EMBO/sd-character-level-ner huggingface dataset:
ER Stress Modulates ATF6α-PDI Mixed-disulfide Complexes A-D HEK293T cells were co-transfected with ATF6α S1P and PDI substrate-trapping mutants and either left untreated (-) or treated (+) for 1 h with 5 µM TG (A), 5 µg/ml tunicamycin (B) or 20 µM MG132 (C) to induce ER stress.Figure 3HEK293T cells were co-transfected with ATF6α S1P and PDI substrate-trapping mutants and either left untreated (-) or treated (+) for 1 h with 5 µM TG to induce ER stress. ATF6α was immunoisolated from cell lysates with mouse anti-ATF6α, separated by SDS-PAGE under non-reducing conditions and ATF6α detected by western blotting using rabbit anti-HA. Blots indicate that mixed-disulfide complexes between ATF6α and PDI enzymes are regulated by ER stress. M, D and O refer to ATF6α monomer, dimer and oligomer respectively. *indicates positions of the ATF6α-PDI enzymes mixed-disulfide complexes. HEK293T cells were co-transfected with ATF6α S1P and PDI substrate-trapping mutants and either left untreated (-) or treated (+) for 1 h with 5 µg/ml tunicamycin to induce ER stress. ATF6α was immunoisolated from cell lysates with mouse anti-ATF6α, separated by SDS-PAGE under non-reducing conditions and ATF6α detected by western blotting using rabbit anti-HA. Blots indicate that mixed-disulfide complexes between ATF6α and PDI enzymes are regulated by ER stress. M, D and O refer to ATF6α monomer, dimer and oligomer respectively. *indicates positions of the ATF6α-PDI enzymes mixed-disulfide complexes. ** indicates hypoglycosylated ATF6α. Data shown are representative of three independent experiments. HEK293T cells were co-transfected with ATF6α S1P and PDI substrate-trapping mutants and either left untreated (-) or treated (+) for 1 h with 20 µM MG132 to induce ER stress. ATF6α was immunoisolated from cell lysates with mouse anti-ATF6α, separated by SDS-PAGE under non-reducing conditions and ATF6α detected by western blotting using rabbit anti-HA. Blots indicate that mixed-disulfide complexes between ATF6α and PDI enzymes are regulated by ER stress. M, D and O refer to ATF6α monomer, dimer and oligomer respectively. *indicates positions of the ATF6α-PDI enzymes mixed-disulfide complexes. ** indicates hypoglycosylated ATF6α. Data shown are representative of three independent experiments. HEK293T cells were co-transfected with ATF6α S1P and PDI substrate-trapping mutants and either left untreated (-) or treated (+) for 1 h with 5 µM TG to induce ER stress. Samples from (A) were rerun and probed with anti-sera raised against respective PDI enzymes as indicated (D). *indicates positions of the ATF6α-PDI enzymes mixed-disulfide complexes.
Figure 5A Representative photographs of GMR-GAL4; (CGG)90-EGFP fly eyes expressing manipulations of eIF4B and eIF4H. (B) Quantitation of GMR-GAL4, (CGG)90-EGFP eye phenotypes with eIF4B/H manipulations (Mann-Whitney U test with Bonferroni corrections for multiple comparisons; n=26-55/genotype). Data Information: For all panels, ns=non-significant, * P≤0.05, ** P≤0.01, **** P≤0.0001 for the specified statistical test. All panels present data as means ± SD (compiled from ≥3 replicates). C, D Expression of plasmid-based AUG-NL and +1 (CGG)100 NL-3xF reporters (C), or co-transfected AUG-FF reporters (D), following knockdown of EIF4B or EIF4H. Black asterisks refer to comparisons between siEGFP- and siEIF4B/H-treated cells; pink and blue asterisks refer to comparisons between siEIF4B- (pink) or siEIF4H- (blue) treated cells expressing AUG-NL-3xF and those expressing +1 (CGG)100 (two-way ANOVA with Tukey's multiple comparisons test, n=9/condition). E Expression of plasmid-based AUG-NL-3xF and (CGG)100 +1 NL-3xF reporters with and without over-expression of EIF4B, EIF4H, or both (two-way ANOVA with Dunnett's multiple comparisons test; n=20/condition). Asterisks refer to comparisons between cells over-expressing either EGFP or EIF4B, EIF4H, or EIF4B and EIF4H and expressing the same reporter. Data Information: For all panels, ns=non-significant, * P≤0.05, ** P≤0.01, **** P≤0.0001 for the specified statistical test. All panels present data as means ± SD (compiled from ≥3 replicates).
Figure 3Young"s modulus for matrices previously remodeled by PSCs; control n=126 and tamoxifen n=145. Lines and error bars indicate mean±s.e.m. Four experimental repl Representative images of H&E staining showing AsPC1 cancer cell invasions in matrices previously remodeled by control PSCs (top) or tamoxifen treated PSCs (bottom), scale bar 50 μm AsPC1 invasion quantification, the central box represents values from the lower to upper quartile. The middle line represents the mean. The vertical line extends from the minimum to the maximum value, n=30 control and n=40 tamoxifen
Figure 9(A) Representative immunofluorescence images of ZW10 localization to unaligned KTs in Drosophila S2 cells expressing either SpindlyWT-, SpindlyS499A- or SpindlyS499D-EGFP. Insets display magnifications of the outlined regions, which highlight late congressing KTs. CID was used as a KT reference. (B) Graph represents the percentage of ZW10 levels at late congressing and non-oriented (not oriented parallel to the spindle axis) KTs for cells shown in (A). ZW10 levels were determined relative to CID and all values were normalized to the control mean fluorescence intensity, which was set to 100% (n≥72 KTs from at least 26 cells for each condition, n=4 independent experiments). Data information: Statistical analysis was calculated using a Kruskal-Wallis test for multiple comparisons. p values: *, <0.05; **, <0.01; ****, <0.0001 . Data are shown as mean ±SD. Scale bar: 5 μm. (C) Selected frames from live imaging analysis of chromosome congression in Drosophila S2 cells expressing SpindlyWT-EGFP, SpindlyS499A-EGFP, SpindlyS499D-EGFP or SpindlyS499D-EGFP in a ZW10-depleted background. The first frame shows EGFP-positive cells. Chromosome alignment was followed in vivo by direct visualization of KT-associated PoloWT-mRFP signal. Time 0 refers to nuclear envelope breakdown (NEBD), metaphase refers to full KT alignment and Anaphase onset refers to onset of chromosome segregation. (D) Quantification of the time spent in prometaphase (from NEBD until last KT alignment at the metaphase plate) for cells shown in (C) (n≥10 cells for each condition, n≥2 independent experiments). Data information: Statistical analysis was calculated using a Kruskal-Wallis test for multiple comparisons. p values: *, <0.05; **, <0.01; ****, <0.0001 . Data are shown as mean ±SD. Scale bar: 5 μm.
Determination of 5-AzadC + SAHA treatment schedule in vitro and ex vivo.Figure 3A-D J-Lat 8.4 cell line was mock-treated or treated with 5-AzadC and/or SAHA for different periods of time as indicated. Samples were harvested at indicated times. Viral production was measured by quantifying p24 antigen production in culture supernatants (A, B) A-D J-Lat 8.4 cell line was mock-treated or treated with 5-AzadC and/or SAHA for different periods of time as indicated. Samples were harvested at indicated times. metabolic activity was assessed by a WST-1 assay (C, D). Means and standard errors of the means from three independent biological duplicates (n=6) are indicated. The result obtained with mock-treated cells was arbitrarily set at a value of 100% (C, D). E From data of ex vivo cultures of CD8+-depleted PBMCs isolated from 24 HIV+ patients presented in Appendix Table S2, the extracellular HIV-1 genomic RNA levels for each LRA treatment are represented. One night after cell purification, cells were mock-treated or simultaneously treated with 5-AzadC (1µM) and/or SAHA (1µM). Six days after treatment, the concentration of viral RNA in culture supernatants was determined (in copies/ml). The results were reported as the actual HIV RNA copy numbers/ml or as an estimated value calculated as 50% of the smallest value when HIV RNA was not detected in order to assign a log value. Means are represented.
The N-terminal end of Armi plays an important role in the departure of Piwi−pre-piRISC from Yb bodies.Figure 6Armi-Flag (Armi-F) WT and the ∆N34 mutant were expressed in OSCs where endogenous Armi and Zuc had been depleted by RNAi (Zuc KD + Armi KD). Immunoprecipitation and subsequent western blotting show that Armi-F WT but not Armi ∆N34 mutant bound with Gasz in the cells. Both Armi WT and ∆N34 mutant interacted with Piwi to similar extents. Luciferase-Flag (Luc-F) was used as a negative control. Armi-Flag (Armi-F) WT and the ∆N34 mutant were expressed in OSCs where endogenous Armi and Zuc had been depleted by RNAi (Zuc KD + Armi KD). Armi-F WT (green) localized onto mitochondria (red), whereas Armi-F ∆N34 mutant (green) localized to Yb bodies (see also Fig EV6D). The scale bar represents 5 μm. DAPI (blue) shows the nuclei. In vitro pull-down assays show that both Armi-Flag (Armi-F) WT and ∆N34 mutant directly bind with GST-Gasz∆C113 but not with GST. Armi-F WT and mutant were immunopurified from Schneider 2 (S2) cells under harsh conditions. GST and GST-Gasz∆C113 were visualized by CBB staining, while Armi-F WT and mutant were detected by western blotting using anti-Flag antibodies. Immunoprecipitation and subsequent western blotting shows that Armi-Flag (Armi-F) but not Flag-Armi (F-Armi) strongly bound with Gasz in Zuc/Armi-depleted OSCs (Zuc KD + Armi KD). Piwi bound with both Armi (F-Armi and Armi-F). Luciferase-Flag (Luc-F) was used as a negative control.
Hydrogen-deuterium exchange kinetics reveal long-range nucleotide- and/or membrane binding driven conformational changes in dynamin.Figure 1(A) Crystal structure of a dynamin monomer (PDB ID: 3SNH) and corresponding color-coded linear domain architecture of dynamin. The PHD is shown in both the closed (3SNH, solution structure) and open (3ZYS, cryo-EM reconstruction of membrane-bound dynamin) states. The approximate locations of terminal residues flanking different functional domains are identified with black arrowheads. The positions of the L1SP and L2SP loops are identified by red arrowheads. Changes in HDX behavior for dynamin analyzed (B) in solution the presence GMPPCP, (C) in solution in the presence of GDP•AlF4-, (D) membrane-bound, or (E) membrane-bound in the presence of GMPPCP and mapped onto the structures of dynamin monomers. Color coding (see scale below) shows the extent of solvent exchange relative to apo dynamin in solution. All HDX MS data shown represents an average of 3 replicates and are color coded from red to blue with warm colors representing increased conformational dynamics (red being the highest D2O uptake) and cool colors representing decreased conformational dynamics (blue being the lowest D2O uptake). Grey indicates no statistically significant change between the two states. Quantitative data on the individual peptides used to generate this map are provided in Appendix (Figure S1-S4).
Figure 4A ScV1-H subunit interactions. Region of electron density (contoured at 1.2 σ) outlined by the box in the side view of ScV1 shown to the right. The H subunit N-terminal domain (HNT) is bound to the N-terminal α helices of peripheral stalk EG1 while its globular C-terminal domain (HCT) is bound at the base of the catalytic hexamer. B Interaction between HNT and peripheral stalk EG1. View of HNT as in panel A rotated 110° along the horizontal, showing the α helical HEAT repeats of HNT forming a pocket for binding the N-termini of peripheral stalk EG1. HNT's α helices forming the pocket are labeled as in (Sagermann et al, 2001). Pro197 on HNT (red sphere) has been previously shown to cross-link subunit E (Jefferies Forgac, 2008).C Interaction between HCT and the base of the catalytic hexamer. The H subunit inhibitory contacts at the base of the ScV1 are mediated by a loop in HCT (highlighted in magenta) that is seen to contact two short segments of the C-terminal domain of the subunit G from the open catalytic site (AB) 1 (highlighted in purple) and two turns of the N-terminal a helix of the central rotor subunit D (highlighted in blue). D HCT binds to the open (AB) 1 pair. View of ScV1 looking along the central rotor subunit D towards the cytoplasm, showing the position of the H subunit binding at the interface of the open A/B pair (AB) 1. The catalytic hexamer is shown in surface representation and the central rotor, peripheral stalks and subunit H are shown as ribbons.
Figure 4(A) Examples of sororin (dotted arrows) and SMC3ChIP-seq and BrdU DIP-seq data from early S-phase as compared to G2-phase using the Integrated Genome Browser (IGB; Nicol et al, 2009).(B) Alignment of sororin, BrdU and BrdU-negative control sequencing bins in early S-phase to humanchromosome 18 (GRCh37/hg19). Bars correspond to called peaks, curves to read enrichments after smoothing (k=25 kbp).(C) Quantification of the co-occupancy of BrdU incorporation and sororin localization in early S-phase and sororin in G2-phase depicted as p-value distributions calculated with IntervalStats (Chikina Troyanskaya, 2012) and compared to randomized controls. Sororin peaks were identified as overlapping peaks of 4 samples in early S-phase and 2 samples in G2-phase, using only the common peaks.(D) Quantification of the co-occupancy of SMC3 in early S-phase and in G2-phase with BrdU incorporation in early S-phase.
Cytoplasmic Cdc14 is required for cytokinesis+CDC14 and +CDC14‐NLS cells were grown at 35.Figure 2A +CDC14 and +CDC14‐NLS cells were grown at 35.5°C for 5 h and sonicated, and differential interference contrast (DIC) images were acquired. B Cells from a synchronous culture (as in Fig B) were treated with zymolyase to remove the cell wall. The fraction of remaining budded cells is shown over time (n = 100). C Cells expressing the plasma membrane marker GFP‐Spo2051-91, exemplifying the indicated bud neck phenotypes and stages of cytokinesis.D Bud neck phenotypes of synchronized GFP‐Spo2051-91‐expressing +CDC14 or +CDC14‐NLS cells during the progression of a cell cycle (n > 75). E +CDC14 or +CDC14‐NLS cells expressing Spc42‐YFP and GFP fusions with the indicated cytokinetic protein were synchronized in G1 using α‐factor. Upon release at 35.5°C, cells were imaged every 2 min and the time from anaphase onset to the indicated events was recorded. Error bars represent SD; Student's t‐test was applied to analyze differences between the indicated conditions; **P ≤ 0.01; ****P ≤ 0.0001. For representative image sequences, see Supplementary Fig S2C.
Figure 4An mRNA-miRNA reaction network with one miRNA binding site on the target mRNA (mmi-1 Model). An mRNA-miRNA reaction network with two miRNA binding sites on the target mRNA (mmi-2 Model). Gray box indicates the condition that allows bistability (with Eq A depicted in panel B). We generated 10000 parameter sets, with values for a1, a2, b1 and b2 randomly drawn from a uniform distribution over the interval (0.125, 16). All other parameters, except the control parameter for bifurcation analysis and the scaled dissociation rate constant (KkR0/sR = 10 − 5), were set to 1. Bifurcation diagrams demonstrate the steady states calculated for 3348 bistable systems out of 10000 parameter sets. Red curve: stable steady state with high amounts of free mRNA. Black curve: stable steady state with low amounts of free mRNA. Blue curve: unstable steady state. One representative system is highlighted with thick curves, and other systems are represented by thin curves. Light green arrow denotes parameter sets in which mRNA degradation is not enhanced by complex formation. In these systems, a steady state is established in the presence of high amounts of miRNA but with total mRNA levels not less than 1. Phase plane analysis with three representative parameter sets. Left: cooperative mRNA degradation (a1 = b1 = b2 = 1, a2 = 10). Middle: cooperative miRNA degradation (a1 = a2 = b2 = 1, b1 = 10). Right: target-mediated miRNA degradation (TDMD) (a1 = b2 = 1, a2 = b1 = 1.8). All other parameters are equal to 1, except KkR0/sR = 10 − 5. Dark red and black: stable steady states. Light blue: unstable steady state. Blue curve: nullcline of ODE for RT. Orange curve: nullcline of ODE for rT (Eq A in Figure 4B). Gray arrows: representative directions in the vector field. Gray curve: separatrix. To obtain the nullclines vector field, coordinates in the RT, rT space were used as the initial conditions for ODEs in Eq A. The corresponding values of C1 and C2 were obtained by solving the algebraic equations in Eq A with the initial conditions. Resulting values for the right-hand side of the ODEs were used to determine the positions of the nullclines.
Figure 5A Western blot of HEK293 cells expressing GFP-tagged gephyrin or G375D using GFP-specific and polyclonal gephyrin antibodies. Note that both gephyrin variants are stably expressed and show no degradation in the cells. Asterisk in the poly-gephyrin blot shows endogenous gephyrin. B SDS-PAGE of E. coli expressed and purified 6His-gephyrins and CD spectra. Mean residue ellipticity of gephyrin and G375D show profiles with minima at 208 nm and 222 nm. C Size-exclusion chromatography of purified 6His-gephyrin and isolated E-domain variants. Full-length gephyrins and isolated E-domains were loaded onto a Superose 6 gel filtration column and elution of the protein was monitored by measuring absorbance at 280 nm (mAU, milliabsorbance units). D Co-immunoprecipitation of HEK293 cell lysates expressing myc-tagged gephyrin together with GFP-tagged gephyrin or G375D using myc-specific antibodies. Control IgG were used to show specificity of the assay.
Neuroprotection of cellular L-BMAA model by diverse GSK-3 inhibitors.Fig 4A) Diverse GSK-3 inhibitors selected showing their inhibition kind. B) Cell viability measured in the experiment with or without inhibitors.
Replication stress induces loss of acetylated histones.Figure 5(A) Representative western blots of histone acetylation levels of HeLa cells obtained at the indicated time points after UV irradiation (16 J/m2), HU/AraC treatment (100 mM/10 μM) or MMC treatment (10 µg/ml). Blots were stained with α-acetyl-lysine (top panel), α-histone H4 (middle panel) and α-tubulin (bottom panel) (B) Quantification of average histone acetylation signal, normalized to loading control. N≥3. Error bars represent SEM. Significant differences, calculated with T-test, between treated and mock conditions are indicated with * (p<0.1), ** (p<0.05) and *** (p<0.01) (C) Representative Western blots, stained with the indicated antibodies of HeLa cells pre-treated o/n with an S-phase inhibitor (PHA 767491 hydrochloride, 10 μM) and lysed at indicated time points after UV (16 J/m2) (D) Quantification of the α-acetyl-lysine signals, normalized against histone H4 levels, N= 5, error bars represent SEM. Significant differences, calculated with T-test, between UV-irradiated and inhibitor treated conditions are indicated with ** (p<0.05)
Figure 5(A) Schematic of the experimental strategy to assess the effects of COCO in the Oxygen-induced Retinopathy model. (B) Retinal flat mounts of P17 OIR mice injected with PBS, COCO or Flt1Fc are stained with IB4. Red areas highlight vascular tufts. (C) Quantification of neovascular tuft area in P17 OIR. **P=0.0021 (PBS vs COCO); *P=0.0122 (PBS vs Flt1Fc); (n=6). (D) Quantification of central avascular area in P17 OIR; (n=6). (E) Schematic of the experimental strategy to assess the effects of COCO in the Laser-induced Choroidal Neovascularization (CNV) model. (F) Choroidal flat mounts of adult mice subjected two weeks prior to Laser-CNV injected with PBS, COCO or Flt1Fc stained with IB4 (green) and phalloidin (red). Scale bar, 100 μm. (G) Quantification of CNV surface area. *P=0.0210 (PBS vs COCO); *P=0.0113(PBS vs Flt1Fc); (n=12-14 burns from 5 animals/group).
TT-seq analysis and transcript annotation in human cancer cells.Figure 1(A) Experimental design. TT-seq data of fourteen cell lines representing seven different human cancer types: brain (M0569J, U-118 MG), breast (BT-20, MDA-MB-231), colorectal (GP5d, HCT 116, LoVo), kidney (769-P, 786-O), prostate (DU 145, PC-3), and uterine cancer (SK-UT-1) as well as leukemia (Jurkat (Michel et al., 2017), K562 (Schwalb et al., 2016). For TT-seq, newly synthesized RNA is metabolically labeled with 4-thiouridine (4sU) for five minutes and labeled RNA is isolated, and processed as described with minor modifications Libraries of total fragmented RNA (RNA-seq) and labeled RNA are sequenced. (B) Definition of transcript classes and putative enhancer RNA (eRNA) annotation. Top: non-coding RNAs are classified into four categories: upstream antisense RNA (uaRNA), convergent RNA (conRNA), antisense RNA (asRNA) and intergenic RNA. asRNAs and bidirectional intergenic RNAs originating from GenoSTAN enhancer state regions (Zacher et al., 2017) are classified as putative eRNAs (green). Bottom: exemplary genome browser view of TT-seq coverage in HCT 116 colorectal cancer cells and transcript annotation at the KLF6 locus (hg38; chr10:3,758,830-3,815,253; Due to the high expression of KLF6 the TT-seq coverage is cut at 200 to allow for better visualization of the surrounding eRNA signal. H3K4me1 ChIP-seq, H3K27ac ChIP-seq and DNase I-seq signal from ENCODE (C) Definition of putative enhancer regions. For non-overlapping (left) and overlapping (middle) bidirectional intergenic RNAs the region between the TSSs of each pair is extended by 500 bp in both directions, and defined as putative intergenic enhancer region. For asRNAs (right) the region covering 750 bp upstream of the TSS to 500 bp downstream is defined as putative intragenic enhancer region. (D) Number of putative enhancer regions per cell line. Bar depicts the mean. Diamonds represent individual numbers in each cell line and are color-coded according to cancer type (E) Heat maps of DNase I signal for enhancer regions (rows). For intergenic enhancers (left) the signal is aligned at the upstream (relative to + strand) enhancer edge, shown from 4 kbp upstream to 5 kbp downstream, and ordered by increasing length of enhancer regions. For intragenic enhancers (right) the signal was aligned at the upstream and downstream enhancer edges and shown within ± 4 kbp. Exemplary data from LoVo colorectal cancer cells. (F) Heat maps as in (E) showing composite ChIP-seq peak coverage from 326 TFs