EMBO/sd-character-level-ner huggingface dataset:
Functional characterization of SlHAK20.Figure 3A Phylogenetic tree of the HAK/KUP/KT transporter family clade IV. The phylogenetic tree was constructed based on the amino acid sequences using the neighbor-joining method with MEGA5 in dicots and monocots. The expression pattern of SlHAK20 in tomato. The GUS activity was examined in seedling (left) and root (right) of 21-days-old SlHAK20TS-21pro:GUS transgenic tomato (B). Bars, 2 cm. The expression pattern of SlHAK20 in tomato. Cross-sections of hypocotyl (C) of SlHAK20TS-21pro:GUS transgenic plants. The scale bar is 400 μm in (C) The expression pattern of SlHAK20 in tomato. Cross-sections of root he scale bar is 200 μm in (D). E Subcellular localization of SlHAK20-YFP in tomato. The SlHAK20Hap1-YFP (left) and SlHAK20Hap2-YFP (right) were localized in the plasma membrane. YFP, yellow florescence protein. FM4-64, a lipophilic styryl compound as a red fluorescent marker of plasma membrane. Bar, 20 μm. F SlHAK20 complements the K+ uptake-defective yeast mutant assayed using a dose-response method. The K+ uptake-defective yeast mutant R5421 transformed with SlHAK20Hap1, SlHAK20Hap2, and vector (p416-GPD) were cultured in AP medium containing indicated concentrations of K+ for 24 h. The data points represent three replicates. G Na+ uptake analysis of yeast cells expressing SlHAK20. Shown are the Na+ concentrations in the medium over the time of culture of the ANT3 yeast cells transformed with SlHAK20Hap1, SlHAK20Hap2, or empty vector. The initial NaCl concentration in the medium was 60 μM. The data are shown as the means ± SD (n = 4). H Na+ uptake kinetic analysis of SlHAK20 in yeast cells. The curves show concentration- dependent Na+ inﬂux in yeast expressing SlHAK20Hap1 or SlHAK20Hap2 in the presence of 0, 5, 10, 20, 30, 40, 50, 60, 100, 150 or 200 μM NaCl in the medium. The data are expressed as mean ± SD (n = 3).
Figure 4Cell cycle analysis by flow cytometry of BT474 and BTRH cells (50,000 events) treated with EV20/MMAF (10 nM) for the indicated times. BT474 and BTRH cells were treated for the indicated days with EV20/MMAF (10 nM), lysed and the amount of different proteins analyzed by Western. Numbers below the blots show the quantitation of the signal of each band, referred to time 0. Action of EV20/MMAF on mitotic spindle formation. BT474 and BTRH cells were treated (10 nM, 48 hours), fixed, stained for β-tubulin (green) and DAPI (blue). Scale bar: 7.5 μm. Quantitative analysis of mitotic cells with abnormal spindles treated or not with EV20/MMAF (10 nM, 48 hours) in BT474 and BTRH cells. Bars represent the mean + SD of two independent experiments, calculated as follows: (number of mitotic cells with abnormal spindles/total number of mitotic cells) ×100 (%). Cell death induction by EV20/MMAF (10 nM) analyzed by double annexin V and PI staining in BT474 and BTRH cells (50,000 events). Time-course analysis of the effect of EV20/MMAF (10 nM) on cleaved-caspase-3, analyzed by Western blotting in BT474 and BTRH cells. GAPDH was used as a loading control. Samples with intensity values of 1 were used as reference. Fluorimetric analyses of caspase-3 activity in BT474 and BTRH cells treated with 10 nM EV20/MMAF. Data show the mean + SD of triplicates of an experiment that was repeated twice.
Replication fork progression is unsteady after Chk1 loss.Figure 1Labelling scheme and representative DNA fibers from U2OS cells transfected with siLuc or siChk1. Western Blot of Chk1 in U2OS cells, 48 h after transfection with siRNA. Actin was used as loading control. Control (siLuc) and Chk1-depleted (siChk1) U20S cells were labeled with CldU and IdU for 10 and 30 min, respectively. Mean (+SEM) track lengths (top) and CldU/IdU ratios (bottom) are shown. Data come from 6 independent experiments and a total of 579 (siLuc) and 590 (siChk1) fibers were scored. Western Blot of phospho-serine-296-Chk1 (p-Chk1) in U2OS cells. Cells were treated 1.5 h with the Chk1 inhibitor Gö6976 (Chk1i) or DMSO, irradiated with 100J/m2 UV to induce high levels of p-Chk1, and collected 1.5 h afterwards. Actin was used as a loading control. U2OS cells treated with DMSO (control) or Chk1i were labeled with CldU and IdU for 10 and 30 min, respectively. Data come from 3 independent experiments and a total of 304 (DMSO) and 312 (Chk1i) fibers were scored.
Figure 4A) Density maps identifying iDHSs and showing DNase-Seq and H3K4me2 ChIP-Seq peaks in order of increasing DNase-Seq tag count signal for CD4 TB+ compared to TB cells. Also depicted are the locations of 6823 major DHSs that are 5.5 fold induced and a subset 1217 these iDHSs that are 11 fold induced. On the right is the log2 TB+/TB fold change in expression of the closest gene to the corresponding DHS. B) Average DNase I profiles of the 1217 iDHSs in TN (+/- PMA/I) and TB (+/-PMA/I) (left), and in TM (+/-PMA/I) (right). C) Average DNase I and H3K27ac profiles of the 1049 dDHSs in TN and TB (+/-PMA/I) which are 4 fold diminished after stimulation. D) The genomic distribution of the 1217 iDHSs. E) Barplot showing the number of TM-specific genes with an iDHS within 150 kb of the TSS. F) Barplot showing the median distance between the TSS and the closest iDHS of the TM specific genes grouped according to the fold induction in TM after 2 hours plus PMA/I compared to TM. Genes which had a TSS greater than 1 Mb from an iDHS were excluded. G) Barplot showing the number of TM specific genes which have an iDHS within 150 kb of the 683 pDHSs. H) Median distances from the closest pDHS to the closest iDHS grouped according to the fold induction in TM after 2 hours plus PMA/I compared to TM. I) UCSC genome browser shot of the Th2 Il4/Il13/Rad50 locus showing DNaseI-Seq and ChIP-Seq. Black and red arrows represent pDHSs and iDHSs respectively. The values above the arrows indicate the distance in kb from the Il4 promoter.
∆SxIP allele reveals impaired kinetochore localization of Dam1c and a delay in cell cycle progression A: Serial dilution assay of yeast strains with different Duo1-6xFlag alleles in a wild type or Dad1-GFP strain background.Figure 3A: Serial dilution assay of yeast strains with different Duo1-6xFlag alleles in a wild type or Dad1-GFP strain background. Growth phenotypes of the respective yeast strains were analyzed by spotting serially diluted amounts of cells on YEPD plates and incubation at different temperatures. B: FACS analysis of the DNA content of strains with Duo1WT-6xFlag or Duo1∆SxIP-6xFlag alleles with untagged Dad1. Samples were taken from log phase cultures grown at 30 °C. Cells with a 1C DNA content are in G1 phase, a 2C DNA content is characteristic of mitotic cells. Cells with an intermediate DNA content are in S phase. C: Representative images from fluorescence live cell microscopy of Dad1-GFP strains with Duo1WT-6xFlag or Duo1∆SxIP-6xFlag. Displayed cells are in either metaphase (left) or anaphase (right). Scale bar: 2 μm. D: Quantification of signal intensities of Dad1-GFP at metaphase and anaphase kinetochore clusters of Duo1WT-6xFlag and Duo1∆SxIP-6xFlag strains. Bars represent the mean +/- standard deviation. Each spot represents the value measured for a single kinetochore cluster. Values were normalized to the mean value of metaphase clusters of the Duo1WT-6xFlag strain. A one-way ANOVA test with Sidak's multiple comparisons test was used to calculate p values. n ≥ 76 kinetochore clusters. E: Quantification of extra-large (XL) budded cells with short inter-kinetochore distance. Only cells with buds that were grown to more than half of the mother cell's size were counted. Mean +/- standard deviation of three independent experiments are displayed. p-value was calculated by an unpaired t-test. F: Analysis of cell cycle progression of yeast strains with different Duo1 alleles. Accumulation and degradation of Pds1 after release from α factor arrest was analyzed by western blot (left). An antibody against Pgk1 was used to confirm equal protein amounts in samples. The DNA content of the cell population at indicated time points after release from α factor arrest was analyzed by FACS (right). Cells in G1 phase and mitosis are characterized by a 1C and 2C DNA content, respectively. An intermediate DNA content is detected for cells in S phase. G: Pds1 signal intensities from western blots shown in F were quantified and corrected for Pgk1 signal of the corresponding sample. Finally, the highest signal intensity (45 minutes after release) was defined as 1.00 and values for other time points were normalized accordingly.
Figure 2A. Immunoblot analysis of chromatin fractions of U2OS cells transfected with indicated siRNAs and treated or not with HU. B. U2OS cells treated with HU in the presence or absence of ATR inhibitor (ATRi) were fractionated and immunoblotted with indicated antibodies. Relative RADX levels on chromatin were quantified and normalized to histone H3. See also Fig EV2A. C. Representative images of U2OS cells stably expressing indicated GFP-RADX alleles that were subjected to laser microirradiation, fixed with methanol/acetone immediately afterwards and immunostained with γH2AX antibody. D. As in (C), except cells were fixed with paraformaldehyde 20 min after DNA damage infliction and immunostained with PCNA antibody. E. Representative images from PLAs with GFP and RPA2 antibodies in U2OS and U2OS/GFP-RADX cell lines labeled with EdU for 30 min and then fixed or exposed to HU or CPT for 4 h before fixation. F. Quantification of data in (E) by QIBC (n≥3000 cells per condition; data from a representative experiment are shown). See also Fig EV2D. All scale bars, 10 μm.
Figure 7A) Citrate synthase activity in spinal cord homogenates at disease end stage. Results are expressed as mean ± SEM; n= 8 (4 males and 4 females) mice per group; *p= 0.024, by paired Student's t test. Activities are expressed as nmoles of substrate oxidized per minute per mg of protein. B) Cytochrome oxidase (COX) activity at end stage. Results are expressed as mean ± SEM; n = 8 (4 males and 4 females) mice per group; *p= 0.032, by paired Student's t test. Activities are expressed as nmoles of substrate oxidized per minute per mg of protein. C) qPCR (fold change) of PGC1α mRNA normalized by β-actin mRNA at 130 days of age. Results are expressed as mean ± SEM and as fold change of the Non Tg; n= 8 (4 males and 4 females). No statistically significant differences were found between G93A and PKO/G93A (p= 0.250 by paired Wilcoxon's test); *p= 0.035 paired Friedman's test with Dunn's correction (Non Tg vs. G93A). No other statistically significant differences were found. D) qPCR (fold change) of PGC1α mRNA normalized by β-actin mRNA at end stage. Results are expressed as mean ± SEM and as fold change of the Non Tg; n= 8 (4 males and 4 females). No statistically significant differences were found between G93A and PKO/G93A (p= 0.613 by paired Student's t test). **p= 0.0058 by paired Friedman's test with Dunn's correction (for Non Tg vs. G93A comparison) and ***p<0.0001 by paired one-way ANOVA with Tukey's correction (for PKO vs. PKO/G93A comparison). Parkin knockout improves PGC1α expression at 130 days (C) but the effect is lost at end stage (D). E E, F) Representative Western blots (E) and quantification (F) of PARIS at disease end stage. Protein levels are normalized by β-actin and results are expressed as mean ± SEM and as percent of Non Tg; n= 8 (4 males and 4 females). No statistically significant differences were found between G93A and PKO/G93A by paired Wilcoxon's test (p= 0.99). PARIS levels are unchanged in SOD1-G93A mice relative to Non Tg at disease end stage.
Figure 1(A) C57BL/6 mice were inoculated with 6x105 B16Bl6 parental or dnTNF-R1 cells on day 0 and treated daily with 7µg mTNF p.l. from day 10. Tumor growth is shown as mean tumor size index (TSI) + SEM (n = 6). The line under the graph represents the treatment period. (B) B16Bl6 tumor growth after daily p.l. treatment with 7µg mTNF in C57BL/6J wild-type (WT), TNF-R1-/-, conditional TNF-R1 reactivation knockout (p55cneo/cneo), or p55cneo/cneo mice with Cre expression in endothelium (Flk1Cre) or all cells (DelCre). Tumor growth is shown as mean TSI + SEM (n = 7 for WT and Flk1Cre, 8 for DelCre, 9 for TNF-R1-/- and 10 for p55cneo/cneo). The line under the graph represents the treatment period. (C) PCR analysis for the detection of wild-type, conditional knockout (cneo) or reactivated (lox) TNF-R1 allele in whole tissues, lung endothelial cells (LECs) and lung single-cell suspension depleted of CD31+ cells. (D) Toxicity of a single i.v. bolus injection of the indicated dose of mTNF. Mean rectal body temperature + SEM and cumulative survival rates are shown (n = 4 for Flk1Cre 10µg; 6 for DelCre 10µg, Flk1Cre 10µg and p55cneo/cneo 15µg; 7 for WT 10µg; 9 for WT 8µg, 10 for DelCre 20µg; and 5 for all other groups). For continuity of the temperature graphs, dead mice were included with a temperature of 20°C.
Mice were analysed 2 days after TAC (a–c).f2Mice were analysed 2 days after TAC (a-c). a, Haematoxylin and eosin-stained heart sections. Scale bar, 100 µm. b, AZAN-Mallory-stained sections. Scale bar, 100 µm. Mice were analysed 2 days after TAC (a-c). c, Immunohistochemical analysis using antibodies to CD45, CD68, Ly6G and CD3. Scale bar, 100 µm.
Figure 8A. Yeast strains (200 cells) containing chromosomal Flag-tagged rbd2, rbd2-G244R, rbd2-G246R or rbd2-S130A in rbd2Δ background and sre1Δ yeast were grown on rich medium in the absence or presence of cobalt chloride (CoCl2). B. Western blot of phosphatase-treated, whole cell lysates from strains in (A) grown for 3 hr in the presence or absence of oxygen was probed with anti-Sre1 or anti-Flag IgG. P and N denote Sre1 precursor and cleaved nuclear forms, respectively. C. Sre2 cleavage was assayed by western blot using anti-Sre2 serum or anti-Flag IgG of phosphatase-treated, whole cell lysates from the indicated strains. N denotes Sre2 cleaved nuclear form. D. Whole cell lysates from indicated yeast strains carrying a plasmid expressing 3xFlag-Sre2MS were analyzed by western blot with anti-Flag antibody. Two independent isolates, A and B, are shown for each strain. Asterisk denotes non-specific band. E. Diagram of four Rbd2 chimeras fused to the SHP-box from S. pombe Der1-like protein (SPBC365.08c). The SHP-boxes of Rbd2 and Der1-like protein are shaded red and green, respectively. Asterisks denote point mutation in the SHP-box. F. Yeast strains containing Flag-tagged rbd2-SHP, rbd2-SHP*, rbd2-G246R-SHP, and rbd2-G246R-SHP* were generated in rbd2Δ background by chromosomal integration of the rbd2 fusion proteins depicted in (E). Indicated yeast strains were then assayed for Sre1 cleavage as in (B). G. Indicated strains were assayed for Sre2 cleavage as in (C). H. Wild-type, sre2Δ or strains containing chromosomal Flag-tagged rbd2, rbd2-G246R or rbd2-S130S in rbd2Δ background (lane 7-12) were treated with bortezomib (Bz) for 2 hr, and phosphatase-treated, whole cell lysates were analyzed by western blot with anti-Sre2 serum or anti-Flag IgG.
Increased VEGF-A in the RPE causes neovascular AMD-like pathologies by inducing RPE barrier breakdown through activation of Flk1-signaling in the RPE.Figure 6RPE-specific overexpression of VEGF-A in VMD2Cre+/WTROSA-STOPfl/fl-VEGF-A164mice (VEGF-ARPEmice) results in CNV at sites of VEGF-A overexpression (Cre+ patches (white nuclei); red arrow), while Cre- areas with normal VEGF-A expression show no CNV (white arrow). Neovessels are CD31+ (red). VEGF-A-induced CNV lesions and RPE barrier breakdown in VMD2Cre+/WT ROSA-STOPfl/fl-VEGF-A164mice resemble those seen in VEGF-Ahypermice (B.-C., arrows; phalloidin staining in green). Scale bars 100 μm. D. Genetic inactivation of Flk1 specifically in the RPE in VEGF-Ahypermice (VMD2Cre+/WTFlk1fl/flVEGF-Ahypermice) prevents RPE barrier breakdown and CNV lesion formation. Choroidalflat mounts of 5 months old VEGF-Ahypermice and VMD2Cre+/WTFlk1fl/flVEGF-Ahypermice are shown. β-gal shows uniform VEGF-A expression in these mice (green). Crestaining (red) shows co-localization of β-gal and Crestaining in the nuclei of RPE cells in VMD2Cre+/WTFlk1fl/flVEGF-Ahypermice (red areas of staining occur in CNV lesions of VEGF-Ahypermice due to labeling of anti-mouseIgG and not Cre, as the anti-mouseIgG secondary antibodies were used to detect the mouse anti-Cre antibody). Phalloidinstaining (white) reveals extensive RPE barrier breakdown and CNV lesions (arrow) in VEGF-Ahypermice, which is not observed in VMD2Cre+/WTFlk1fl/flVEGF-Ahypermice. Scale bar 100 μm.
a) Left, the structure of THPN.figf1(a) Left, the structure of THPN. Right, THPN specifically induces the cell death in several melanoma cell lines but not in any other types of cancer cell lines. The cells were treated with THPN for 48 h, as indicated; cell survival was determined by flow cytometry. '-' represents vehicle treatment only. (b) THPN-induced cell death is prevented by the autophagic inhibitors wortmannin (100 nM) and chloroquine (20 μM) but not by the apoptotic inhibitor zVAD (20 μM) or the necrotic inhibitors butylated hydroxyanisole (100 μM) and necrostatin-1 (30 μM). The cells were pretreated with each inhibitor for 1 h separately before the THPN treatment (20 μM, 48 h). Ctrl, control. (c) THPN induces autophagy in melanoma cells. Top, THPN (20 μM, 24 h) induces a change in the localization of GFP-LC3 fusion proteins from a ubiquitous, diffuse pattern to cytoplasmic puncta. Serum starvation (SS, 24 h) was used as a positive control (scale bars, 10 μm). Middle, the GFP-LC3-positive cells with puncta were calculated from five randomly picked regions. Bottom, the conversion of LC3-I to LC3-II induced by THPN was determined by western blotting. Images of full blots are shown in Supplementary Figure 7. All of the data are presented as the mean ± s.e.m. of three independent experiments. *P 0.05, **P 0.01, ***P 0.001; NS, not significant.
a, Phase partitioning of Apg8.figf1a, Phase partitioning of Apg8. Wild-type and Δapg4Δapg8 cells producing Apg8FG and Δapg7 cells were subjected to phase partitioning (see Methods). b, MALDI mass spectrum of His6-Apg8. Purified His6-Apg8 (predominantly Apg8FG-X and a small amount of Apg8FG) was extracted with 0.4% β-octylglycoside from a polyacrylamide gel and desalted with ZipTipC4(Millipore) before mass measurement. The principal MH+ ion signal at m/z 15,003 is shifted in mass by about +635 from the MH+ value corresponding to His6-Apg8FG. c, MS/MS spectra of the C-terminal peptides of Apg8FG-X (top) and Apg8FG (bottom). C-terminal fragments were generated from Apg8FG-X and Apg8FG (see Methods). Each fragment that was observed only as non-18O-labelled signals was subjected to MS/MS. The MS/MS spectra, deconvoluted with MaxEnt3, were interpreted by SeqMS software26. Arrows show the sequences from the N and C terminus based on y″m and bl ions, respectively (where m and l denote positions counted from the C and N terminus) that were produced by cleavage of peptide bonds during MS/MS. Amino acids shown in three-letter code denote immonium ions. The nomenclature of these ions is as in previous work28.
The ATG16L1-binding motif is present with a similar ATG16L1-binding capacity in other molecules.Figure 8(A) Alignment of the region that includes the motif in NOD2 with the same area in NOD1. The relevant amino‐acid stretch is boxed. Individual amino‐acid highlighting (black) in NOD2 indicates the residues identified by the Prosite algorithm as part of the motif. In NOD1, all residues that could be part of an eventual motif are highlighted to indicate that they form an incomplete motif. (B) Amino‐acid region in TLR2 that includes the motif. Highlighting indicates residues identified by Prosite as part of the motif. (C, D, E, G, H) 293T cells were transfected with the indicated constructs, lysed and subjected to GST immunoprecipitation with agarose beads coupled to glutathione. Shown are WBs against the indicated molecules. (C) The N‐terminal CARD of NOD2 (NOD2-CARD1-HA), but not the CARD of NOD1 (NOD1-CARD-HA), co‐precipitates with GST-ATG16L1. (D) The ID of TLR2 (HA-TLR2-ID) co‐precipitates with GST-ATG16L1. (E) Mutated versions (MUT) of NOD2-CARD1-HA and HA-TLR2-ID (as shown) do not co‐precipitate with GST-ATG16L1. (F) Amino‐acid sequences of T3JAM and DEDD2 including the motif. Residues identified by the Prosite algorithm are highlighted. (G) Wild‐type (WT) versions of HA-T3JAM and HA-DEDD2 (as indicated) co‐precipitate with GST-ATG16L1. (H) Mutated versions (MUT) of HA-T3JAM and HA-DEDD2 do not co‐precipitate with GST-ATG16L1. Asterisks indicate irrelevant bands in C, D and G.Source data for this figure is available on the online supplementary information page.
a, NCOA4 depletion rescues ferritin degradation upon 9 h of iron chelation in PA-TU-8988T cells.f3a, NCOA4 depletion rescues ferritin degradation upon 9 h of iron chelation in PA-TU-8988T cells. Relative FTH1 levels (n = 3, biological triplicate) for each chelator are quantified. Bars and error bars represent mean values and standard deviation (s.d.), respectively. **P 0.01 and *P 0.02 comparing FTH1 levels between different cell lines (one-sided t-test). b, Deferoxamine mesylate (DFO) chelation time course in U2OS cells. DFO added at time 0, two NCOA4 antibodies are used for immunoblotting (top panel, Sigma antibody; second panel, Bethyl antibody). c, GFP-NCOA4 (green) co-localizes with endogenous LC3B (red) and endogenous ferritin (blue) in U2OS cells subjected to DFO chelation in the presence of lysosomal protease inhibitors for 6 h. Scale bar, 20 µm. d, Immunostaining of PA-TU-8988T cells subjected to DFO chelation in the presence of lysosomal protease inhibitors for 9 h. Scale bar, 10 µm. Punctate ferritin fraction was quantified from >100 cells per cell line from two independent experiments (biological duplicate). Bars and error bars represent mean values and s.d., respectively. ***P  0.001 using a one-sided t-test. e, Lysates from PA-TU-8988T cells as in a were analysed using indicated antibodies (lanes 1-3, untreated), with quantification based on at least three independent experiments (biological triplicate). Bars and error bars represent mean values and s.d., respectively. *P  0.05 using a one-sided t-test. f, PA-TU-8988T cells stably expressing shGFP or shNCOA4-1 were treated with H2O2 and cell viability was measured at 72 h. Bars and error bars represent mean values and s.d., respectively, of technical triplicates. ***P 0.001 using a two-sided t-test.
Figure 3(A-A''') Immunostaining of discs expressing ptc>RFP with antibodies to Rpk and ATPα showing elevated levels of both proteins anterior to the compartment boundary. White arrowheads indicate approximate position of the A-P compartment boundary. (B-C') L3 discs that are temperature-sensitive for hh following shift to the restrictive temperature for 12 h show loss of increased expression of Rpk (B, B') and ATPα (C, C') anterior to the compartment boundary. Controls shown in Figure EV2. White arrowheads indicate approximate position of the A-P compartment boundary. (D-E''') Clones of cells expressing a constitutively activated form of the transcription factor Ci show elevated Rpk and ATPα expression in the anterior compartment (white box) as well as the posterior compartment (yellow arrowhead). (E-E''') A single clone in the A compartment is shown at higher magnification. (F-F') A clone of cells in the anterior compartment expressing ci3M following incubation in DiBAC. (G-G') A clone of cells in the anterior compartment expressing ptc RNAi following incubation in DiBAC. The clones also express RFP.