EMBO/sd-character-level-ner huggingface dataset:
Figure 3A Naïve CD4+ T cells (black bars, throughout) were isolated from peripheral blood and activated in vitro with antibodies against CD3 and CD28 (red bars, throughout). B Phosphorimaging analysis of CD45 RT-PCR products generated with primers extending from the exon 2/3 junction through exon 7 in the presence of dCTP a-32P. Percent exon 5 inclusion was determined as the sum of the exon 5-containing CD45 variants versus the sum of all CD45 variants. C MedIP with antibodies specific to 5hmC (left) or 5mC (right) followed by qPCR with the indicated CD45 primers in naïve and activated CD4+ T cells. D CTCF-ChIP in naïve and activated T cells and relative qPCR for CD45 DNA. Rabbit IgG ChIP served as control. E qRT-PCR for TET family members normalized to the average of RPS16, Bact, and Ubch5b transcripts in naïve and activated CD4+ T cell cDNA. F TET1 and TET2 Western Blot in nuclear lysates from naïve and activated CD4+ T cells and p84 immunoblot as a loading control. G TET1-ChIP in naïve and activated T cells and qPCR for CD45 DNA, relative to mouse IgG control ChIP.
Figure 7Isolated islets from mice were treated for 3 days with recombinant mouseIGFBP1 protein, respectively. (A-B) Representative confocal images of control and Igfbp1-treated mouse islets. gcg+ins+ cells are indicated by arrows. Scale bars: 25 μm. (C) Quantification of the percentage of gcg+ins+ bihormonal cells per ins+ cells in mouse islets after treatment with increasing concentrations of IGFBP1. P=0.7304, 0.0023, 0.0013, 0.0410, respectively. (D) Quantification of β-cell proliferation in mouse islets after treatment with Igfbp1; data displayed as relative values so that the proliferation markers EdU and Ki67 can be compared. Isolated islets from human donors were treated for 3 days with recombinant human IGFBP1 protein, respectively. (E-F) Representative confocal images of control or IGFBP1-treated human islets. A gcg+ins+ cell is indicated by an arrow. For clarity, individual colors for denoting insulin (green), glucagon (red), and DAPI (blue) are also shown separately. Scale bars: 15 μm. (G) Quantification of the percentage of gcg+ins+ bihormonal cells per ins+ cells in human islets from 5 donors. P=0.0036. Co-expression of insulin and glucagon is indicative of transdifferentiation, which could reflect either α cells transdifferentiating to β cells or vice versa. (H) Our proposed mechanism by which the extracellular signals insulin, IGFs and IGFBP1 can regulate α- to β-cell transdifferentiation.
ATFS‐1‐mediated mitochondrial stress suppresses the rpl‐43 phenotype A Phsp‐60::GFP is weakly expressed in wild‐type animals.Figure 4A Phsp‐60::GFP is weakly expressed in wild‐type animals. B egl‐46(RNAi) causes upregulation of Phsp‐60::GFP. C Percentage of indicated mutant animals with different levels of SQST‐1::GFP aggregates. S: strong. M: medium. N: none. >30 animals were examined in each group. D Levels of SQST‐1::GFP in various genetic mutants. 200 young adult animals for each genotype were collected for analysis. E, F Loss of function of atfs‐1 suppresses the upregulation of Phsp‐60::GFP in egl‐46(n1126) mutants. (E): DIC image of (F). G, H The dramatic decrease in the number of SQST‐1::GFP aggregates in the intestine in rpl‐43(bp399); egl‐46(RNAi) mutants (G) is restored by simultaneous loss of function of atfs‐1(gk3094) (H). I, J GFP::LGG‐1 forms numerous puncta in the intestine in egl‐46(RNAi) (I), gfi‐1(RNAi) (J) animals.
Figure 2Response to trastuzumab (50 nM, 5 days) of BT474, BTRH, patient derived cells (PDX118) and their trastuzumab-resistant derivatives TR1 and TR2. HER2, HER3 and pHER3 levels in BT474, BTRH, PDX118, TR1 and TR2. pHER3 levels were analyzed by a specific anti-pHER3 antibody. Effect of EV20/MMAF on BT474, BTRH, PDX118, TR1 and TR2. Cells were counted after 5 days of treatment with the indicated doses of EV20/MMAF. Effect of trastuzumab (50 nM, 5 days) in HER2 positive breast cancer cell lines (SKBR3, BT474, BTRH, MDA-MB-361, HCC1419, HCC1569 and HCC1954). The blue line indicates the threshold (established from BTRH) used to consider a cell line resistant to trastuzumab. Analyses of HER2, HER3 and pHER3 in the cells used in (D). Numbers below the blots show the quantitation of the signal of each band, referred to SKBR3 cells (intensity of band in SKBR3 cells=1). pHER3 levels were analyzed by a specific anti-pHER3 antibody. Effect of EV20/MMAF on the cell lines used in (D). Cells were counted after 5 days of treatment with the indicated doses of EV20/MMAF. Knockdown of HER3 in BT474 cells. Control and HER3-targeted short hairpin sequences were transduced in BT474 cells and HER3, pHER3 and pAKT levels analyzed by Western. Levels of pHER3 were analyzed by a specific anti-pHER3 antibody. Effect of HER3 knockdown on the antiproliferative action of EV20/MMAF analyzed by cell counting after 5 days of treatment with the ADC.
CryoEM structure and HDX-MS changes in K+ bufferA.Figure 6A. CryoEM structure of EAAT1WT trimer with tranDs and scaDs depicted in magenta and teal, respectively. Non-protein extra density corresponding to lipid/detergent molecules is depicted in grey. B. Comparison of EAAT1WT (left) and EAAT1CRYST (right) structures in inward- and outward-facing states, respectively. Several TMs were removed for clarity. C. TranDs from EAAT1WT (magenta) and EAAT1CRYST (orange) structures are overlapped using HP1 as reference. D. K+-induced HDX increase (blue) and decrease (pink) in EAAT1CRYST is mapped on Na+/transmitter-bound tranD structure. Unchanged regions (orange) and those outside HDX-MS sequence coverage (grey) are also shown.
Genomic Compartments are Largely preserved upon Transcriptional Inhibition A) Hi-C interaction heatmaps at 500kb (left) resolution showing all of chromosome 11, and at 100kb resolution (right) showing chr11: 79-115Mb for control and 24hr ActD treated cells.Figure 4A) Hi-C interaction heatmaps at 500kb (left) resolution showing all of chromosome 11, and at 100kb resolution (right) showing chr11: 79-115Mb for control and 24hr ActD treated cells. The arrows point out to perturbed long-range interactions at shorter scales. B) Pearson correlation heatmaps showing the genomic interactions at 500kb resolution for chromosome 4, with the 1st principal component (1st PC) below for control and 24hr ActD treated cells. C) Violin plot showing the first eigenvalues (calculated by the trans-chromosomal data) in control and ActD treated cells. p-value: Wilcoxon rank-sum test. In the violin plots, the horizontal bands represent the median, the error bars represent +/- 1.5 x IQR values, and the outer shapes represent the density of the data points. The figures represent data generated from the pooled Hi-C datasets with 2 biological replicates. D) Saddle plots showing the compartmental interactions for both cis- and trans-contacts across the conditions. E) Scaling plot generated by 500kb resolution Hi-C data showing the interaction frequency as a function of genomic distance.
Bruce suppresses autophagy at region 2 within germarium and in dying stage 8 egg chambers.Figure 5.(A) The germarium in well-fed BruceE81 flies showed an increase in LTR staining (red) compared with wild-type well-fed flies shown in Fig. 3 B (top right). DAPI staining (white) of nuclei is shown on the right. (B) In well-fed wild-type flies, mid-oogenesis nurse cell death is a rare event. Lack of Bruce function resulted in an increase in dying stage 8 egg chambers (arrows) in ovaries under well-fed conditions, and these degenerating stage 8 egg chambers had numerous LTR (red) punctate dots. DAPI staining (white) is shown on the right. Bars: (A) 20 μm; (B) 50 μm.
Fig.Figure 8Left panel. Transient overexpression of human FLT3/ITD mutation resulted in axis-duplication and dorsalization abnormalities in zebrafish accompanied with upregulation of embryonic morphogen Fst. Upregulation of FST was consistently found in FLT3/ITD-transgenic zebrafish, Flt3/ITD knock-in mice, FLT3/ITD-mutated AML cell lines and primary AML samples in vitro and in vivo. Right panel. A novel FLT3/ITD-p90RSK-CREB-FST signaling cascade was demonstrated in human AML. FST is a promising biomarker and therapeutic target for human FLT3/ITD+ AML.
Figure 6(A-G) Ablation of Sst-Cre::AviliDTR positive sensory neurons by injection of diphtheria toxin (DTX). Immunostaining of DRG from Sst-Cre::AviliDTRmice with an antibody against the diphtheria toxin receptor and IB4 in the absence of diphtheria toxin (A-C) and after systemic injection of diphtheria toxin (D-F). (G) Quantification of diphtheria toxin receptor positive neurons before and after injection of diphtheria toxin. (H-K) Examples of calcium flux in dissociated DRG upon application of histamine (H), chloroquine (I), IL-31 (H-K) Examples of calcium flux in dissociated DRG upon application of histamine (H), chloroquine (I), IL-31 (J) and 5HT1F agonist Ly344864 (K). (L) Quantification of percent responding neurons to the indicated pruritogens before or after systemic injection of diphtheria toxin in Sst-Cre::AviliDTRmice. Scale bars, 50 μm, asterisk denotes p<0.05, n=4-6 mice, Student's t-test, error bars indicate SEM.
Hh and lipid metabolism regulators mediate the effects of glial htlACT on NB proliferationA) Pan-glial (repo-GAL4) htlACT overexpression causes upregulation of hh, fasn1 and lsd2 transcripts (n = 3 biological replicates pooled from 20 brains for each genotype; for each biological replicate, we ran 3 technical replicates for each PCR reaction).Figure 6A) Pan-glial (repo-GAL4) htlACT overexpression causes upregulation of hh, fasn1 and lsd2 transcripts (n = 3 biological replicates pooled from 20 brains for each genotype; for each biological replicate, we ran 3 technical replicates for each PCR reaction). The lipogenesis genes (acc, lipin), and lipolysis gene bmm transcripts are not significantly altered. We utilised rpl32 as a reference gene in these experiments, as it is not altered by htlACT overexpression. The data are represented by log2 fold change relative to the control (repo-GAL4> w1118). B) Schematic depicting lipogenesis and lipolysis. Lipogenesis begins with de novo synthesis of fatty acids by carboxylation of cytosolic acetyl-CoA via acetyl-CoA carboxylase (ACC) and elongation of fatty chain via fatty acid synthase (Fasn, red). Dietary-derived and de novo-generated fatty acids are converted into fatty acylCoA, which re-localizes to ER and participates in triglyceride (TAG) synthesis with glycerol-3 phosphate (G3P). This process is mediated by a series of enzymes: glycerol-3-phosphate acyltransferase (GPAT), acylCoA acylglycerol-3-phasphte acyltransferases (AGPAT), Lipin (a phosphatidate phosphatase), diacylglycerol acyltransferase (DGAT, red). TAG is translocated from the ER to the core of the intracellular organelles called LDs. On the surface of LDs, a triglyceride lipase called Brummer (Bmm), and its inhibitor Lsd-2, antagonistically control TAG storage. C-G) Representative images showing that Hh staining normally localised to a ring like structure (yellow arrows), becomes delocalises to the glial cytoplasm upon htlACT overexpression, quantified in (E) (n=4, 6 brain lobes). Glial cells are marked with repo-GAL4>GFP. (E-F') are zoomed in images of (C-D'). H) Cortex glial (NP2222-GAL4>) overexpression of two independent hh RNAis significantly rescue EdU incorporation defects caused by htlACT overexpression (n=10, 16; 7, 12; 14, 11 brain lobes). I) Knockdown of NB Hh signalling pathway (dnab-GAL4> UAS-ciRNAi) rescues NB EdU incorporation defects induced by glial htlACT overexpression (repo-LexA> LexAop-htlACT). Induction of ciRNAi in NBs alone increases NB EdU incorporation (n=8, 13; 10, 10; 30, 26; 12, 7 brain lobes). J) The NB EdU incorporation defects due to cortex glial (NP2222-GAL4>) overexpression of htlACT, is significantly rescued by overexpression of RNAis against fasn1 and lsd2, compared to corresponding control RNAis (n=10, 16; 25, 17; 14, 8 brain lobes).
Western blot analysis of autophagy related proteins.Figure 4(A) The LC3-II level is reduced after 3 or 24 hr exposure to SIV-infected microglia supernatant and this decrease effect is blocked when is pretreated with rapamycin (2 µM). The protein level of elongation complex Atg12-Atg5 is also reduced, and again the effect is blocked in the presence of rapamycin. The protein level of p62 is increased for similar conditions with SIV-infected microglia supernatant, and the increase is blocked in the presence of rapamycin. GAPDH protein was used in these experiments as the loading control. One representative experiment of n = 4 is shown. (B, C and D) Ratios between LC3-II, Atg12-Atg5, p62, respectively, normalized to GAPDH. Data are reported as mean±SEM (n = 4). *** P<0.001. (B, C and D) Ratios between LC3-II, Atg12-Atg5, p62, respectively, normalized to GAPDH. Data are reported as mean±SEM (n = 4). *** P<0.001. (B, C and D) Ratios between LC3-II, Atg12-Atg5, p62, respectively, normalized to GAPDH. Data are reported as mean±SEM (n = 4). *** P<0.001.
Figure 7(A-B) Cell surface binding to endogenous neurexins. (A) Recombinant Fc-tagged LRRTM2 was added to day 10 hippocampal neurons expressing CA10 or control (empty vector) by lentiviral transduction. Surface-bound protein was quantified as the Fc signal of and normalized to a staining for endogenous neurexin (n=4 cultures; three fields imaged and averaged per well). Scatchard analysis for LRRTM2 revealed an apparent Kd of 29.5 nM and a Bmax of 0.49 nM for control and Kd 35.3 nM, Bmax 0.28 nM upon expression of CA10; significant (p=0.0006) by non-linear regression. Data shown as mean ± SEM of biological replicates (n=4). (B) Increasing amounts of recombinant, Fc-tagged Nlgn1 was added to immature hippocampal neurons expressing CA10 or control (empty vector) by lentiviral transduction, and analyzed as in (A). The apparent Kd for Nlgn1 was 186 nM and Bmax 1.65 nM for control and Kd 223 nM, Bmax 1.49 nM when CA10 was overexpressed. The difference was not significant (p=0.20) by non-linear regression. Data shown as mean ± SEM of biological replicates (n=4). (C-D) Artificial synapse formation assays. (C) Confocal images of hippocampal neurons transduced to express CA10-FLAG or control (empty vector) and co-cultured with HEK293 cells expressing GFP, Nlgn1-YFP, LRRTM2-YFP or TrkC-YFP. Cells were stained with antibodies against the synaptic marker SV2 (red) and somatodendritic MAP2 (blue); GFP/YFP is shown in green. Scale bar 20 μm. (D) Quantifications of SV2 signal overlapping GFP/YFP-positive cells. Data shown as means ±SEM with the n, reflecting number of independent cover slips analyzed, indicated in each bar with the total number of cells imaged in parenthesis. Comparisons were made by one-way ANOVA with Holm-Sidak's posthoc test for multiple comparisons between the indicated groups (GFP condition was omitted from analysis due to unequal variance; ***, p<0.001. (E-F) Analysis of cerebellin(Cbln)-induced artificial synapse formation. (E) Confocal images of hippocampal neurons transduced to express CA10-FLAG or control (empty vector) and co-cultured exposed to anti-V5-coupled beads pre-incubated with recombinant V5-tagged Cbln1 or empty anti-V5 beads. Cells were stained with antibodies against the synaptic marker synapsin (green), somatodendritic MAP2 (blue); beads are colored red and indicated by arrowheads. Scale bar 10 μm. (F) Quantifications of the synapsin signal overlapping red beads. Data shown as means ±SEM (n=3 independent experiments, each data point corresponding to 58-171 analyzed beads), with comparisons made by two-way ANOVA with Holm-Sidak's posthoc test for multiple comparisons between the indicated groups.
hnRNP L depletion results in increased apoptosis in SU-DHL-4 cells.Figure 6A qPCR of selected transcripts in SU-DHL-4 cells under conditions of BCL2 knockdown, hnRNP L knockdown, or non-targeting control siRNA Nucleofection (n ≥ 4 independent experiments). Bars indicate mean ± SD. Significance was determined compared to NT using two-tailed Student's t-tests. Data information: *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Quantification of protein reduction in knockdown experiments and a representative western blot. Each point represents a trial (n ≥ 5 for a given condition). Mean with standard deviation are indicated on the plot. Target protein levels from all biological replicates were normalized to MATRIN3, with statistical analysis as in panel (A). Data information: *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. C FACS-based apoptosis analysis of SU-DHL-4 cells Nucleofected using the indicated siRNAs and exposed to 5 μM MCL1 inhibitor for 24 hours. Quantification of relative change in each quadrant is provided where points represent individual experiments (n ≥ 5 for a given condition). Mean with standard deviation are indicated on the plot and statistical analysis was performed as in panel (A). A representative FACS experiment is shown with the percentage of cells in each gate indicated. Data information: *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001.
a) General domain structure of human VPS34 kinase comprising the helical domain (yellow) and the kinase domain (green).figf2(a) General domain structure of human VPS34 kinase comprising the helical domain (yellow) and the kinase domain (green). The ATP-binding pocket and active site lie between the amino-terminal lobe and the carboxy-terminal lobe of the kinase domain. (b) Close-up view of the VPS34 active site with PIK-III bound in the ATP-binding pocket. PIK-III forms two hydrogen-bond interactions between the compound's donor/acceptor moiety and the backbone amide and carbonyl oxygen of residue Ile 685. A prominent hydrophobic pocket, bounded in part by the side chains of Phe 612, Pro 618 and Phe 684 accommodates the cyclopropyl functional group. An extended solvent-mediated hydrogen-bonding network bridges interactions between the PIK-III aminopyrimidine moiety and the side chains of Asp 671 and Asp 644. (c) The binding properties surface of the VPS34 active site is superimposed on the solvent-accessible surface. Green surfaces are regions with hydrophobic binding properties; note the hydrophobic nature of the active site floor and the deep hydrophobic pocket in which the cyclopropyl moiety fits. Blue and red surfaces represent hydrogen bond donor and acceptor regions, respectively. PIK-III is shown bound in the active site. (d) Superposition of the PIK-III-bound VPS34 pocket surface with a mesh (white) mapping the human PI(3) Kα active site. The displacement of the PI(3) Kα hydrophobic pocket away from the hinge (relative to that of VPS34) is readily apparent.
Figure 1(A, B, C). RNA-seq transcriptional profiles across the modified locus, as well as immunoblots assessing protein expression of Lon and FtsH are shown for ΔIndLon (A), ΔIndFtsH (B), and ΔIndLon_FtsH (C) grown under inducing or depleting conditions (48h and 72h of depletion for Lon and FtsH, respectively). Symbols +/- indicate inducing or depleting conditions. LC, loading control. WT, wild-type. A schematic representation of the DNA rearrangements in the lon and ftsH locus is also shown for each strain. The ftsH inducible platform inserted by transposon delivery is shown for ΔIndFtsH and ΔIndLon_FtsH strains. The Pxyl/tetO2 inducible promoter is highlighted with a red bent arrow and the terminator sequence used to isolate the promoter is represented by a hairpin structure. The tetR repressor gene and the resistance markers cat and tetM are indicated in purple, blue and red, respectively.
Figure 4(A) Primary airway epithelial cells were isolated from tracheas of Atg7+/+ and Atg7-/- mouse and were evaluated by immunoblotting. (B) Sections of tracheas from Atg7+/+ and Atg7-/- mouse were evaluated by immunofluorescence microscopy following staining of nuclear DNA with DAPI (Blue), ciliated epithelial cell marker β-tubulin IV (green) and Atg7 (red). Scale bars: 20 µm. (C,D) Atg7+/+ and Atg7-/- mice were intranasally injected with 3 x107 CFU of WT, ΔS, ΔTY or ΔSTY P. aeruginosa. After 24 hours, lungs (C), spleens and livers (D) were harvested, homogenized and diluted for CFU counting. Each data point represents bacterial count (Log10 CFU/g) from individual mouse. The horizontal lines represent the mean value from each group (6 mice each group), error bars represent standard deviation. The significance of differences between different drug treatment was determined using One-way ANOVA and Dunn's multiple comparisons test., NS: not significant; *p ≤ 0.05 (E,F) Atg7+/+ and Atg7-/- mice were intranasally injected with PBS with or without 1 x109 CFU of ΔTY (E) ΔSTY (F). Mice survival was monitored for 10 days post infection. Kaplan-Meyer survival curves were made based on the observation of 12-18 mice/group from two independent experiments. The P-values were calculated using the log-rank test (ΔTYAtg7+/+ vs ΔTYAtg7-/-, P = 0.2186; ΔSTYAtg7+/+ vs ΔSTYAtg7-/-, P=0.0145).
Figure 8(A) MTT assays performed at different time points with HeLa and TRIM16KO cells, untreated or treated with H2O2 (400 µM, 2h). Data, mean ±SD, n=3, *p < 0.05 (Student's unpaired t-test). (B) Images of HeLa and TRIM16KO cells, untreated or treated with As2O3 (2.5 µM, 4h). Scale bar: 400 µm. Flow cytometry analysis of HeLa and TRIM16KO cells stained with Annexin-V/Propidium Iodide (double staining), untreated or treated with (C) As2O3 (2.5 µM, 4h) Flow cytometry analysis of HeLa and TRIM16KO cells stained with Annexin-V/Propidium Iodide (double staining), untreated or treated with (D) H2O2 (400 µM, 2h) Flow cytometry analysis of HeLa and TRIM16KO cells stained with Annexin-V/Propidium Iodide (double staining), untreated or treated with (E) puromycin (5 µg/ml, 6h) Flow cytometry analysis of HeLa and TRIM16KO cells stained with Annexin-V/Propidium Iodide (double staining), untreated or treated with (F) MG132 (20 µM, 8h). (G) Immunoblot blot analysis of HeLa and TRIM16KO lysates of cells treated with 5 µM of As2O3 for different durations as indicated and probed with antibodies as shown. L.E, Low exposure; H.E, High Exposure. Arrowheads indicate the cleaved form of PARP-1 and Casapse-3.
Figure 4A-F Representative kymographs of EB1-GFP comets in the proximal dendrites of ddaC neurons at 96 h AEL. The horizontal arrow indicates the direction toward the soma, and the vertical arrow indicates the time. In contrast to the wild type (A), the number of EB1-GFP comets was significantly increased in efa612/GX6w- mutant neurons (B). The comet length and speed were significantly decreased in Efa6FL- and Efa6Nterm-overexpressing neurons, but not in Efa6ΔMTED-overexpression neurons (C-F). G-J Quantification of EB1-GFP orientation, number, track length and speed.