EMBO/sd-character-level-ner huggingface dataset:
BRCA1+/+, BRCA1-/- and BRCA1-/-/TP53BP1-/- RPE-1 cells were treated with 10 μM pyridostatin (PDS) or 2 μM olaparib for 2 days. Whole-cell extracts were immunoblotted as indicated. KAP1, IRF3 and STAT1 phosphorylation sites are shown in red. SMC1 and GAPDH were used as loading controls.
Mean H2Bac profiles at TSS relative to gene expression obtained from RNA-seq data. Expressed genes were separated into 4 groups (Q1 to Q4, 6483 genes/group), Q1 representing the less expressed genes (0-25%) and Q4, the most expressed genes (75-100%). Profiles were established with SeqMiner centered on TSS ±2Kb. C Mean H2Bac profiles established with SeqMiner for all genes at TSS (top) and gene bodies (bottom) for the 4th quartile (most expressed genes) in the two replicates (rep1 and rep2). Significance (Benjamini, p<0.05) of the annotations associated with both TSS (green) or Gene profiles (red) are shown (DAVID, KEGG Pathways)
O, Illustration depicting criteria for random section analysis (not drawn to scale). Ribbon-associated synaptic vesicles (SVs, green) in the first row ≤80 nm from ribbon (R) and membrane-proximal vesicles (yellow) in ≤25 nm membrane-to-membrane distance from the plasma membrane (blue) and ≤80 nm from each side of the presynaptic density.
IF of NSMCE2 foci in MMS‐treated Nsmce2+/+ and Nsmce2SD/SD MEFs. Scale bar, 2.5 μm.
Downregulation of ADAR1 levels in MCF7 cells was done using specific siRNAs. Cells were then subjected to hypoxia treatment for 5 hrs. The extent of knockdown and gene alterations was monitored by western blotting using antibodies targeting LIMD1, VHL, PHD1, PHD2, ADAR1 and HIF-1α, as indicated β-actin was used as loading control.
(D) Schematic representation of yki locus and its two isoforms: Yki2 (includes exon 3 and contains 2 WW domains) and Yki1 (skips exon 3 and contains 1 WW domain).
(A) Immunoblot analysis. Primary hepatocytes prepared from Nrf2f/f and Nrf2f/f; Alb-Cre mice were infected with adenovirus expressing GFP or NBR1 for 48 hrs. Cytosolic and nuclear fractions were prepared and subjected to immunoblot analysis with the indicated antibodies. Data shown are representative of three separate experiments. Bar graphs indicate the quantitative densitometric analysis of the indicated proteins relative to actin (cytosolic proteins) or Lamin B (nuclear proteins). Data are shown as means ± s.e. *P < 0.05, and **P < 0.01 as determined by Welch's t-test.
MDM were treated with 5μM ETO for 18h, infected with HIV-1 BaL and DNA isolated 4, 6, and 18 h post‐infection for qPCR. (n = 3, mean ± s.e.m.; (ns) non‐significant, paired t‐test).
H. Viable cell relative to starting cell numbers were determined after 24 h or 48 h treatment with vehicle (-) or 4HT on uncoated plastic surfaces. Means ± SEM (n=3).I. Viable cell relative to starting cell numbers were determined after 24 h or 48 h treatment with vehicle (-) or 4HT on collagen1-coated surfaces. Means ± SEM (n=3).
(B) FDB muscle fibres were transfected by electroporation in vivo. Eight days later adult fibres were isolated and placed in cell culture. Adult fibres were loaded with TMRM (5 nM) for 30 min at 37°C. TMRM accumulates in the mitochondria that maintain mitochondrial membrane potential. Oligomycin (Olm, 5 μM) and the protonophore FCCP (4 μM) were added at the indicated time points. TMRM staining were monitored in at least 10 fibres per construct (*P0.001).
(b) Colocalization is expressed in terms of Mander's coefficient M1 to indicate the proportion of LC3 intensities that also contain ATG9A intensities. A representative experiment of three independent experiments is shown, in which at least 33 cells were analysed per condition. Error bars indicate s.e.m., and ***P0.001 by 2-tailed Student's t-test.
(A) A diagram that outlines the steps of the genetic screen. ATXN1 reporter cell line produces RFP-conjugated ATXN1 and YFP internal control independently from a same transcript, and ATXN1 levels were monitored by measuring the ratio of red to yellow fluorescence intensity. 1) ATXN1 reporter cell line was transduced with lentivirus harboring shRNAs of 858 ubiquitination-related genes. 2)-3) The cells with the 5% lowest and 5% highest fluorescence ratio were sorted. 4) Illumina sequencing revealed enriched shRNAs in the two groups compared to the non-sorted group. 5) Identifying the genes targeted by these enriched shRNAs revealed 39 positive regulators including ZBTB7A and ZBTB7B, and 13 negative regulators of ATXN1 protein levels
(C) Fstl1 promoted fibroblasts proliferation was diminished by PD98059. PD98059 (5nM) was added 30 min prior to recombinant Fstl1 stimulation (50ng/ml). The cells were cultured for 48 hours, and EdU was added into media at 4 hours before harvest. Error bars represent mean ± SEM (n=4-10, per treatment group). Statistical analysis was performed by one-way ANOVA and Tukey multi comparison test. Two independent experiments were performed.
(i) Luciferase activity in TH1 cells transiently transfected with a vector containing a luciferase reporter under the control of the Lamp2 proximal promoter and stimulated for 0, 12 or 24 h with plate-bound anti-CD3 and anti-CD28; results are presented relative to those of resting cells (time 0).
A - Schema of experimental design. At 40 days in culture, human cerebral organoids were electroporated with one of the indicated DNA plasmid combinations, followed by 7 additional days in culture prior to analysis.
A. Model of centromere strength via CENP-C recruitment supported by DNA sequence and CENP-B. KT = kinetochore, MT = microtubules. IAA = auxin Cen = centromere. Blue arrows represent CENP-B boxes. Upon IAA addition, AID-tagged CENP-A is degraded.
L Lysates from MEFs stably expressing GFP-CbpCT4 or GFP in the presence or absence of rapamycin or chloroquine were subjected to SDS-PAGE and immunoblotted using antibodies against LC3 or p62.
C. Wild type and GAL-Mia40 cells were transformed with plasmids that express Yellow Fluorescent Protein (YFP) under the control of a minimal promoter containing the Hsf1-driven heat shock element (HSE). Cells were grown at 30 or 37°C before fluorescence was measured. Mean values and standard deviations of three replicates are shown.
(A) Schematic illustrating strategy to rescue DKO NPC differentiation defect by simultaneous lentiviral overexpression of Flag-Sox4 and Flag-Sox11 with two lentiviruses. Created with Biorender.com.
(M, N). The conformational change of the P-loop of the inactive state (M) and the active state of PTP-MEG2 in response to NSF-pY83 segment binding (N). The disruption of the charge interaction between R409 and D335 resulted in the movement of the main chain from G408 to K411.
(E and F) Effects of RNF26 knockdown on virus-induced K11-linked polyubiquitination of endogenous MITA. In (E), THP-1-Flag-Ub-K11O-RNF26-RNAi or control cells (2×107) were infected with SeV or HSV-1 for the indicated time points or left uninfected followed by IP under denatured conditions with anti-MITA. The immunoprecipitates were analyzed by immunoblots with anti-Flag (upper panels) or anti-MITA (lower panels). The whole cell lysates were analyzed by immunoblots with antibodies against the indicated cellular or viral proteins. In (F), 293-HA-Ub-K11O cells (2×107) were transfected with a control or RNF26-RNAi plasmid (10 µg each). Twelve hours after transfection, puromycin (1 µg/mL) was added into the culture medium. The cells were selected for twenty-four hours and infected with SeV or left uninfected for the indicated time points followed by IP under denatured conditions and immunoblot analysis as in (E). All experiments were repeated for at least three times with similar results.
E, F: EM images of negatively stained Dam1c alone (E) and in the presence of Bim1 (F), at low salt conditions. The final concentrations of Dam1c and Bim1 were 0.3 µM and 0.1 µM, respectively. The scale bars are 100 nm, boxes mark representative complexes shown as magnified inserts.
SseL interacts with p62 and autophagic substrates.(A and B) Co-immunoprecipitation from RAW264.7 macrophages infected with ΔsseL mutant bacteria expressing SseL-HA or SseLC/A-HA for 10 h. (A) Infectedcell lysates and immunoprecipated fractions were probed with anti-HA, anti-ubiquitin and anti-p62 antibodies. (B) At 7 h post-infection, infected cells were subjected to mock, 3-MA or starvation (Stv) treatments for 3 h before harvesting and processing as described in (A). Cell lysates and immunoprecipiations were probed with anti-HA and anti-ubiquitin antibodies.
D. FACS based mitophagy flux analysis performed in HeLa cell line stably expressing HA-Parkin, mt-mKeima, and a GFP fusion of the indicated sensor. Cells were treated with OAQ (oligomycin, antimycin, Q-VD) for indicated times and analyzed by FACS for lysosomal-positive mt-mKeima (561nm). The average of three independent experiments is presented. Representative FACS data from experiments (GFP control and AS3_67) are shown in the lower panels. Error bars indicate standard deviation. Significance calculation: 2-way anova of one-way anova. n.s.= not significant. *** indicates p-value of 0.0001.
D. Tyrosine phosphorylation of LORE in vivo. Two-week-old 35S:LORE-FLAG complementation plants were subjected to immunoprecipitation with anti-FLAG agarose beads. A23 (40 μM) was used to inhibit the protein phosphorylation, and the treatments were same as in (A). Protein abundance of precipitated LORE-FLAG was detected by immunoblotting. The tyrosine phosphorylation was probed by anti-pTyrosine (α-pY) antibody. Similar results were observed in three biological replicates.
Immunisation with cGAMP-VLPs enhances accumulation of Tfh cells in the draining lymph node. T follicular (Tf) cells were identified by flow cytometry as CD4+CD44+CXCR5hiPD1hi cells and were further subdivided into Tfr cells (FoxP3+) and Tfh cells (FoxP3-). the percentages of Tf, Tfh and Tfr cells within CD4+ cells are shown in (B).
(b) Cell cycle analysis. Normal Jurkat T cells and cells overexpressing ATG5 were cultured for 48 h in the presence and absence of 3-MA. Induction of G2- or M-phase arrest was seen as a consequence of ATG5 overexpression independent of treatment with 3-MA, as normal Jurkat T cells exhibited no G2/M arrest following 3-MA treatment. Representative flow cytometry diagrams are shown (n=3). Moreover, induction of autophagy by Beclin 1 overexpression or starvation failed to produce cell cycle arrest (Supplementary Fig. S4).
A) Representative mEPSC traces recorded from control and PTX3-treated (1µg/ml; 48hrs) neurons. B) mEPSC frequency quantitation (Hz, Ctr=0.618±0.069; PTX3=1.991±0.313; Number of neurons: Ctr=22, PTX3=16; 3 independent experiments, Mann-Whitney test. Data are presented as a distribution plus mean±SEM). C) mEPSC amplitude quantitation and cumulative probability distribution of mEPSC amplitudes (pA, Ctr=12.76±0.813; PTX3=16.07±0.709. Number of neurons: Ctr=22, PTX3=16; 3 independent experiments, Mann-Whitney test. Data are presented as a distribution plus mean±SEM. Cumulative probability distributions are analyzed by Kolmogorov-Smirnov test
D. Binding kinetics of UVR8ValPro/AlaAla pre-monomerized by UV-B versus the COP1 WD40 domain obtained by GCI experiments. Sensorgrams of UVR8 injected are shown in red, with their respective 1:1 binding model fits in black. The following amounts were typically used: ligand - COP1 (2000 pg/mm2); analyte - UVR8ValPro/AlaAla +UV-B (highest concentration 2 μM). ka = association rate constant, kd = disassociation rate constant, Kd = dissociation constant.
(b) HEK293 cells were transfected with TFEB and influenza hemagglutinin (HA)-tagged full-length AR25 (FL) or a C-terminal AR mutant (m1, m2, m3, m4). Immunoprecipitation (IP) was performed with anti-TFEB antibody, followed by immunoblotting (IB) for TFEB and AR. Untransfected HEK293 cells served as a negative control, and no AR was observed with IgG antibody only (not shown).
c, The DNA binding activity of TWIST1 (c) were evaluated in anti-GFP-mCherry ChIP-ChIP immunoprecipitates by RT-PCR in A549 cells. Red, Hprt1 promoter (-190-+40 bp, negative control). (10% input of each groups were pull down and applied to qPCR) Data are presented as mean ± SD in bar graph (p < 0.001, Student's t‐test) of 3 independent experiments, each performed in triplicate. Data information: Each experiment was repeated at least three times.
Cystic encephalomalacia evident in the right inferior frontal lobe secondary to a historical intraparenchymal haemorrhage.
(E) Phenotypic characterization of organotypic models made with HaCaT WT or GALNT1/2/3 KO keratinocytes. IHC of tissue sections stained for differentiation marker keratin 10 (upper panel) or proliferation marker Ki67 (lower panel). Scale bar - 50 μm. Red arrows - flattened cells; red asterisks - K10-negative region in suprabasal/granular layers; purple asterisks - pyknotic nuclei; green asterisks - increase in Ki67 positive cells.
(B) Microneutralization assay of FD20 against authentic SARS-CoV-2 Wuhan isolate (hCoV-19/China/CAS-B001/2020). The protective effect of antibody was assessed using cells infected with a high viral dose (2,000 TCID50 mL-1). Data are from a single experiment performed in octuplicates. Numbers in brackets indicate IC50 values in nM.
The illustration highlights the position of multiple-tethered (M, brown) and docked (D, light orange) SVs relative to PD (pink), indicating a depletion upon prolonged stimulation in wild-type.
A: MEFs from Pitrm1+/+, Pitrm1+/-, Mfn1-/-, Mfn2-/-, as well as h-PITRM1 R183Q and h- PITRM1 WT were exposed to Aβ1-40 peptide for 18h. Note that Aβ1-40 signal was still evident in Pitrm1+/- and h-PITRM1 R183Q cells.B: quantification of three independent experiments similar to those shown in panel A. Statistical analysis was by unpaired, two-tail Student's t test. **p<0.01, ***p<0.001.
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)
A Platelets were isolated from wild-type (WT) or Asm-deficient (Asm−/−) mice by density-gradient centrifugation, and 1 × 107 platelets were incubated with 1 × 105 B16F10 cells. The samples were lysed, and Asm activity was determined. In unstimulated samples (time point of co-incubation 0 s), tumor cells and platelets were admixed after lysis.
B Representative FACS plots of Treg (CD127lowCD25+FoxP3+) populations after T cell differentiation on media with or without cytokines and antibody supplements (anti-CD3, IL-2 and TGF-b1).
H. FBXW7 siRNA or control siRNA and HA-Ubiquitin were transfected into 293T cells, after 36 hours, cells were treated with MG132 with or without Wnt-3a (50 ng/ml) for six hours. Then lysate of the cells was performed with IP using FoxM1 antibody followed by IB with indicated antibodies.
(c) Suppression of autophagy by γHV68 vBcl-2. At 18 h posttransfection with GFP-LC3 vector, NIH3T3.vector, NIH3T3.vBcl-2 and NIH3T3.vBcl-2AAA, cells were incubated under normal or starvation conditions for 4 h. Subsequently, GFP-LC3 was detected using an inverted fluorescence microscope and autophagy was quantified as mean ± s.d. of combined results from three independent experiments.
ChIP-qPCR analysis of TET1, TDG and XRCC1 at gene promoters in mESCs (24 h 5 µM RA). Relative enrichments to an unbound control region (Chr2neg). n ≥ 3. Means with standard deviation (SD). *: P≤0.05; **: P<0.001; ***: P≤0.0001, by two-tailed Mann-Whitney test.
A HeLa cells were transfected with control or TIAR siRNAs for 48 hours prior to treatment with 0.4 µM APH. Cells were fixed at regular time intervals, and p(S10)-H3-positive cells were quantified by flow cytometry (mean ± SE, n = 3).
ovarian cancer cell lines PEO1, OVCA429, SKOV3, HEYA8 expressing different levels of AXL, and transduced with Empty vector control "E", or OPCML "O", and (L) Densitometry Data in (A) to (N) are representative of at least three experiments with graphs depicting means ± SEM: *P < 0.05, **P < 0.01, and ***P < 0.001 by Student"s t tests
Urine glucose levels of InsA peptide immunized (red: n=10 for d28, d32 and n=8 for d30; green: n=5 for d28, n=4 for d30, n=3 for d32) and CI (grey, n=3) mice were monitored at indicated days post immunization. Dots represent individual mice. For 100:1: representative data are shown for two independent experiments with total n=10. Mean ± SD, statistical significance was calculated by using repeated measure ANOVA test, ***P < 0.001.
(A) Representative immunoblot for GATA4 and actin from neonatal cardiomyocytes treated with control (Ad.Con) or GATA4 expressing adenovirus (Ad.GATA4).
F. Quantitative PCR on DNA immuno-precipitated with AR antibody in ChIP assay showed increased signal for SDHA, SDHB along with PSA after treatment with R1881 compared to androgen deprivation in LNCaP cells. PSA is used as positive control. Data information: ENZA: enzalutamide, CSS: charcoal stripped serum, ODM-201: darolutamide. Data shown as mean ± SD of three independent experiments. Statistical analysis was performed using one-way ANOVA followed by Tukey's test *, p<0.05, **, p<0.01, ****, p<0.0001 compared between groups.
Corresponding quantification of tumor infiltrating cells is displayed (anti-CD68, D), (anti-CSF1R, E) and (anti-CD206, F). Ratio of CD206+ vs. CD68+ cells (G).
A) Schematic representation of hMiro1 sequence highlighting domains and lysine residues (Lys) in its sequence. Key lysine residues reported to be ubiquitinated upon mitochondria damage and mutated to generate the mycMiro15R mutant construct are highlighted.
A Microenvironment t-SNE map separated by menopausal status and colored by expression (red=high expression, grey=undetectable) of selected fibroblast markers (upper panel). The dotted lines indicate the pericyte subsets. Bottom panel shows percentage of cells expressing the markers for the cell clusters
B 293FT-TetR-3xFlagUsp27x or TetR-3xFlag-Usp27xC87A cells treated as in A were analysed using conditions of higher resolution. A band very likely corresponding to phosphorylated BimEL is detectable. Similar results were obtained in n=2 separate experiments.
(B) Orthogonal views of the STC DNA density map with RAG protein rendered transparent. Red and yellow, 12RSS and 23RSS and covalently-linked target site and flank DNA, respectively.
A. Average enrichment of Upf1-23 and Upf1-decapping components in purified Upf1-TAP in the presence (black bars) and absence (blue bars) of NMD4. Pink and dark blue vertical lines highlight proteins of the Upf1-23 and Upf1-decapping complexes respectively. White dots correspond to individual enrichment values obtained in replicate experiments, 6 for Upf1-TAP and 3 for the nmd4Δ condition. Error bars correspond to SD
B. WB of ApoliproteinA4 in jejunum of Pkd2wt/wt and Pkd2ki/ki male mice after one week of HFD. Quantification of bands normalized to loading control and relative to Pkd2wt/wt. n=3.
(d) Tissue lysates were prepared from the liver of fed ob/w or ob/ob mice 4 h after leupeptin administration, and immunoblotting was performed. Numbers below immunoblot bands indicate the fold changes normalized to control bands.
(B) EMSA showing binding of ANAC032 to MYC2, PDF1.2A and NIMIN1 promoter regions (in 5´-DY682-labelled double-stranded oligonucleotides) harbouring ANAC032 binding sites. 1, labelled probe only; 2, labelled probe plus GST protein 3, labelled probe plus ANAC032-GST protein; 4, labelled probe, ANAC032-GST protein and 100 x competitor (unlabelled oligonucleotide containing ANAC032 binding site).
(E) Increases in P0 protein over time also were observed by immunofluorescence analysis at different time points, with P0 protein expression increasing to a greater extent in nerves of mice treated with 4-AP. Scale bar = 200 m.
(A) Schematic representation of the domain structure of IRF2BP1 and IRF2BP2. The primary sequence suggests two putative SUMO-sites that are conserved among different species.
C Pearson correlation coefficients (Pearson R−values) showing no correlation between GFP and PHF1 (PHF1/GFP) at any time points, but negative correlation between TFEB and PHF1 (PHF1/TFEB) starting at 16 h and persisting to 40 h (Student's t−test, n = 4, *P = 0.026 and **P = 0.008 for 16 and 40 h, respectively, comparing R−values for GFP/PHF1 with TFEB/PHF1). The whole view field of eight confocal projection slices per view field, four view fields per time point per transfection was used for analysis. Bar graph represents average ± s.e.m.
B. Diagram of the domain structure of the Cwh43 protein illustrating the location of the damaging CWH43 deletions. Lower panel details the disruptive effect of the k696Asnfs mutation on the C-terminal endoplasmic reticulum export signal (YF), replacing it via a frameshift with a 23 amino acid sequence that contains an endoplasmic reticulum retention signal (KKKS).
Global statistics of protein signal attribution to assembled or monomeric state. The majority of proteins (66 %) and protein mass (55 %) appear in assembled state in SEC-SWATH-MS.
F Recruitment kinetics of GFP-tagged SirT7 after laser-induced microirradiation. (Top) Representative cell at the indicated times after induction of DNA damage (white circle; scale bar 2µm). Images were adjusted to account for photobleaching by normalizing to nucleoplasmic background signal. (Middle) Quantitation of recruitment kinetics at the site of induced damage in the presence of 5M KU-55933 ATM inhibitor (ATMi), 10M Olaparib PARP inhibitor (PARPi), or DMSO. KU-55933 and Olaparib were added 30 min and 1 hour respectively prior to DNA damage (mean ± SEM; sample size: SIRT7-GFP, n=34; SIRT7-GFP + ATMi, n=36; SIRT7-GFP + PARPi=18). (Bottom) Same as (middle) except quantitation of SIRT7-GFP relative intensity within the nucleolus. Data was acquired at five second intervals over a span of five min.
(B) Average MTS prediction scores (MitoFates and TargetP2) for structurally conserved yeast MRPs and their bacterial homologs sorted by the difference between bacteria and mitochondria.
A) srd-1 mutant males were not attracted to the sex pheromone but were attracted to the positive-control chemoattractants, 1:1000 diacetyl and 10 mg/mL pyrazine. This demonstrated that SRD-1 is required and specific for sex pheromone perception. Data information: For each transgenic strain, 3 independent lines were examined. We assayed 400 males from each strain in the sex pheromone chemoattraction assay. Two biological replicates were combined into a single value. Significance was determined using one-way ANOVA with Bonferroni correction: ***P<0.001. Means ± SEM (error bars) are shown.
(E-H) Properties of various GM3 species as a function of BMI: LCFA-GM3 (E), VLCFA-GM3 (F), unsaturated VLCFA-GM3 (G), and α-hydroxyVLCFA-GM3 (h24:0) (H). Colors indicate disease severity: light blue, no abnormal scores (n=25); orange, early-phase obesity (n=74); purple, severe obesity (n=23).
L. Samples in (J) were digested with PsiI+XhoI and analyzed on a denaturing polyacrylamide gel. Stalling points relative to the ICL site are indicated.
Effect of different ETP-47228 and ETP-47037 concentrations during 24 h on proliferation in lung tumor-derived cell line relative to the growth of DMSO-treated cells.
CYP46A1 expression levels in different molecular subtypes from the Rembrandt GBM dataset. Shown are means and SEM (n = 245). ***P < 0.0001. Statistical significance was determined by one‐way ANOVA.
Biotin endocytosis assays of HeLa cells with endogenous DHHC5, over-expression of DHHC5 alone, co-expression of DHHC5 and WT Golga7b, co-expression of DHHC5 and mutant Golga7b or expression of the DHHC5 C-terminal mutant. Biotinylated cell surface proteins endocytosed within 15 minutes were purified using streptavidin resin and blots probed with an antibody to DHHC5. The lack of signal in the pull-down of the DHHC5 C-terminal mutant or when DHHC5 and WT Golga7b are co-expressed indicates that DHHC5 is stabilised at the plasma membrane in these conditions. The robust signal in the pull-down of endocytosed proteins from cells co-expressing DHHC5 and mutant Golga7b shows a high rate of endocytosis of DHHC5 in this condition. N=3, *=p<0.05, paired t test"