D). The distance between the two peak intensities of CREST was measured for 5 kinetochore pairs and averaged for a single cell and plotted. The data are from 4 independent experiments and the mean and SD are indicated. (F) hSgo1 RNAi and rescue with the indicated B56α variants fused to YFP and the Cenp B centromere-targeting domain (CB). Representative images of chromosome spreads are shown. CB targets all the rescue constructs (green) to the centromere. Scale bar, 5μm. (G) Quantification of (F). The distance between the two peak intensities of YFP was measured for 5 kinetochore pairs and averaged for a single cell and plotted. The data are from 3 independent experiments and the mean and SD are indicated.
Figure 1(A-B) WT or MARCO-/- C57BL/6 mice were inoculated subcutaneously (s.c.) in the left rear footpad with 103 PFU of CHIKV or CHIKV E2 K200R. The percent of starting body weight (A) and disease score (B) were recorded daily over 14 days. Mean ± SEM. N=7-12. Data are pooled from three experiments. Two-way ANOVA with Bonferroni's multiple comparison test; ****P < 0.0001. WT or MARCO-/- C57BL/6 mice were inoculated s.c. in the left rear footpad with 103 PFU of CHIKV. Serum collected on days 1, 2, 3, 5, 7, and 14 was analyzed by focus formation assay (FFA), mean ± SD (C) and by RT-qPCR, mean ± SD (D). WT or MARCO-/- C57BL/6 mice were inoculated s.c. in the left rear footpad with 103 PFU of CHIKV. (E) Viral tissue burdens were analyzed at 1 day post inoculation (dpi) by FFA. Mean ± SEM. WT or MARCO-/- C57BL/6 mice were inoculated s.c. in the left rear footpad with 103 PFU of ONNV (F) Tissues and serum were collected at 1 dpi and analyzed by plaque assay. WT or MARCO-/- C57BL/6 mice were inoculated s.c. in the left rear footpad with 103 PFU of RRV (G). Tissues and serum were collected at 1 dpi and analyzed by plaque assay.
Figure 5(A) HEK293T cells (5×105) were transfected with HA-cGAS (200 ng), Flag-STING (200 ng) and Myc-ZDHHC18 (WT or CS mutant) expression plasmids (0, 50, 100, or 200 ng) for 24 h before luciferase reporter assays were conducted. Data information: ZDHHC18(CS): a catalytic mutant with a cysteine-to-serine substitution in the DHHC motif of ZDHHC18 All data are representative of at least two independent experiments. Mean ± SEM from triplicates of technical replicates, unpaired t test; ns, not significant; **, P˂0.01; ***, P˂0.001 (A (B) HEK293T cells (1×106) were transfected with HA-cGAS (500 ng), Flag-STING (500 ng) and Myc-ZDHHC18 (500 ng) expression plasmids for 24 h. TBK1 phosphorylation was detected by immunoblotting. Data information: ZDHHC18(CS): a catalytic mutant with a cysteine-to-serine substitution in the DHHC motif of ZDHHC18 (C and D) HEK293T cells were transfected with the indicated plasmids, and the amount of cGAMP in the lysates was quantified by LC-MS/MS. Data information: ZDHHC18(CS): a catalytic mutant with a cysteine-to-serine substitution in the DHHC motif of ZDHHC18; LC-MS/MS: liquid chromatography-tandem mass spectrometry; L929 cells stably transfected with control shRNA or ZDHHC18 shRNA The knockdown efficiency of ZDHHC18 is shown by RT-qPCR (E) results. Data information: NC: negative control. All data are representative of at least two independent experiments. Mean ± SEM from triplicates of technical replicates, unpaired t test; ns, not significant; **, P˂0.01; ***, P˂0.001 L929 cells stably transfected with control shRNA or ZDHHC18 shRNA efficiency of ZDHHC18 is shown by western blotting (F) results. Data information: NC: negative control. L929 cells stably transfected with control shRNA or ZDHHC18 shRNA were transfected with HT-DNA (2 μg/mL) for the indicated times before RT-qPCR analysis of Ifna4 (G), expression. Data information: HT-DNA: herring testis DNA; NC: negative control. All data are representative of at least two independent experiments. Mean ± SEM from triplicates of technical replicates, unpaired t test; ns, not significant; **, P˂0.01; ***, P˂0.001 L929 cells stably transfected with control shRNA or ZDHHC18 shRNA were transfected with HT-DNA (2 μg/mL) for the indicated times before RT-qPCR analysis of IFNb1 (H), and Cxcl10 (I) expression. Data information: HT-DNA: herring testis DNA; NC: negative control. All data are representative of at least two independent experiments. Mean ± SEM from triplicates of technical replicates, unpaired t test; ns, not significant; **, P˂0.01; ***, P˂0.001 (J) L929 cells stably transfected with control shRNA or ZDHHC18 shRNA were transfected with HT-DNA (2 μg/mL) for the indicated times before immunoblotting analysis with the indicated antibodies. Data information: HT-DNA: herring testis DNA; NC: negative control.
Figure 4A) TDP-43 droplets observed by brightfield and fluorescence microscopy when Oregon green labeled TDP-43 (4 μM, 10% labeled protein) was mixed with no RNA control or GU-repeat RNA oligonucleotides of increasing length at 250 mM salt. As indicated, RNA concentration varied to maintain a constant total number of TDP-43 binding sites. Scale bars, 10 μm. B) The plot shows droplet area quantified as a function of GU-repeat length. Mean and SD of >300 droplets from 3 biological replicates using 3 different protein preparations. C) TDP-43 concentration in the light phase (Cout) as a function of GU (blue) or CA (red)-repeat length quantified by Bradford protein assay. Mean and SD from >5 biological replicates using 2 different protein preparations. (250 mM NaCl, 4 µM TDP-43)