EMBO/sd-character-level-ner huggingface dataset:
SseL deubiquitinates Salmonella- and puromycin-induced ALIS.Figure 6(A) Quantification of large (>2 µm), ubiquitinated aggresome-like induced structures (ALIS) in macrophages. Primary bone marrow-derived macrophages (BMM) were infected with the indicated GFP-expressing S. Typhimurium strains for 10 h, immunolabelled for ubiquitin and analysed by confocal microscopy. A minimum of 50 cells were counted for each bacterial infection per experiment and values are the mean ± SEM of 3 independent experiments. Uninfected cells (uninf) were from the same wells as infected and therefore were exposed to extracellular bacteria. * p<0.05; ** p<0.01. (B) Quantification of the number of puromycin-induced ubiquitin inclusions in cells. HeLa cells were transfected with a vector expressing myc-SseL or myc-SseLC/A for 16 h followed by treatment with puromycin (5 µg/ml) for 4 h and immunolabelled with anti-myc, anti-p62 and anti-ubiquitin. 50 individual cells were counted per condition in each experiment. Values are the means ± SEM of at least 3 independent experiments. ** p<0.01. (C) Single confocal sections of HeLa cells transfected with a vector expressing myc-SseL for 16 h followed by treatment with puromycin (5 µg/ml) for 4 h and immunolabelled with anti-myc (blue), anti-p62 (green) and anti-ubiquitin (Ub, red) (scale bars, 5 µm). The lower panels show a merged image of p62, ubiquitin and myc-SseL. Arrows indicate ubiquitin and p62 aggregates present in untransfected cells and arrowheads indicate cells expressing myc-SseL
a, Polyclonal T-cell development in Atg5–/– to wild-type (WT), or wild-type to wild-type grafts.figf3a, Polyclonal T-cell development in Atg5-/- to wild-type (WT), or wild-type to wild-type grafts. The top dot plots show CD4 and CD8 staining; the bottom dot plots show expression of CD25 and Foxp3 by CD4 SP thymocytes; n ≥ 5. b, Atg5-/- or wild-type lobes were grafted into TCR-HA transgenic recipients. Graft cellularity ± s.d. and the expression of the transgenic TCR on CD4 SP cells were analysed after 6 weeks. The frequency ± s.d. of TCR-HA+ cells among CD4 SP cells was 32.1% ± 3.2% in wild-type to TCR-HA, and 19.6% ± 4.3% in Atg5-/- to TCR-HAlobes; P = 1.8 × 10-8 (n ≥ 14). c, Atg5-/- or wild-type lobes were grafted into AND transgenic mice. Graft cellularity ± s.d. and the expression of the transgenic TCR (Vb3-Va11) on CD4 SP cells were analysed after 6 weeks. The frequency ± s.d. of Vb3-Va11+ CD4 SP cells was 93.5% ± 0.2% in wild-type to ANDlobes (n = 3) and 97.9% ± 1.1% (n = 6) in Atg5-/- to ANDlobes (P = 9 × 10-4). d, Thymi from wild-type or Atg5-/- embryos were grafted into P14, OT-I or HY TCR-transgenic recipients. The dot plots show the expression of the transgenic TCR V-segments on CD8 SP cells. Thymus cellularities ± s.d. are shown in the respective bar diagrams. Data are representative of n = 4 (P14), n ≥ 4 (OT-I) and n ≥ 2 (HY). e, Thymi from wild-type or (BALB/c3C57BL/6) F1 Atg5-/- embryos were transplanted into (BALB/c3C57BL/6) F1 recipients. Six weeks after grafting, expression of the I-Ea52-68-I-Ab complex on cTECs was analysed using the monoclonal antibody Y-Ae. Atg5-/- cTECs are shown in red; wild-type cTECs are shown in blue. Control stainings are shown in orange (Atg5-/-) and green (wild type). Data are representative of two independent experiments.
Figure 4A. Co-IP experiments in HEK293T cells transfected with vector and flag-HDAC3 expression plasmids. Anti-flag antibody was used to put down HDAC3 protein. WB data showed the level of menin and HDAC3 of input samples and output samples. B, C. Different concentrations of vorinostat were used to treat PIME cells (B) and menin null PIME cells (C) for 48 hrs. Pbk expression was examined using WB. D. The control INS-1cells and JunD KD INS-1 cells were subjected to ChIP assay to detect for the binding of menin, HDAC3, JunD, and histone3 acetylation level at the Pbk locus. 1% input denotes the signal level normalized by the equivalent total input DNA (i.e. 1% of sonicated genome DNA) for ChIP assay. Three independent experimets (n=3). *P = 0.0430 (JunD), *P = 0.0480 (Menin), ***P = 0.0008 (HDAC3), *P = 0.0480 (Ac-H3) (two-tailed unpaired student's t-test). ns, not statistically significant difference. E, F. The control INS-1cells and menin KD INS-1 cells induced by CRISPR/Cas9 system were subjected to ChIP assay to detect for the binding of menin, HDAC3, JunD, and histone3 acetylation level at the Pbk locus. Three independent experimets (n=3). *P = 0.0196 (E, Menin), *P = 0.0164 (E, HDAC3), *P = 0.0323 (F, Ac-H3) (two-tailed unpaired student's t-test). ns, not statistically significant difference. G. Control and JunD KD PIME cells were subjected to ChIP assay to detect the binding of JunD, menin, HDAC3, and histone acetylation at the Pbk locus. Three independent experimets (n=3). ***P = 0.0007 (JunD), *P = 0.0486 (Menin), *P = 0.0117 (HDAC3), **P = 0.0012 (Ac-H3) (two-tailed unpaired student's t-test). ns, not statistically significant difference. H, I. PIME cells and menin-null PIME cells were subjected to ChIP assay to detect the binding of menin, HDAC3, JunD (H) as well as histone acetylation level (I) at the Pbk locus. Three independent experimets (n=3). *P = 0.0226 (H, Menin), **P = 0.0084 (H, HDAC3), **P = 0.0041 (I, Ac-H3) (two-tailed unpaired student's t-test). ns, not statistically significant difference. J, K. The effect of T5224 on the binding of JunD, menin, HDAC3, and acetylated histone3 at the Pbk locus in PIME cells (J) or INS-1 cells (K). Three independent experimets (n=3). *P = 0.0490 (J, JunD), *P = 0.0479 (J, Menin), *P = 0.0154 (J, HDAC3), **P = 0.0029 (J, Ac-H3), *P = 0.0104 (K, JunD), **P = 0.0024 (K, Menin), ***P = 0.0002 (K, HDAC3), ***P = 0.0001 (K, Ac-H3), (two-tailed unpaired student's t-test). ns, not statistically significant difference. L. PIME cells were treated with various doses of T5224 for 48 hours, followed by detecting Pbk expression using WB.
Figure 3ChIP-seq tracks of T (in red; input is in blue) around the Oct4 (7.5 kb). DE, distal enhancer; PE, proximal enhancer. The T-binding peak around the Oct4-DE is indicated (arrowhead). Right: ChIP-qPCR for the enrichment of T on the DE and the regions indicated in Fig EV3E. NC, negative control (the locus without T binding). The enrichment is indicated by the percent input with SDs (three independent experiments with two technical replicates). Luciferase assay with T-binding elements (TBEs) around the indicated loci as shown in (A) and Fig EV3E. Relative luciferase activities of sorted cell fractions are shown with SDs. (three independent experiments with two technical replicates). : PCA of global gene expression in low T-inducible ZHBTc4 ESCs stimulated as indicated in Fig EV3G. Right: Heatmap of selected key pluripotency genes derived from mean-centered expression values (log2 scale). Venn diagrams showing the overlap between down- and upregulated DEGs induced by both high T induction and Oct4 deletion in low T-activated condition (fold-change > 1; p < 0.1 by DESeq2). Bottom: Ontology terms enriched in overlapped DEGs. See also Dataset EV3.
Figure 6A Framework for investigation of effects of translation inhibition on UPF1LL activity. B RNA-seq analysis of HEK-293 cells identifies populations of genes that decreased in abundance with puromycin treatment (50 µg/mL for 4hr) and were rescued by UPF1LL-specific knockdown. Indicated are genes that increased in abundance at least 1.4-fold (FDR < 0.05) with UPF1LL-specific knockdown under normal conditions. C RT-qPCR analysis of indicated transcripts following transfection of HEK-293 cells with indicated siRNAs and treatment with 50 µg/mL puromycin for 4hr. Relative fold changes are in reference to vehicle-treated, NT siRNA. Black dots represent individual data points and error bars indicate mean±SD (n = 3 biological replicates). Dashed lines indicate log2(fold change) of ±0.5. PTC+ indicates the use of primers specific to transcript isoforms with validated poison exons
Figure 6(A) Hydrogen peroxide 'acute stress' assay. Δprx1 cells co-transformed with an empty plasmid or a plasmid encoding Prx1, Tsa1, PRDX3, PRDX5, PRDX6, PfAOP or PfAOP-L109M were grown in SD medium lacking the appropriate amino acids for plasmid selection and pre-treated with 0, 1, 5, 10, 25 mM H2O2 for 30 mins. Afterwards, the cells were diluted and a fixed volume was plated on YPD plates. The number of viable colonies was counted after 2 days of growth at 30°C, here represented as a percentage relative to the 0 mM pre-treatment. Error bars represent standard deviation (n = 3-8 biological replicates, with cells taken from independent cultures for each individual biological replicate). Significance was assessed with a Student's 2-tailed, unpaired, t-test. *p < 0.05; **p < 0.01; ***p < 0.001. (B) The efficiency of transfer of oxidation from H2O2 to EGSH strongly correlates with cell death upon exposure to acute H2O2 stress. Data from viability and matrix Grx1-roGFP2 responses during acute H2O2 stress were correlated.
Figure 6U87 cells were treated with LXR623 for 1h, 3h or 5h and subjected to extracellular flux analysis on the Seahorse XFp instrument in the context of a mitochondrial stress assay (A-C). From this assay, coupled respiration was calculated (D-F). U87 cells were treated with LXR623, cholesterol or the combination of both for 24h and subjected to extracellular flux analysis on the Seahorse XFp instrument in the context of a mitochondrial stress assay (G). From this assay, mitochondrial OCR and coupled respiration was calculated (H-I). U87 cells were treated with DMSO or LXR623 20 µM with the indicated time point. Thereafter, whole cell protein lysates were collected and subjected to standard western blot for the expression of the respiratory complexes (OXPHOS), Noxa, and ATF4. A star indicates an unspecific band. U87 cells were treated with DMSO or LXR623 20 µM in the presence or absence of cycloheximide for 7h. Thereafter, whole cell protein lysates were collected and the protein levels of the components of complex V were analyzed by standard western blot. U87 cells were treated with increasing concentration of LXR623 in the presence or absence of cholesterol. Thereafter, whole cell protein lysates were collected and analyzed the protein levels of the respiratory complexes (OXPHOS) by standard western blot. U87 cells transduced with non-targeting or LXRβ shRNA were treated with vehicle or 20 µM LXR623 for 24h. Thereafter, extracellular flux analysis was performed in the context of a mitochondrial stress assay. From this assay, basal respiration and coupled respiration was calculated (M-N). U87 cells transduced with non-targeting or LXRβ shRNA were treated with vehicle or 20 µM LXR623 for 24h. RNA and protein were collected and analyzed for the expression of LXRβ mRNA (O) or LXRβ protein by capillary electrophoresis (P). Shown are means and SD (n = 5). Statistical significance was determined by two-sided student's t.test.
a) Representative electron micrographs of DOX-treated hearts.f5(a) Representative electron micrographs of DOX-treated hearts. Arrows indicate abnormal mitochondria defined by marked swelling, vacuolation (loss of electron density) or disrupted cristae; original magnification × 5,000; scale bar, 2 μm. The magnified photograph represents abnormal mitochondria (blue line) and an autophagic vacuole containing mitochondria (red line); original magnification × 12,000; scale bar, 500 nm. (b,c) Mitochondria incorporated into an autophagic vacuole (b) and abnormal mitochondria (c) were quantified blindly from 8-10 images from different fields (original magnification × 5,000) (n=3 per group). (d) The 8-hydroxy-2′-deoxyguanosine (8-OHdG), popular marker for oxidative DNA damage, in mitochondria DNA from DOX-treated hearts. Results are shown from 6-8 hearts analysed in duplicate. (e) Maximum dP/dt was examined using cardiac catheterization under graded dobutamine infusion in PBS-treated (control) and DOX-treated mice (n=6-8 per group). DKO indicates double knockout. Data are shown as the means±s.d. *P0.05; **P0.01 (two-tailed unpaired Student's t-test).
Figure 8(A) Schema showing the identified tRNA substrates and recognition mechanism of FTSJ1. (B) The translation efficiency of UUU and UUC codons regulated by the interdependence of tRNAPhe(GAA) modifications at positions 32, 34, and 37.
Figure 6(A) A375 cells were infected with retroviruses expressing a control or ZEB1-shRNA. Western blot analyses of ZEB1 and p75 upon ZEB1 knock-down. GAPDH was used as a loading control. High and low exposures (exp) for p75 are shown. (B) Quantitative PCR analyses of MITF, p75, ABCB5 and JARID1B upon ZEB1 knock-down. mRNA expression levels are represented relatively to shRNA control cells (mean ± SD, n=3, Student's t-test). (C) FACS analyses of p75 expression upon ZEB1 knock-down. Bar chart representing the mean percentage of p75-high, int and low cells from 2 independent experiments (Fisher's exact test). (D) Soft agar colony formation assay upon ZEB1 knock-down. Scale bar = 200 µm. Histograms represent quantitative analyses (mean ± SD, n=3, Student's t-test). (E) 2 x 106 Control or ZEB1-shRNA A375 cells were injected subcutaneously into nude mice. The mean tumor volume for 5 mice is represented (± SEM). (Student's t-test). (F) A375 cells were infected with an IPTG-inducible ZEB1-shRNA. Left panel: Western blot analyses of ZEB1 expression ± IPTG (100µM) treatment for 6 days. Actin was used as a loading control. Right panel: 2 x 106 IPTG-inducible shRNA-Control or shRNA-ZEB1A375 cells were injected subcutaneously into nude mice. When the tumor reached 5 mm in diameter, ZEB1 expression was silenced by adding IPTG (10 mM) into the drinking water for 20 days. The mean tumor volume for 5 mice is represented (± SEM). (Student's t-test). (G) SKMEL5 cells expressing an IPTG-inducible ZEB1-shRNA were treated with IPTG (100µM) for 10 days (+IPTG), then IPTG was removed (-IPTG) and ZEB1, MITF and p75 expression levels were analyzed by Western blot and/or quantitative PCR analyses. GAPDH was used as a protein loading control, mRNA expression levels are represented relatively to untreated cells, in which the levels were fixed at 1 (mean ± SD, n=2).
Fig.Figure 1: Displayed (sc)AAV2-based vector backbones) were packaged in AAV serotype 1 capsids. AAV-ITRs are displayed in grey, ∆ITR refers to the mutated ITR-version of scAAVs. The transgene of AAV-pDyn is a codon-optimized version of the full-length human preprodynorphin cDNA enhanced by a WPRE element. Control vectors carry either a truncated, nonfunctional version of the enhanced GFP gene (AAV-ΔGFP), or its functional counterpart (AAV-eGFP; not displayed). Kainic acid mouse model of TLE: Daily EEG recordings obtained from the epileptogenic focus starting from one month after KA injection and spanning the period from 2 days before to 7 days after AAV-pDyn delivery (2x109 gp). higher time resolution of the indicated section of the Day +1 in (B), representing a hippocampal paroxysmal discharge (HPD). A generalized seizure is depicted in Fig. EV1 D and E: The characteristic EEG features of this model, secondary generalized seizures (bars) and HPDs (lines) were reduced in number and in duration by AAV-pDyn (red; n = 3 (from day 60); 7 (till day 30) per time-interval), but not by AAV-ΔGFP or after sham treatment (blue; n = 4 (day 90); 5 (day 30 and 60); 6 (before day 30), per time-interval). The relatively high variability of seizure frequencies and duration is typical for this model and reflects the findings in human mTLE. Injection of norBNI (20 mg/kg; i.p.) results in a transient reappearance of HPDs immediately and 24 hrs after application. One week after norBNI application (washout) suppression of HPDs was re-established. Data obtained from 4 epileptic animals before (black) and after (red) AAV-pDyn delivery (2x109 gp) are depicted. * EEG recordings obtained from the ipsilateral dorsal hippocampus of rats after electrical self-sustained status epilepticus (SSSE) before (black) and after (red) AAV-pDyn delivery (4x109 gp) are depicted. Spike trains with a frequency of at least 2.5 Hz induced by SSSE were markedly reduced by AAV-pDyn (n=4).
Figure 5IFNB1 mRNA induction analyzed by qPCR at the indicated times after BO-110 treatment (0.5 µg/ml) of SK-Mel-147 melanoma cells or HLEC (left and right graphs, respectively). Data correspond to the mean ± SD of three experiments with three technical replicates normalized to vehicle control. Quantification of the impact of BO-110 as single agent or in the presence of the indicated blocking antibodies for type I interferon (IFNB1 or IFNAR1). Upper graphs show the effect of these agents on MDK mRNA levels in SK-Mel-147 melanoma cells. Similar treatments were performed on HLEC for the analysis of VEGFR3 mRNA (middle graphs) and tube formation capacity (lower graphs). Data correspond to the mean ± SD of 3 biological replicates in triplicate. BO-110-driven blockade of the tube-forming capacity of HLEC and rescue with anti-IFNAR1 blocking antibodies. Images correspond to cells plated in matrigel and imaged 8 h after treatment with 0.5 µg/ml BO-110. See also Fig. EV4B, for additional results with anti-IFNAR1 and anti-IFNβ blocking antibodies. Growth of B16 melanoma xenografts in siblings of Ifnar1+/+, Ifnar1+/- or Ifnar1-/- mice. Treatment started 10 days after tumor cell implantation. BO-110 was administered at 0.8 mg/kg, every third day for 2 weeks. N= 6 mice per condition. Graphs show the mean tumor size ± SD at each time point. Statistical significance was determined by Two-Way ANOVA. Histological analyses of lymphatic vessel density by Lyve1 (blue) and Prox1 (purple) in representative lymph nodes of animals in (D) processed at the endpoint of the experiment (4 doses of BO-110 or vehicle control). N= 6 mice per experimental condition.
PA200-proteasome degrades acetylated histones.Figure 7(A) Western blots of HeLa cells pre-treated with the E1 enzyme inhibitor PYR-41 (10 μM) one hour before UV irradiation and lysed at different time points after UV (16 J/m2). Blots were stained with the indicated antibodies and representative blots are shown (B) Quantification of histone acetylation levels, normalized against histone H4 levels, average of 5 experiments. Error bars represent SEM (C) Representative western blots of siControl or siPA200 (2 independent siRNAs, a and b) transfected cells, lysed at indicated time points after UV-irradiation (16 J/m2) (D) Quantification of histone H normalized against tubulin and non-treated levels are set as 1. The average of at least 9 independent experiments and SEM is shown. Significant differences were calculated by T-test and are indicated with * (p<0.1), ** (p<0.05) and *** (p<0.01) Quantification o histone acetylation levels, normalized against tubulin and non-treated levels are set as 1. The average of at least 9 independent experiments and SEM is shown. Significant differences were calculated by T-test and are indicated with * (p<0.1), ** (p<0.05) and *** (p<0.01) (F) Clonogenic survival of HeLa cells treated with different doses of UV-C. Relative survival (in percentage) is plotted against UV-C dose. N≥2, error bars represent SEM. Significant differences, calculated by T-test are indicated with * (p<0.1), ** (p<0.05) and *** (p<0.01) (G) Unscheduled DNA synthesis in non-cycling C5RO cells measured by the incorporation of EdU (20 µM) after UV irradiation (16J/m2) visualized by ATTO594 labelling using Click-chemistry. Levels in siControl cells were set as 1. N=2, >200 cells analyzed, error bars represent SEM
Figure 2A, Quantitative data-independent scanning (DiS) mass spectrometry protein profiles for CI, CIII and CIV. Vertical numbers indicate the BNGE gel slices. Left and right profiles correspond to scaf1+/+ and scaf1Δ1/Δ1 animals, respectively. Red heatmap corresponds to the E-score from two proteotypic Scaf1-derived tryptic peptides spanning sequences ascribed to the CIII interacting site (in green) and to the CIV interacting site (in yellow). Thick blue line, marked with an asterisk, indicates the putative proteolytic site in Scaf1. B, Sequence alignment of Scaf1 protein in mouse and zebrafish. Structural and functional regions previously described in mouse are indicated in shaded grey boxes. Thick blue line indicates the proteolytic processing site in the mouse sequence.
Preclinical orthoxenograft MPNST models to test drug treatment regimens.Figure 7Tumor growth effects of treatment with doxorubicin, sorafenib, rapamycin and combinations thereof in the five MPNST xenograft models. Results are plotted as an average of the log2 ratio of tumor volume at different days relative to the initial value. Statistically significant differences are shown as *P < 0.05 and **P < 0.001 versus control group by the Bonferroni test. For long-term studies, a subgroup of treated mice (n = 3-5 mice/group) was kept alive for a maximum period of 4 months and sacrificed over time when relapsed tumor masses grew as large solid masses (usually 1,500-2,000 mm3). The graph illustrates differences in the time delay (in days) of relapsed tumor masses for the different treatments.
Figure 10(A) The precursor form of wild-type COA7 is synthesized in the cytosol and imported into the mitochondrial intermembrane space by the MIA pathway, where it is involved in the respiratory chain biogenesis. (B) The precursor form of mutant COA7 is import-defective and therefore it is degraded in the cytosol by proteasome. (C) Proteasome inhibition rescues the mitochondrial import of mutant COA7 as well as the biogenesis and function of the respiratory chain.
Figure 2A Representative still images of Alexa488-labeled TDP-43 condensates by spinning disc timelapse confocal microscopy. Wt condensates do not fuse, 5D condensates fuse slowly and 12D condensates readily fuse upon contact and relax into spherical droplets. Bar, 5 µm. B Representative images of FRAP experiments at indicated time-points. Boxes indicate bleached area (half-bleach of condensate). Bar, 5 µm. C FRAP curves after half-bleach of freshly formed Alexa488-labeled TDP-43 condensates. Values represent means ± SD of three independent experimental replicates (n=3) of ≥ 9 droplets analyzed per condition. ***p < 0.0002 by one-way ANOVA with Tukey´s multiple comparison test for area under the curve (AUC) of individual droplets. D Confocal images of Alexa488-labeled TDP-43 aggregates formed in an in vitro aggregation assay (with TEV protease cleavage). Bar, 100 µm. Zoom shows magnified view of aggregates at the 24h time point. Bar, 20 µm. E SDD-AGE followed by TDP-43 Western blot to visualize SDS-resistant oligomers / high molecular weight species of TDP-43-MBP-His6 in an in vitro aggregation assay (without TEV protease cleavage). Asterisks represent monomeric (*), oligomeric (**) and polymeric (***) species. F Input of TDP-43-MBP-His6 variants used in the SDD-AGE assay, detected by Western blot (anti-TDP-43 N-term).
p62 is required for the formation of GFP-LC3–positive punctuated structures in HeLa cells.Figure 6.(A) HeLa cells transiently transfected with GFP-LC3 alone or cotransfected with siRNA against p62, HA-p62, or HA-p62 D69A were either left in normal medium or starved for amino acids for 1 h. The cells were fixed, and p62 was stained using a p62 mAb. More than 200 randomly selected cells for each condition were scored for the cytoplasmic pattern of GFP-LC3 as either diffuse or punctuate. The frequency of GFP-LC3-positive cells with punctuate localization are indicated to the right. (B) Endogenous p62 as well as coexpressed myc-tagged wild-type p62 or a UBA deletion mutant of p62 coimmunoprecipitated with GFP-LC3 from HeLa cell extracts. GFP or GFP-LC3 was immunoprecipitated from total cellular extracts after cotransfecting the indicated constructs. The cell cultures were either left untreated or starved for amino acids for 1 h as indicated. Copurified endogenous or ectopically expressed myc-tagged p62 constructs were detected using the p62 mAb. Bars, 20 μm.