EMBO/sd-character-level-ner huggingface dataset:
Figure 8(A,B) AMG386 (5.6mg/kg s.c.; twice/week) and SHP099 (100mg/kg orally/daily) were administered individually or together to MC38 tumor-bearing mice (control n=10; SHP099: n=15; AMG386: n=10; SHP099+AMG386 n=15). Tumor growth from initiation of treatment to endpoint (left) and tumor weight (right) at endpoint; error bars ± S.D.; P values by analysis of variance with Dunnett's multiple comparison test; *P<0.05, **P<0.01, ***P<0.001 (A). Representative control and treated (n=4/group) MC38 tumor sections through the maximum diameter (B); H&E staining; scale bar: 10mm. (C) Vessel perfusion visualized by FITC-dextran (green); CD31 identifies endothelial cells (red); DAPI (blue) identifies the nuclei; representative (of 3 mice/group) confocal images (scale bar: 50μm); arrow heads point to FITC-dextran extravasation. (D-G) Quantification of "vascular sleeves" (CD31-Collagen IV+); n=4 tumors/group (D); cleaved caspase-3+/CD31+ cells; n=4 tumors/group; at least 1000 CD31+ cells counted/sample (E); tumor vascular area; n=7 tumors/group (F); Ki67+ tumor cells; n=4 tumors/group; at least 1000 CD31+ cells counted/sample (G); error bars: ± S.D. P values by analysis of variance with Dunnett's multiple comparison test; *P<0.05, **P<0.01, ***P<0.001. (H) Representative images depicting Ki67+ cells in control and treated MC38 tumors; representative images (scale bar: 50μm) of 4 tumors/group. (I) Quantification of necrotic tumor area in control and treated mice; 4 tumors/group; error bars: ± S.D. P values by analysis of variance with Dunnett's multiple comparison test; *P<0.05, ***P<0.001. (J-L) Quantification of p-STAT3, p-ERK and FOXO1 in tumor CD31+ endothelial cells in control and treated mice; (n=3 tumors/group); CD31+ cells counted in J: control (n=3,842), SHP099 (n=21,212), AMG386 (n=12,786), combined SHP099+AMG386 (n=11,746). CD31+ cells counted in K: control (n=12,125), SHP099 (n=8,033), AMG386 (n=3,916), combined SHP099+AMG386 (n=4,230). CD31+ cells counted in L: control (n=16,076), SHP099 (n=6,555), AMG386 (n=13,715), combined SHP099+AMG386 (n=7,260). P values by analysis of variance with Dunnett's multiple comparison test; *P<0.05, **P<0.01, ***P<0.001. (M) Schematic representation of the effects of AMG386 and SHP099 on signaling pathways active in the tumor vasculature.
Figure 1A The gene and protein structures of the Dof domain of CDF4. Cysteine residues conserved in the DOF domain are indicated in red, and the conserved DOF Zn-figure DNA-binding domain is found in CDF4. B Trans-activation activity assays of CDF4 in Arabidopsis protoplasts. The full-length ORF of CDF4 and its four deletion constructs Dof (gray boxes) represent the DNA-binding domains of CDF4. Negative control was the effector vector without gene inserts. The effector vector used is shown in the left panel. The GAL4 transient expression assays were performed using Arabidopsis protoplasts, shown in the right panel. Deletion of the CDF4 N-terminal domain compromised the transcriptional repression by CDF4 in our assay. Three independent experiments were conducted. Values are given as mean ± SD, n=3. *p<0.05 by Student's t test. C Localized expression of CDF4 in a senescing rosette leaf. The ~50% senesced rosette leaf was cut into three parts as indicated by the red lines. The level of CDF4 mRNA was determined in the three sections of the senescing leaf. Base; Middle; Tip. Three independent experiments were conducted. Values are given as mean ± SD, n=3. D CDF4 expression level at various developmental stages of rosette leaves. YL, young leaf; NS, fully expanded, non-senescent leaf; ES, early-senescent leaf, with 1/4 leaf area turned yellow; LS, late-senescent leaf, with >50% leaf area turned yellow. Three independent experiments were conducted. Values are given as mean ± SD, n=3. E Effect of plant growth hormones (1 mM SA, 1 μM ABA, 10 μM JA and 10 μM 1-aminocyclopropane -1-carboxylic acid (ACC) on CDF4 expression. Three independent experiments were conducted. Values are given as mean ± SD, n=3. F Effects of abiotic stress treatments on CDF4 expression. Rosette leaves were treated with salt (100 mM NaCl), drought, H2O2 (2mM) or darkness. Three independent experiments were conducted. Values are given as mean ± SD, n=3. G Effects of SA, H2O2, and dark treatment on CDF4 expression in the aba2-1 mutant background. AtACT2 was used as an internal control. Three independent experiments were conducted. Values are given as mean ± SD, n=3.
Figure 4A Scheme of the fluorescence-based unwinding assay to monitor UPF1 translocation in real time An RNA substrate harboring a high-affinity PTBP1 binding site is incubated with highly purified UPF1ΔCH in the absence and presence of equal molar amounts of highly purified PTBP1. Upon the addition of ATP, UPF1 translocation results in a decrease in fluorescence due to displacement of a labeled, duplexed oligonucleotide and subsequent quenching by a trap strand. B UPF1SLΔCH translocation along an RNA substrate harboring a high-affinity PTBP1 binding site in the absence and presence of PTBP1. Time to 50% unwound and relative total % unwound at end-of-assay (1200 sec) are indicated. Results of four technical replicates are shown for each dataset and represent at least three independent experiments. Shaded region indicates SD. C Results as in (B) but with UPF1LLΔCH.
Figure 4A Hormone-inducible nuclear import assay, representative still images of GCR2-EGFP2-TDP-43 Wt, 12D and 12A before and during import triggered by addition of dexamethasone. Images were live recorded by spinning disc confocal microscopy. Bar, 20 µm. B Quantification of the hormone-inducible nuclear import measured during a total time course of 50 min. Values represent the mean fluorescence intensity of GCR2-EGFP2-TDP-43 in the cytoplasm for three independent replicates ± SEM (≥ 42 cells per condition). C Phosphomimetic 12D TDP-43 is competent in autoregulating TDP-43 expression. SDS-PAGE followed by TDP-43 Western blot showing downregulation of endogenous TDP-43 through autoregulation (60) after 48 h expression of Wt, 12D and 12A variants in HeLa cells. TDP-43 was detected using rabbit anti-TDP-43 C-term antibody (Proteintech), Histone H3 (rabbit anti-Histone H3 antibody, Abcam) was visualized as a loading control. * denotes an unspecific band. D Electrophoretic mobility shift assays (EMSA) of TDP-43-MBP-His6 variants (Wt, 12D and 12A) in a complex with TDP-43 autoregulatory RNA binding site (60). All TDP-43 variants form TDP-43-RNA complexes equally well. E Splicing analysis by RT-PCR of known TDP-43 splice targets (SKAR exon 3 and Bim exon 3) in HeLa cells. Silencing of endogenous TDP-43 by siRNA leads to altered splice isoforms of SKAR and Bim (second vs. first lane). These splicing alterations can be rescued by re-expression of TDP-43 Wt, but also 12D or 12A variants, demonstrating that phosphomimetic TDP-43 is fully competent in regulation splicing of these TDP-43 splice targets.
a) Representative examples for ChIP-sequencing traces obtained from NPCs using an α-Miz1 antibody.f3(a) Representative examples for ChIP-sequencing traces obtained from NPCs using an α-Miz1 antibody. Input traces are shown as negative control. For each gene, the intron/exon structure is shown below the traces with exons indicated as vertical bars and the transcriptional start site (TSS) at the left. An enlarged picture of the peak region is shown on the right. (b) Miz1 binds predominantly to core promoters. The panel shows a histogram of the percentage of promoters with the indicated distances between the peak of the Miz1-binding site and the next TSS. Of all the peaks, 140/261 (54%) are located in core promoters (±1.5 kb). (c) Miz1 binding is conserved across different cell types and species. The plot shows a Venn diagram documenting the overlap in Miz1-bound promoters between murine NPCs and human MDA-MB231 cells (a mammary epithelial tumour cell line). The P-value was calculated using a hypergeometric test. Note that different α-Miz1 antibodies (H190 and G18) were used for the analysis of mouse and human Miz1-binding sites. (d) Miz1-binding sites contain a conserved sequence element. Sequences (±50 bp) around all 261 Miz1-binding sites observed in NPCs were analysed by MEME-ChIP ('Motif Analysis of Large DNA Data sets'). (e) Electrophoretic mobility shift assay documenting binding of Miz1 to the conserved sequence element ('Miz1-binding motif'). Nuclear extracts from control HeLa cells ('Mock') or cells ectopically expressing Miz1 were incubated with radioactive oligonucleotides spanning the Miz1-binding motif of the Ambra1 and Vps28 promoters. Where indicated, binding reactions included a 25-fold excess of non-labelled oligonucleotides ('Competitor'), the indicated mutants thereof or of an oligonucleotide containing an E-box sequence from the Ubap2 promoter. Supershift assays were performed using α-Miz1 (H190, G18 and C19), α-Myc (N262) or control antibodies. *Non-specific band. The sequences of the oligonucleotides used are shown above the panel; mutated nucleotides are shown in bold.
Figure 3(A) Representative images from ARPE-19 cells treated with DMSO and NSC668394. All cells were fixed and stained with anti-LC3 (green) and anti-LAMP1 (red) antibodies and nuclei were counterstained with DAPI (blue). Scale bar 5 μm. (B) The plot shows the quantification of the numbers of LC3/LAMP1-positive vesicles per cell (n ≥ 100). Values represent means ± s.e.m from 3 independent experiments. Student's t-test (NSC668394 vs DMSO) ***p ≤ 0.005. (C) Representative Western blots of LAMP1, EZRIN, EZRIN-pT567, CLN5, TRPML1, SQSTM1/p62, CTSD and LC3 proteins from DMSO or NSC668394 treated cells. Note an increase of both proCTSD and its maturation CTSD heavy chain (hc). The plot shows the quantification of the indicated proteins normalized to the GAPDH loading control. Bar graphs represent mean values ± s.e.m. of at least n = 6 independent experiments. Mann and Whitney test (NSC668394 vs DMSO) *p ≤ 0.05, ***p ≤ 0.005. (D) Representative images of RFP-GFP-LC3 assay in ARPE-19 cells transiently transfected with RFP-GFP-LC3 and treated with DMSO or NSC668394. Nuclei were counterstained with DAPI (blue). Scale bars: 5 μm. (E) Box plots showing quantitative analysis of RFP+GFP+ puncta (Autophagosome) and RFP+GFP- puncta (Autolysosome ) (n ≥ 100 cells) from 3 independent experiments. Box limits represent 25th percentile and 75th percentile; horizontal lines represent medians; whiskers display min. to max. values. Student's t-test (NSC668394 vs DMSO) *p ≤ 0.05. (F) Endolysosomal Cathepsin activity was revealed by incubation with cathepsin B (CTSB) Magic Red substrate for 20 min and images of the living cell collected on the HC analysis. Scale bar 10 μm. (G) Graph showing the mean values of intensity of cresyl violet fluorescence ± s.e.m. of representative data (n ≥ 100 cells) from 3 independent experiments. Mann and Whitney test (NSC668394 vs DMSO) *p ≤ 0.05. (H) Cathepsin B (CTSB) activity in lysates from NSC668394-treated and DMSO-control ARPE-19 cells. Graph showing the percentage of values ± s.e.m. of CTSB activity from 4 independent experiments. Mann and Whitney test (NSC668394 vs DMSO) *p ≤ 0.05.
Figure 3(A) The developmental phenotype of the arid2/3/4 mutant as compared to the wild type. Two-week-old seedlings grown on MS medium are shown. (B) The developmental phenotype of the pwwp1/2/3+/- mutant as compared to the wild type. One-month-old plants grown in soil are shown. The effect of the arid2, arid3, and arid4 mutations on the silencing of solo LTR, SDC, and FWA as determined by qPCR. Showing are results of three biological replicates with standard deviations. The effect of the pwwp1, pwwp2, and pwwp3 mutations on the silencing of solo LTR, SDC, and FWA as determined by qPCR. Showing are results of three biological replicates with standard deviations. (E) The effect of the trb1 mutation and the TRB2 knockdown (TRB2-KD) on the silencing of solo LTR, as determined by qPCR (right panel). The transcript level of TRB2 was evaluated to confirm the TRB2 knockdown (left panel). Error bars represent standard deviations of three biological replicates.
a, Decreased phosphorylation of mammalian TOR substrates in U87 cells treated with octyl &agr-KG or oligomycin.f4a, Decreased phosphorylation of mammalian TOR substrates in U87 cells treated with octyl alpha-KG or oligomycin. Similar results were obtained in HEK-293 cells, normal human fibroblasts and mouse embryonic fibroblasts (data not shown). P, phospho. b, Increased autophagy in animals treated with alpha-KG or RNAi for atp-2 or let-363. Photographs were taken at ×100 magnification. c, GFP::LGG-1 puncta quantified using ImageJ (Methods). Data show results of 2-3 independent experiments. Bars indicate the mean. ****P  0.0001; NS, not significant (t-test, two-tailed, two-sample unequal variance). d, alpha-KG levels are increased in starved worms. **P  0.01 (t-test, two-tailed, two-sample unequal variance). Mean ± s.d. is plotted. e, Model of α-KG-mediated longevity.
Figure 4(A) Lung ECs were isolated from EC- miR-483 transgenic rats (Tg) and their wild-type littermates (WT), n = 3 in each group. MiR-483-3p/-5p levels were measured by qPCR. Data information: Values are expressed as mean ± SEM. (B) Serum from WT and Tg rats (n = 6) were collected. MiR-483-3p and -5p associated with CD144 were enriched by immunoprecipitation with anti-CD144. Levels of miR-483-3p/-5p were measured by qPCR. Data information: Values are expressed as mean ± SEM. Lung ECs isolated from miR-483-Tg rat and WT littermates were exposed to normoxia or hypoxia (0.2% O2) for 24 hr. Proliferation of ECs were measured by flow cytometry Data information: Values are expressed as mean ± SEM. Data are representative of three independent experiments Statistical test: t-test (*P < 0.05 vs. respective controls or between the indicated groups Lung ECs isolated from miR-483-Tg rat and WT littermates were exposed to normoxia or hypoxia (0.2% O2) for 24 hr. migration of ECs were measured by wound healing assay Data information: Values are expressed as mean ± SEM. Data are representative of three independent experiments Statistical test: t-test (*P < 0.05 vs. respective controls or between the indicated groups Tg and WT rats were injected with saline or MCT. Expression levels of TGF-β, TGFBR2, β-catenin, CTGF, IL-1β, and ET-1 mRNA and protein in lung tissues were measured by qPCR and Western blot, respectively (E). Data information: Values are expressed as mean ± SEM. Tg and WT rats were injected with saline or MCT. Levels of miR-483-3p/-5p in serum and lung tissues were measured by qPCR (F). Data information: Values are expressed as mean ± SEM. Tg and WT rats were injected with saline or MCT. Ago1- or Ago2-associated miRNAs (G) were enriched from the isolated lung tissues by immunoprecipitation with anti-Ago1 or anti-Ago2 and quantified by qPCR (n = 3×3, samples were pooled from three animals for each assay, and 3 independent experiments [a total of 9 animals] were performed). Data information: Values are expressed as mean ± SEM. Tg and WT rats were injected with saline or MCT. Ago1- or Ago2-associated mRNAs (H) were enriched from the isolated lung tissues by immunoprecipitation with anti-Ago1 or anti-Ago2 and quantified by qPCR (n = 3×3, samples were pooled from three animals for each assay, and 3 independent experiments [a total of 9 animals] were performed). Data information: Values are expressed as mean ± SEM.
Figure 9A. An example of the time-course experiments for the osmostress-induced Hog1 phosphorylation. The yeast strain KY603-3 (ΔS/O/H/M ssk2/22Δ STE11-Q301P) was stimulated with 1.0 M NaCl for the indicated times, and the percentage of phosphorylated Hog1 (Hog1-P) was determined using a Phos-tag band-shift assay. Compilations of the time-courses of osmostress-induced Hog1 activation in (B) KY603-3 (ΔS/O/H/M ssk2/22Δ STE11-Q301P), For clarity, time-course curves are shown in two panels for lower and higher ranges of NaCl concentrations. Data information: For each data point, n=1 or more. Compilations of the time-courses of osmostress-induced Hog1 activation in (C) TM257 (ssk2/22Δ), and (D) TM142 (wild-type). For clarity, time-course curves are shown in two panels for lower and higher ranges of NaCl concentrations. The color chart below (D) indicates the concentrations of NaCl used. Data information: For each data point, n=1 or more. E. Effects of Hog1 kinase activity and osmostress-induced glycerol accumulation on the time-courses of Hog1 activation. Cells were stimulated with 0.4 M NaCl (left panel) or 1.6 M NaCl (right panel) for the indicated times, and the percentage of phosphorylated Hog1 was determined using Phos-tag band-shift assay. Strains used were: TM142 (WT), KT254 (gpd1Δ gpd2Δ stl1Δ), and TM232 (hog1Δ) carrying pRS416-HOG1-K52S K53N. K/S K/N, K52S K53N; gpd1/2, gpd1Δ gpd2Δ. Data information: (E) Error bars are SEM (n=3 or more). F. A model of the negative and positive feedback regulations in the HOG pathway. The upstream osmosensors are Sln1 and the complexes composed of Sho1, Opy2, Hkr1, and Msb2. The molecular identity of the downstream osmosensor, which enhances the signaling between Pbs2 and Hog1, is currently unknown.
Figure 7A, B qPCR analysis of Tnf mRNA (A) and Il6 mRNA (B) in splenic cells from C57BL/6 mice infected with WT or mutant Mtb stains for 0-20 days. Data are representative of one experiment with two independent biological replicates (mean ± SEM of n = 6 mice per group). *P < 0.05 and **P < 0.01. Data were analyzed according to one-way ANOVA using GraphPad Prism 7. C Bacterial load in lungs from C57BL/6 mice treated as in A. Data are representative of one experiment with two independent biological replicates (mean ± SEM of n = 6 mice per group). *P < 0.05 and **P < 0.01. Data were analyzed according to one-way ANOVA using GraphPad Prism 7. D H&E staining of lungs and livers of C57BL/6 mice treated as in A. Arrows indicate puncta of cellular infiltration. Scale bars, 200 μm. E Proposed model depicting Mtb PknG-mediated host innate immune suppression. During mycobacterial infection, PknG binds to ATP to catalyze a unique two-step ubiquitination cascade through unconventional E1 and E3 enzyme activities with the release of ADP instead of AMP, ultimately leading to degradation of TRAF2 and TAK1 and suppression of host innate immune responses.
Figure 2A. H&E staining of mouse organ sections. Scale bars: spleen, colon 100 μM, bone marrow - 50 μM. B. Incidence of pathological findings (number of mice with pathology/total number of mice). C. H&E staining of liver sections. Yellow arrow - a mitotic hepatocyte; blue arrows - lymphocyte infiltration. Scale bars - 100 μM.
Figure 9A Summary of molecular interconnections underlying cortical wave self-organization. B Diagram of different profiles of cortical patterns and corresponding cell morphology. C Upper right illustrates a simplified case where the wave propagates along cell cortex without inducing membrane deformation such as waves at the basal surface of an adherent cell. Individual actin filaments do not dislocate laterally, only elongate perpendicularly to the cell membrane generating retrograde flow (green arrow). STEN activity propagates along the cortex as waves (blue arrows), and leads to CEN activation promoting new actin polymerization (bright green). The successive polymerization events among filaments thus spread along the cortex in the form of waves (also blue arrows). After STEN moves away, actin polymerization ceases, and existing filaments gradually depolymerize. Lower panels show that waves with different properties result in different types of protrusions.
Figure 4(A) Scheme of the domain architecture of WAVE1 variants immobilized to lipid-coated beads. Nucleation is always driven from wild-type WAVE PWCA (60% total), while other variants (40% total) do not activate the Arp2/3 complex (Figure EV 4A,B) but can affect filament elongation (B) Widefield epifluorescence images (Alexa488-actin) of bead-attached dendritic networks following kinetic arrest after 10 min of network growth for spike-in experiments (i.e. 60% his10-darkCherry-PWCA, 40% his10-Cherry-W; molar percentage) using indicated WAVE1 variants. Conditions are 5μM profilin-actin, 200nM CP and 50nM Arp2/3. For all conditions, actin network assembly was quenched by adding 5M excess Latrunculin B and phalloidin 10 minutes after initiating the reaction. (C) Average network growth rates for indicated WAVE1 variants or mCherry as a negative control (N= 57-73 beads per condition). Errors bars represent SD. (D) Scheme of domain architecture of surface-immobilized WAVE1 variants (top panel), time averages (middle panels, 7 min total) and representative kymographs (lower panel) from multi-color TIRF imaging of polymerization from profilin-actin on WAVE1-coated coverslips. Conditions are 2µM actin, 3µM profilin, 5nM Alexa488-UTRNN. (E) Average barbed end polymerization rates for indicated WAVE1 variants. Errors bars represent SD.
PC2_L677A/N681A (AA) mutant is a new GOF channel.Figure 2A. Cryo-EM structure of PC2. Left: bottom view of PC2 homomeric tetramer showing the pore-lining S5-S6 from each subunit assemble into the channel pore. Right: side view of S5-S6 from two subunits showing the L677 and N681 (in red) contribute to the restriction at the lower gate. F604P (in cyan) on S5 and the three residues (in green) forming the selectivity filter are also indicated. The structure shown here is previously reported with Protein Data Bank (PDB) code 5T4D [20]. A grey dashed line indicates the path of the ion flow. B. Representative I-V curves (left) and a scatter plot and bar graph (right) showing that the PC2_AA is a GOF mutant and gave rise to a larger current than PC2_F604P. Scatter plot and bar graph shows the average current sizes at +60 mV. The cations included in the bath solution, 100 mM Na+ and 2 mM Ca2+ in this case, are indicated by the thick-lined boxes here and in all the following figures. Oocyte numbers for bar graph are indicated in parentheses. Data are presented as mean ± SD in bar graph (***P < 0.001, Student's t-test). C. Representative currents of PC2_F604P and PC2_AA mutants in the divalent ion-free bath solution, which contains 100 mM Na+. D. Representative currents of indicated WT and mutants of PC2 in a bath solution containing 70 mM Ca2+. E. Calcium-activated chloride channel (CaCC) blocker MONNA (10 μM) or niflumic acid (NFA) (1 mM) partially blocked the current recorded from the PC2_AA-injected oocytes in the 70 mM Ca2+ bath solution.
Manipulation of ERK activation dynamics rewires cell fate.Figure 7ARepresentative micrographs of tubulin‐stained PC‐12 NS‐1 subclone cells stimulated with sustained and multi‐pulse GF regimes. Scale bar = 20 μm.B, CQuantification of neuronal differentiation fate. (B) Total neurite outgrowth per cell normalized to the major axis length of cell soma for sustained and multi‐pulse GF regimes. (C) Differentiation ratio calculated as the fraction of cells with normalized total neurite outgrowth per cell larger than 1. At least 5,000 cells quantified in at least 2 independent experiments for each condition. Boxplots with median, interquartile (Box), and 1.5 IQR (Whiskers) are shown.