EMBO/sd-character-level-ner huggingface dataset:
HEK293 cells were transfected with α11.2, α2δ-1, and β2A and cultured for 22-24h before cell attached patch recording in 110 mM Ba2+. Single CaV1.2 channel recordings of CaV1.2 WT and K1647A. Holding potential (HP) was -80 mV and test potential (TP) 0 mV. Shown are 10 consecutive sweeps from representative experiments (see Fig. EV 4 for more sweeps). Dot plots and means + SEM for availability (i.e., likelihood that a sweep had at least one event; c), NPo (d), Po (e), unitary current amplitude i (e), and the mean of the current of a single channel at any point in time calculated from the ensemble averages of each experiment (g) (**p<0.01, ***p<0.001 compared to WT; one-way ANOVA with Bonferroni post-hoc test (c) or Welch ANOVA with Tamhane T2 test (d-g); n = 7 to 35 see Table 5 for more details).
(A) Tissue-specific ChIP-seq was done by isolating nuclei from specific tissues (shown in red) by expressing the Escherichia coli biotin ligase (BirA) and the biotin ligase recognition peptide (BLRP) fused with a nuclear envelope-targeting sequence in the tissue of interest. This allows the isolation of nuclei from the tissue of interest using streptavidin magnetic beads.
C-D) Correlation of FcγR activation data from A with (C) patient SLEDAI or (D) conventional SLE disease markers (α-dsDNA levels indicated as IU/ml or C3d concentrations indicated as mg/L). Left panel: FcγRIIIA; right panel: FcγRIIB/C. FcγR activation from a dose-response curve as in A was calculated as area under curve (AUC) for each SLE patient represented by individual dots. AUC from SLE patients are expressed as fold change compared to the healthy control mean (n=4). SLE patients with α-dsDNA levels below 50 IU/ml and C3d values below 6 mg/L were excluded. One-tailed Spearman's. Dotted lines show 95% confidence bands.
A B. SEC-MALS analysis of His-GFP-spMis18fl and His-spMis18ΔC, respectively. Refractive index (RI, left Y-axis) and molecular mass (kDa, right Y-axis) profiles show that both His-GFP-spMis18fl (predicted MW of a monomer is 51.97 kDa) and His-spMis18ΔC (Predicted MW of a monomer 21.8 kDa) predominantly exist as tetramers (187.6 kDa and 78.6 kDa, respectively) with a small population of dimer (128.5 kDa and 45.8 kDa, respectively).
(E) Representative confocal images of control siRNA and IRGM siRNA transfected THP-1 cells processed for IF analysis with Ubiquitin (green) and TOM20 (red) antibodies. Scale bar, 10 µm.
(E) In vivo analyses of the Atg12 and Atg5 mutants for studying the functional significance of the non‐covalent Atg12‐Atg5 interactions. Yeast cells with or without starvation were lysed and subjected to urea SDS-polyacrylamide gel electrophoresis (PAGE) followed by western blotting. Ape1, aminopeptidase I; mApe1, mature form of Ape1; PE, phosphatidylethanolamine; prApe1, preform of Ape1; WT, wild‐type.
(D) Cell extracts were subjected to SDS-polyacrylamide gel electrophoresis followed by immunoblot analysis using anti‐GFP antibodies. GFP, green fluorescent protein; mAPI, mature aminopeptidase I; pAPI, premature aminopeptidase I; WT, wild type.
esg-Gal4 midguts expressing mCherry and a svb::GFP rescue mini-gene, consisting of svb-cDNA tagged by GFP (green) and driven by E and 7 svb enhancers (see Fig 1). Samples were stained for GFP (green), mCherry (red) and Prospero (white). B' shows closeup views.
e, The percentage of phagosomes (n ≥ 50 per group) with GFP-LC3 in wild-type and Tlr2-/- macrophages. Means and ranges are shown (4 mice per group were used).
(D) MG activity was assessed as in panel C upon single or double knockdown of RBBP6 and hnRNP L. Data information: the data represent the mean ± S.D. from one representative experiment in which each transfection condition was performed in triplicate (n=3). The experiment was reproduced in two additional, independent experiments Statistical significance was calculated using ANOVA with Tukey's multiple comparisons test. ****p<0.00005; ***p<0.0005, **p<0.005, *p<0.05.
(A) Autophagic clearance of α-synuclein inclusions was verified by immunocytochemical analysis with anti-phosphorylated α-synuclein (P-αSyn) and anti-LC3 (LC3) antibodies. Confocal images reconstructed in the z-axis along the white lines are shown in the right panel (Z-axis). Blue, DAPI. Scale bar, 10 µm. Phosphorylated α-synuclein-positive inclusions were sequestrated into LC3-positive autophagosomes.
G. 30-days old fly eye brain complexes stained with neuronal marker Elav (blue) and poly-Ub antibody (red). The right panel is the enlarged images of the boxed regions at the left side
Pleural fluid cell pellets and supernatants from 45 patients (called ASK #) with pleural effusion from Munich, Germany (Klotz et al, 2019a and 2019b) were subjected to digital droplet polymerase chain reaction (ddPCR) for the detection of mutant (MUT) copies of KRAS codon 12/13 (KRASG12/13) and KRAS codon 61 (KRASQ61), as well as copies of TP53 and TERT. Diagnoses were benign pleural effusion (n = 5), lung adenocarcinoma (LUAD; n = 16), MPM (n = 12), as well as other extrathoracic cancers (n = 12). The assays were designed for detection of down to 1:20000 copies using EKVX (KRASWTTP53G610T), A549 (KRASG12STP53WT), NCI-H460 (KRASQ61HTP53WT), NCI-H3122 (KRASWTTP53E285V), and NCI-H3255 (KRASWTTP53G560-1A) human LUAD cells as controls. TP53/TERT Any number of KRAS-mutant droplets detected in any sample (KRAS plot in A) and any patient that failed to achieve normal TP53 ploidy by any sample (TP53 plot in A) was deemed altered.
(B) In vitro binding assay for thalidomide and 5-hydroxythalidomide. Interaction between bls-CRBN and FLAG-GST-IKZF1, -SALL4 and -PLZF in the presence of DMSO, thalidomide or 5-hydroxythalidomide (3.125, 6.25, 12.5, 25, 50, 100 or 200 µM) was analysed using the AlphaScreen technology. All relative AlphaScreen (AS) signals are expressed as a relative luminescent signal with a luminescent signal of DMSO as one. Error bars denote ± standard deviation (n = 3).
Immunoblotting analysis of signalosome components by immunoblotting in BCL9, CLTC or in both BCL9 and CLTC knockdown mitotic cells with CHX. Quantification of the stability of several LRP6 signalosome components by immunoblotting of mitotic cells with CHX.
HeLa CCL2 cells stably expressing control vector only (+vector), β-catenin WT (+WTR), S60A mutant (+S60AR), or S60D mutant (+S60DR) were generated using lentiviral infection and then infected with β-catenin knockdown lentivirus (shβ-catenin). A dot density graph with a box plot shows the percentage (%) of cells with cytokinesis-defective phenotypes (C). The box plot represents: central blue band, Median; box, Interquartile range; upper whisker, Maximum observation below upper fence; lower whisker, Minimum observation above lower fence; dot, Outlier. Each symbol in the graph represents a percentage of more than 30 cells from three independent experiments. ***, p<0.001 (one-way ANOVA); Scale bars, 10 μm.
(F) At 7 d post-infection, aliquots of supernatant taken before the addition of WST-1 were tested for lactate dehydrogenase (LDH) spectrophotometrically using the LDHPLUS assay. For the last hour cells were incubated with WST-1, and the reduction of the WST-1 reagent to its formazan product was monitored spectrophotometrically.
(A-B). qRT-PCR analysis of the relative expression of ICAM-1 and EPCR mRNA in non-treated PLB985 cells (control), ICAM-1 knock-down (ICAM-1kd) and EPCR knock-down (EPCRkd) PLB985 cells.
D Expression of SOX9 protein by immunoblotting of in vivo activated HSCs extracted from WT mice following CCl4 injections compared to olive oil control.
Median CSF protein abundance distribution as calculated from MS-intensities of quantified peptides of each protein. The top ten most abundant proteins and hemoglobin proteins are highlighted.
(C) ADP-Glo assay showing the effect of added CK1-7 on Hrr251-394, Hrr251-394:Mam187-191, and Hrr251-394:Mam187-191 R131A.
A, Venn diagram showing a comparison between TSN2_NS, TSN2_HS and TSN2_NaCl protein pools.
Resting mitochondrial calcium levels in control (ctrl) and mitochondrial-targeted Akt D/D (mt-Akt D/D)-expressing HeLa cells, evaluated through ratiometric imaging of the mitochondrial-targeted GCaMP6m (n=4 independent experiments; 43-47 cells)
Haematoxylin and eosin staining of sections representative of CntlKD, BNIP3KD or ATG5KD B16-F10 tumors. Necrotic areas are highlighted with an asterisk (*). Quantification of TUNEL+ tumor area (representing the fraction of dying/dead cell population) of CntlKD, BNIP3KD or ATG5KD B16-F10 tumors (n= 5 per condition). Quantification of the Ki67+ cells within the tumor area (representing the fraction of proliferative cell populations in non-necrotic areas) of CntlKD (n=7), BNIP3KD (n=6) and ATG5KD (n=8) B16-F10 tumors.
G A hypothetical model how Fragile X-related proteins spatialy regulate nucleoporin condensation. FXR proteins (blue) interact with cytoplasmic soluble Nups (red circles) and dynein (green) and facilitate their localization to the NE during early G1. This function of FXR proteins inhibits formation of aberrant cytoplasmic Nup assemblies, the Cytoplasmic nucleoporin Granules (CNGs), contributing to the equilibrium of NE-NPCs and driving the G1-specific protein export and maintenance of nuclear shape and cell cycle progression. Silencing of FXR proteins, for instance in FXS patients, leads to the accumulation of CNGs, nuclear atypia, protein export defects, and defects in cell cycle progression, which may contribute to the pathology of FXS.
(D) Overlap of miCLIP dataset generated in S2R+ cells defining m6A modified transcripts with Fmr1 and Ythdf mRNA targets determined by TRIBE in S2R+ cells. The indicated P-value was determined by hypergeometric test.
HeLa cells stably expressing GFP-Parkin with mock treatment (Control) or depleted of FUNDC1 (FUNDC1 KD) were incubated for 16 h with ethanol (Vehicle) or 10 μM CCCP (+CCCP), and analyzed by IB using the indicated antibodies.
A Schematics of AB diblock copolymers with different lengths of blocks and examples of the microphases (sphere and cylinder) that they form. Different blocks are shown in different colors (magenta and cyan).
I VCaP cells were transfected with the indicated siRNA as described in (B). After transfection, VCaP cells were cultured for 2 weeks. The colonies formed were stained and photographed.J Quantification of the data from (I). n = 3, *P < 0.0001. Error bars indicate SD.
Jurkat cells were exposed to either DMSO, or dieldrin in DMSO for 30 min. RNA-seq was then performed on cDNA libraries prepared after depletion of ribosomal RNAs. Jurkat T cells were treated with either DMSO (vehicle) or 100μM dieldrin for the indicated times. Abundance of IL2 mRNA was assessed by RT-qPCR. Data shown are means +/- SEM from four independent experiments. Significance (p-value) was estimated using the two-sided student t test appropriate for small sample numbers. ***: p-value<0.001.
(B). Venn Diagrams comparing DEGs with FDR<0.05 in three different cell lines after treatment with lurbinectedin shows the overlapping among the three DEG analyses. 3374 genes are commonly downregulated in the three cell lines, independently of their ASCL1 or NEUROD1 expression levels. Over-representation analysis of the common genes downregulated in the three cell lines after lurbinectedin treatment (blue table).
(K) Sphere formation assay results of MGC-803 cells treated with sEVs as indicated for 7 days. Scale bar = 100 µm (n = 3 biological replicates; mean ± SEM; **P = 0.0019 and **P = 0.0050; two-tailed unpaired Student's t-test).
Cartoon illustration that Snf1 signaling in ∆V and ρ0 cells upregulates glycolysis and represses ribosome biogenesis to optimize energy metabolism.
Effect of PP2A knockdown on Ago2 phosphorylation and miRNA-Ago2 binding in RAW 264.7 cells infected with Ld. Cells were co-transfected with siRNA and FH-Ago2 expression plasmid. Phosphorylated Ago2 levels were detected after 4 hrs of infection in siCon or siPP2A transfected cells (H).
M. The relative EBV production in C666 cells transfected with YTHDF1-specific siRNAs (siY1-1 and siY1-2) or the siNC control was measured by qPCR. The DNA from the cell-free culture supernatant was harvested at 24 h after siRNA transfection. The number of EBV copies in the supernatant of the cells was quantified by qPCR using specific primers targeting BamHI-W fragment region of EBV. The mean value of EBV copies in the siNC cells was normalized to 1.
D. Schematic representing the glutathione reductase (GR)-coupled assay to measure the GPX-like activity of Vs.
(D) Growth of wild type, phm8 and pnp1/urh1 strains in the switch from glucose to glycerol+ethanol. The x axis represents hours after starvation and the logarithmic y axis represents optical density (A600) in (B and D) and percentage of live cells calculated by dividing the number of colonies formed after starvation by the number of colonies formed before starvation in (C) (mean±range of N=2 biological replicates).
Figure 7. Model of RING dimerization in different TRIM ligases.TRIM25 and TRIM32 use different mechanisms to allow their respective RING domains to dimerize: In TRIM25 the propensity to dimerize is low and happens in an intramolecular fashion possibly aided by E2~Ub and substrate binding. It is possible that the B-box domains may contact the CC region to support dimerization. The RINGs of TRIM32 are constitutive dimers even in the absence of the rest of the remainder of the protein or additional binding partners. Tetramerization of TRIM32 may be important for the recognition of low affinity substrates or may allow co-localization of different binding partners.
(F) Single tube qPCR experiments showing the down-regulation of the synaptic genes NLGN3, SLC6A, TRIM9, SYT1, SYNGR1, and SNAP91 in ALSC9orf72 MN (Welch's t-test). n=3 independent cultures for each hiPSC line. Data information: *p<0.05; **p<0.01; ***p<0.001. Error bars represent SEM. Arrow indicates the structure displayed at higher magnification. Exact p-values are reported in Appendix Table S1.
Wild-type and cep41-deficient Tg(kdrl:eGFP) zebrafish embryos were fixed for immunostaining with Ac-Tub- or GT335-specific antibodies at 28 hpf in (C). The rectangles indicate the arterial cilia (AC) and venous cilia (VC) in zebrafish endothelial cells (EC). Magnified representative images are displayed in the right panels. Scale bars, 40 μm. Quantification of the labeled cilia observed in equivalent fields of view are presented graphically in (D). Data are shown as median of three independent experiments (n ≥ 20 embryos per condition).
E In situ hybridization of Col3a1 on femur sections. Col3a1 was expressed in the perichondral (a) and periosteal (b) regions, but not in the bone marrow. B: Bone; BM: Bone marrow; Peri: Periosteum; Endo: Endosteum. Scale bars were 100 µm. Arrowheads indicated the periosteum.
Schematic representation of neuron remodeling of γ-KCs and its regulation by the nuclear receptors EcR and UNF. p: axon peduncle; m/v: medial and vertical lobes.
Overexpression of lnc-MARCKS enhances IL-6 expression. RT-qPCR analysis of THP1 cells transduced with lentiviruses expressing empty vector (E.V.) or lnc-MARCKS, treated with PMA overnight, and left unstimulated or stimulated with Pam3 for 8 h. IL-6 expression (G).
B, heatmap of log10 scaled PBMC community frequencies for all samples (n = 42 patients × 2 timepoints = 84 samples). Clinical variables are depicted for all samples across the top of the heatmap; convalescent post-symptom onset anti-CHIKV antibody titer and viral titer (which was measured during the acute phase) are both in units of log10 dilutions. Hierarchical clustering (using complete linkage) was applied to both samples (X axis) and communities (Y axis). Four major clusters of communities and two major clusters of samples (largely separating acute and convalescent samples) are highlighted.
Melanoma cells were transfected with the GFP-tagged WT MICU1. MICU1-GFP immunocomplexes were precipitated with a GFP antibody and analyzed with PAS (phospho-Akt substrate) antibody by western blotting
(D) Cortical neurons expressing Mito‐GFP (green) were exposed to aged or fresh 200 μM SNOC solution. After 2.5 h cultures were loaded with hydroethidine. Images are representative of more that 20 neurons analyzed (in D) for each condition from at least three independent experiments. Scale bar, 10 μm.
(B) Total protein breakdown of cells starved on nitrogen‐free medium is significantly reduced in aut2Δ and aut7Δ cells compared with wild‐type cells. pep4Δ (pra1Δ) cells are almost completely impaired in starvation‐induced vacuolar protein breakdown. Cells were pulse‐labeled with [35S]methionine and chased on non‐radioactive starvation medium. Aliquots were taken at the indicated times and acid soluble small peptides generated by proteolysis were determined according to Straub et al. (1997).
d, Autophagic flux of +/−KRas cells stably expressing GFP-LC3. Bafilomycin treatment rescues GFP (−KRas+Baf) (n = 3); representative flow cytometry of two tumours.
The liver tissues were collected from WD/CCl4-treated mice expressing KLF10 or control (C) Expression of CD36 in whole livers and plasma membrane fractions. Protein levels were normalized to the β-actin or ATP1a1, respectively. n=5. Results represent three independent experiments. Data information: * P < 0.05, ** P < 0.01, *** P < 0.001. Results are shown as mean ± SD. Student's t test
A. Nucleosomes (N) decorating a relaxed plate (P1); insets in the upper right show additional examples. Nucleosomes are not visible as individual units in typical compact plates (P2). Short compact interdigitated solenoids are shown in the main image (S) and in the bottom-left insets. Data information: Scale bars: 50 nm
B. CMTs visualized by the MAP4-GFP reporter (left) and scheme of the CMT orientations (right) in epidermal cells of distinct growth zones (TZ, EZ and DZ). Individual cells monitored at two time points (0 and 60 min). Dark blue arrows mark the cell expansion direction. Scale bar 10 µm.
Statistical analysis of stx mutant stxd77 sleep profile compared with control w1118. (G) total sleep (From left to right mean±SEM: 359.0±12.91, n=64; 444.5±10.98, n=74; 478.9±10.76, n=64; 458.1±13.98, n=74). (H) sleep bout duration (From left to right mean±SEM: 21.01±1.312, n=64; 37.63±2.988, n=74; 58.81±5.202, n=64; 61.0±6.555, n=74), (I) number of sleep bouts (From left to right mean±SEM: 20.23±0.627, n=64; 16.38±0.693, n=74; 11.66±0.605, n=64; 11.93±0.556, n=74). (J) sleep latency (From left to right mean±SEM: 52.20±5.496, N=7; 22.76±2.621, N=9).
(B) Left Panel: Fusion was quantified by GFP area/ number of nuclei and normalized to D614G for each of the transfected variant S proteins. Right Panel: Representative images of Vero GFP-split cells 18h post-transfection, GFP (Green) and Hoechst (Blue). Top and bottom are the same images with and without Hoechst channel. Scale bars: 200 µm.
D,E Quantitative analysis (D) and representative images (E) showing increased density of presynaptic terminals in the hippocampal mossy fibers of AD mice infected with AAV-mCherry-Snapin. Blue indicates the signal of DAPI staining. The percentage area of SYP-labeled presynaptic terminals was quantified and normalized to that of control AD mice injected with AAV-mCherry (D). Data information: Data were quantified from a total number of 40-44 imaging slice sections of three pairs of mutant hAPP Tg mice with AAV injection.
Western blot detection of ATM, p-CHK2 Thr68, CHK2, p-Beclin 1 Ser90, p-Beclin 1 Ser93 and Beclin 1 in H1299 cells transfected with indicated shRNA in normal medium or after HBSS starvation (F)
g, Density gradient fractions of NRK cells starved for times shown and blotted for LAMP2 or Cathepsin D. Middle panel shows 8-h fractions 7 and 9 analysed by TEM. Fraction 10  is the  gradient top.
C. GST, GST-Snf7 and GST-snf7OPEN were immobilized on GT-resin and incubated with buffer(-) or recombinant heh2(1-308)-His6. Bound proteins separated by SDS-PAGE were visualized by coomassie stain and by Western blot with anti-His6 antibody and ECL detection (WB; top panel). Middle panel shows indicated cropped region of gel where contrast has been increased. Numbers on side of gel show position of molecular weight (MW) markers.
(B) HMGB1 levels in the medium of CT26 cells treated with BoxA or 1μM MTX for 24 hours (n=3). Data information: bars and error bars represent mean ± SD; statistics: One-way Anova with Dunnett's post-test, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.
Mouse mammary organoids were isolated from 19-20 weeks old C57Bl/6 mice that were either on normal (ND) or high fat diet (HFD) (n=3). Organoids were cultured in collagen for 2 days after which the number of invasive protrusions per each organoid was determined microscopically. NCT502 (10 μM) reduces the number of invasive protrusions in organoids isolated from mice that were either on G) ND or H) HFD. Error bars represent mean ± SD from 3 independent experiments where 30 organoids were counted per each mouse (labelled with a different symbol shape).
(F) Representative confocal images of Stat3-/- and MLS-Stat3 cells stained with anti-Stat3 and anti-ATAD3 antibodies. Merge image shows co-localization between Stat3 and the nucleoids marker Atatd3 (R=0.72 for MLS-Stat3 cells, R=0.22 in Stat3-/-). DAPI serves as nuclear counterstain. Scale bar, 10 μm.
Intracellular levels of Aβ1-42 are significantly increased in AP-2µ KO axons (WTAβ1-42: 0.65±0.08, KOAβ1-42: 0.91±0.10, p=0.046, WT=38 and KO=38 neurons, N=3 biological replicates). Scale bar: 5 µm.
D Representative microCT images of trabecular bone from the femoral metaphysis of female Sp7-Cre;Usp34f/f and littermate control mice. Scale bar, 500 μm. E, F Von Kossa stainin
(E) The truncated HOIL-1[233-510; I358R] mutant was assayed as in (B). (F) The truncated HOIL-1[233-510; F381A/E383A] mutant was assayed as in (B).
(D) Survival of cells containing upregulated antibiotic resistance reporter induced by dCas9 or dCas9-AsiA with gRNA-H3 under kanamycin selection (2.5 μg/ml).
Glucose tolerance test (GTT) after 25 weeks on NC or HFD. GTT was performed following an overnight fast Data information: values shown are mean ±SD. * = p < 0.05; ** = p < 0.01; *** = p < 0.001; **** = p < 0.0001 (two-way ANOVA with multiple comparisons).
(B) Number of enhancers per promoter, for all promoters that were paired to at least one enhancer with the CN approach. Fractions are shown for enhancers per promoter per cell line (light grey) and over all cell lines (dark grey). (C) As in (B) for pairs obtained with the CW approach.
Immunoblot detection of Hydroxylated HIF-1α (HIF-1α-OH), total HIF-1α, LDHA and ACTIN protein levels from normoxic lysates of B16-F10 cells cultured alone or in the presence of the PHD2 inhibitor IOX2 (100µM) for 24h.
(B) The N-lobe kinase linker of PI4KB can be efficiently phosphorylated by PKA. Recombinant PKA at different concentrations (0, 7, 34, 168, or 840 ng) was incubated with recombinant (E. coli) wild-type PI4KB (20 μg) for 1 hour with 200 μM ATP and the amount of phosphorylation was followed using mass spectrometry. Relative abundance of Ser496 phosphorylated PI4KB was calculated using the relative intensity (total area) of the phosphorylated vs non-phosphorylated peptide (486-506).
B. In vitro pull-down experiment. Indicated proteins (purified from Sf21 cells, 5-fold molar excess of MCAK over Fbxw5/Skp1) were mixed with competing E.coli lysate and precipitated via Ni-NTA agarose. Proteins were washed, eluted in SDS sample buffer and analysed by SDS-PAGE.
SARS-CoV-2 was first treated with trypsin or thermolysin and then allowed to infect A549* and Vero cells (MOIs ~0.2 and 0.3) in the continuous presence of SB412515 (cathepsin L inhibitor). Infected cells were quantified by flow cytometry as described in Fig. 1D, and data were normalized to samples where SB412515 had been omitted. n = 4 biological replicates.
C The cell viability was assessed by CellTiter-Glo after 24 hours of treatment (means ± SEM, n=4 biological replicates).
D Specific qPCR for HS3SH4, GPC6, and VCAN ITSs after ChIP using an anti-H3K27ac antibody in TRF2-overexpressing or -knockdown BJcl2 cells. Data represent fold enrichment of the H3K27ac level compared with the total H3 level. Data information: data represent n = 3 independent experiments with mean ± SEM