EMBO/sd-character-level-ner huggingface dataset:
Figure 7(A) Colorectal tumors generated in Apcmin/+ mice following treatment with PBS, E. coli DH5α (E. coli), fadA-deletion mutant US1 (US1) or F. nucleatum 12230 (Fn). Each symbol represents one mouse. Horizontal lines represent mean values. Representative tumors formed in the mouse colon are shown on the right, pointed by blue arrows on the right. *p<0.05, **p<0.01 (one-way ANOVA). (B) ANXA1 mRNA levels in Apcmin/+ mouse colonic tumors and normal colonic tissues as measured by real-time qPCR. Each symbol represents one mouse. Horizontal lines represent mean values. *p<0.05, **p<0.01 (two-way ANOVA). (C) Positive correlation between fadA gene copy numbers (x-axis) and ANXA1 mRNA levels (y- axis) in F. nucleatum-induced Apcmin/+ mouse colonic tumors (n=34; Pearson's correlation). Each dot represents the average of qPCR results performed in duplicates (D) Abundance of fadA in the paired normal and adenocarcinoma tissues from human CRC patients (n=18) expressed as ratio of fadA over total 16S rRNA genes determined by qPCR. Each symbol shows the average of duplicate qPCR results from one patient. Horizontal lines represent mean values. **p<0.01 (paired t-test). (E) ANXA1 mRNA levels in paired normal and adenocarcinoma tissues from CRC patients (n=18). Each symbol shows the average of duplicate qPCR results from one patient. Horizontal lines represent mean values. **p<0.01 (paired t-test). (F) Positive correlation between fadA gene copy numbers (x-axis) and ANXA1 mRNA levels (y-axis) in human colorectal adenocarcinoma tissues (n=18; Pearson's correlation). Each dot represents the average of qPCR results performed in duplicates. (G) Confocal microscopy analysis of paired normal and carcinoma tissues from two colon cancer patients. The frozen sections were incubated with rabbit anti-AnnexinA1 polyclonal antibodies and 5G11 mouse anti-FadA monoclonal antibodies. The slides were then stained with Alexa Fluor®680-conjugated donkey anti-rabbit and Alexa Fluor®555-conjugated goat anti-mouse, washed, and covered in mounting medium containing DAPI. The scanning confocal microscopy mages were taken with a Nikon Ti Eclipse inverted microscope at 200x magnification for the normal tissues and 400x for the carcinomas. Scale bar, 50 μm. Co-localization of FadA (red) and Annexin A1 (green) was observed in carcinomas but not in the paired normal tissues.
Figure 5ABCE1 binds to 70S/80S ribosomes containing mRNA, tRNA in the P-site (not shown), and an A-site factor (a/eRF1 after canonical termination; a/e Pelota during stalled ribosome recognition) to form pre-splitting complexes. Here, NBSII is primed in a semi-closed state and anchored to ribosomal RNA via hinge 2. ATP occlusion and tight closure of NBSII triggers an allosteric chain within ABCE1 leading to a tight closure of NBSI. Consequently, the FeSD is displaced and the parallel hinge-opening rearranges ABCE1 in the ribosomal subunit cleft. Thereby, the subunits are split apart and the FeSD is repositioned at h44. During and/or after the splitting process, the A-site factor dissociates and mRNA and tRNA are recycled (not shown). At the post-SC, ABCE1 occludes two ATP molecules in the NBSs. ATP hydrolysis is a prerequisite for NBS opening and dissociation of ABCE1 from the SSU. Black arrows indicate domain movements within ABCE1.
Eliminating protein activity using a combination of CRISPRi, sRNA, and proteases.Figure 3(a) Schematic showing three levels of repression. A small guide RNA (sgRNA) directs deactivated Cas9 (dCas9) to block transcription from a promoter. sRNA binds to the mRNA and promotes degradation by recruiting Hfq. The mf-Lon protease targets a tag (blue) added to the protein. The protease is also targeted to itself to reduce toxicity (Methods) (b) Reduction of fluorescence of RFP by the different mechanisms of repression after 18 hours of growth (Methods). The inducers are either 1 mM IPTG (sRNA) or 25 μM DAPG (sgRNA, mf-LON). sgRNA and mf-LON are co-transcribed on a single transcript, processed by ribozymes (Appendix Figure S13). (c) The dynamics of repression by each of the mechanisms is shown. Empty circles are uninduced and black circles are induced (1 mM IPTG or 25 µM DAPG) at the 2-hour time point (dashed line) (Methods) (d) Metabolic pathways to acetate in E. coli are shown along with the targeted enzymes. The impact of either knocking out these enzymes or knocking them down by the various mechanisms after 20 hours are shown. On the left are shown empty strains containing either no acetate pathway modifications (WT; MG1655∆glnL), a double deletion (∆pta∆poxB), or single knockouts and protease tag modifcations (∆poxB pta::pdt3, ∆pta poxB::E170). On the right are shown knockdowns of pta (blue) and poxB (red), tested in strains ∆poxB pta::pdt3 and ∆pta poxB::E170, respectively (e) Design of a PoxB mutant that can be targeted by the SuMMV protease. The structure of PoxB is shown (PDB: 3ey9) (Neumann et al, 2008), highlighting the amino acid sites selected for degron insertion (in red) (f) The impact on acetate production for a double mutant where poxB is complemented on a BAC (Methods). Note that complementation causes a ~3-fold reduction in acetate compared to WT in part d. N-ter and E170 refer to the location of the SuMMV tag. (g) Reduction of acetate production by the expression of SuMMV from an DAPG-inducible promoter (- no inducer, + 25 µM). The PoxB variant with the degron inserted at position E170 is either carried on a BAC or at its native location in the genome
Figure 6A. Schematic representation of ACT1 with indicated TRAF6-binding domain, SEFIR domain and location of phosphorylation sites identified in our MS analysis B. Analysis of ACT1 structure using three different algorithms predicting disordered regions. Detected phosphorylation sites are all in the disordered region of the protein. ACT1 KO cells were reconstituted with either wild type ACT1 or indicated mutants or ACT1 with all nine identified phospho-sites mutated to alanines (9ST mut). Cells were stimulated with IL-17 (500 ng/ml) as indicated and lysates were analyzed by immunoblotting. * indicates nonspecific band. ACT1 KO cells were reconstituted with either wild type ACT1 or indicated mutants or ACT1 with all nine identified phospho-sites mutated to alanines (9ST mut). Cells were stimulated with IL-17 (500 ng/ml) as indicated and lysates were analyzed by immunoblotting. * indicates nonspecific band. E. ACT1 KO cells expressing either ACT1(WT) or ACT1(9ST mut) were stimulated with SF-IL-17 (500 ng/ml) as indicated or were left unstimulated and IL-17 was added post lysis. IL-17RSC was isolated via anti-Flag immunoprecipitation and analyzed by immunoblotting. F. ACT1 KO cells were reconstituted with either ACT1(WT) or ACT1(Δ20-380). Cells were stimulated with IL-17 (500 ng/ml) as indicated and lysates were analyzed by immunoblotting. * indicates nonspecific band. G. Analysis of iBAQ intensity of ACT1 normalized to iBAQ intensity of IL-17RC. Data are composite of all 14 MS analyses of IL-17RSC from wild type ST2 cells performed in this study. Mean + SEM is shown.
a) Top, FAK−/− cells were treated with 200 nM dasatinib for 24 h and then fixed and stained for anti-Src antibody (green).figf4(a) Top, FAK−/− cells were treated with 200 nM dasatinib for 24 h and then fixed and stained for anti-Src antibody (green). Solid arrows indicate Src at the cell periphery; dashed arrows indicate Src in autophagosomes. Scale bars, 20 μm. Bottom, quantification of dasatinib-treated cells with Src in puncta. Data are presented as mean±s.d. and significance is P0.001 (n=3). Middle, lysates from cells treated with dasatinib were immunoblotted with anti-Src and anti-pTyr-416-Src and lysates from cells treated with dasatinib and chloroquine were immunoblotted with anti-LC3B and anti-actin antibodies. Densitometry was carried out to calculate the increase in the amount of LC3B-II relative to actin for each condition after treatment with chloroquine and is presented as a percentage increase for the immunoblot shown. (b) Top, FAK−/− cells were transiently transfected with SrcY527F-GFP or with Src-251-GFP (green) and stained for anti-LC3B (red). Scale bars, 20 μm. Bottom, quantification of cells with co-localization between Src and LC3B. Data are presented as mean±s.d. and significance is P0.001 (n=3). (c) Left, FAK−/− cells stably re-expressing wild-type FAK, FAKY397F or FAKY4F-Y9F were fixed and stained for anti-FAK (red) and anti-pTyr-416-Src (green). Solid arrows indicate co-localization at adhesions and dashed arrows indicate active Src in autophagosomes. Scale bars, 20 μm. Right, lysates from these cells were also immunoblotted with anti-FAK and anti-actin antibodies. (d) Left, wild-type FAK, FAKY397F or FAKY4F-Y9F cells were transfected with RFP-GFP-LC3. Solid arrows indicate co-localization and dotted arrows indicate its absence. Scale bars, 20 μm. (e) Lysates were immunoblotted with anti-LC3B and actin antibodies in the absence and presence of chloroquine. Densitometry was carried out to calculate the increase in the amount of LC3B-II relative to actin for each cell type after treatment with chloroquine and is presented as a percentage increase for the immunoblot shown. Uncropped images of blots are shown in Supplementary Fig. S9.
p53 constrains NFATc1‐dependent promotion of EMT phenotype and cancer stem cell‐like properties through induction of miRNA‐200c A Expression analysis of indicated miRNAs upon adenoviral re‐expression of p53 in KP∆NC tumor cells, detected by real‐time PCR (P  0.Figure 7A Expression analysis of indicated miRNAs upon adenoviral re‐expression of p53 in KPΔNC tumor cells, detected by real‐time PCR (P 0.05). B Protein expression of EMT marker genes after adenoviral re‐expression of GFP‐tagged p53 in KPΔNC tumor cells. C, D Protein (C) expression levels of EMT and stemness marker genes upon miRNA‐34a and miRNA‐200c overexpression using specific mimics in KPNC tumor cells. Representative results from at least three independent experiments are shown (means ± SD; P 0.05). C, D mRNA (D) expression levels of EMT and stemness marker genes upon miRNA‐34a and miRNA‐200c overexpression using specific mimics in KPNC tumor cells. Representative results from at least three independent experiments are shown (means ± SD; P 0.05). E Western blot analysis of EMT marker expression in KNC cells following depletion of miR‐200c. F ChIP experiments showing NFATc1 binding along with H3K27 acetylation mark at the selected Snai1 enhancer region as well as Sox2 and RNA polymerase II binding at Snai1 promoter after miR‐200c overexpression in KPNC tumor cells. Means ± SD are shown from one out of three independent experiments. G ChIP analysis in KP∆NC tumor cells after adenoviral GFP‐tagged p53 overexpression. NFATc1 and H3K27ac binding are illustrated on Snai1 enhancer, and Sox2 and RNA polymerase II binding are demonstrated on Snai1 promoter. Means ± SD are shown from one out of three independent experiments. H Quantification of KP∆NC tumour cell-derived spheres after adenoviralGFP‐tagged p53 overexpression.
The kinase-dead ULK mutants inhibit autophagy.Figure 2.(A) In vitro kinase assay of endogenous ULK1. MEFs were cultured in complete or starvation medium for 60 min. ULK1 kinase activity was determined as described in Materials and methods. Relative kinase activity is shown. Data are the mean ± SE of five independent experiments. (B and C) NIH3T3 cells were transiently transfected with HA-ULK1, HA-ULK2, or their kinase-dead mutants and subjected to immunofluoresence microscopy using monoclonal anti-HA and polyclonal anti-Atg16L1 antibodies for primary staining and Alexa Fluor 488-conjugated goat anti-mouse IgG and Alexa Fluor 568-conjugated goat anti-rabbit IgG antibodies for secondary antibodies. Transfected cells are indicated by arrows. Bars, 20 μm. (D) NIH3T3 cells stably expressing GFP-ULK1K46N and GFP-ULK2K39T were cultured in starvation medium for 120 min. The cells were subjected to immunofluorescence microscopy using anti-Atg16L1 antibody. Bar, 20 μm. (E) NIH3T3 cells were transfected with the retroviral vectors encoding HA-ULK1K46N or with the corresponding empty retrovirus (mock). They were cultured in complete or starvation medium for 120 min. Cell lysates were then analyzed by immunoblot analysis with the indicated antibodies.
Abnormal localization of LAMP2A at trafficking vesicular compartments in CTNS-deficient cells.Figure 6Immunofluorescence analyses of endogenous LAMP2A, ER (Grp78/94) (A), which is expanded in cystinotic cells (white arrow), VAMP7-positive vesicles (B) and Rab11a-positive structures (C) was performed using wild-type or Ctns−/− fibroblasts as described in Materials and Methods. At least 2 independent experiments were performed for each vesicular trafficking or ER marker. (B, C) Results are expressed as the percentage of LAMP2A that colocalizes with VAMP7 or Rab11. For Rab11/LAMP2A colocalization, 29 wild-type and 15 Ctns−/− cells were quantified. For VAMP7/LAMP2A colocalization (white arrows), 49 WT and 57 Ctns−/− cells were analyzed. Data are expressed as mean ± SEM. *P < 0.001 (unpaired t-test). Scale bars: 20 μm.
Figure 1A. Schematic representation of the experimental procedure. B. IHLs were isolated from the liver of Tie2-GFP mice intrasplenically injected with NaCl (n=3) or with 5x103 CT26 at the specified time points (day 25 post-injection n=6, day 35 post-injection n=5). The number of GFP+ TEMs was estimated by flow cytometry as 7AAD-/CD45+/CD11b+/CD11c-/GFP+ cells per liver; data pooled from three independent experiments; mean values are shown; error bars=S.E.M.; p-values were calculated by Mann-Whitney test. C. Representative images (top panels) and corresponding H&amp;amp;E micrographs (bottom panels) of the liver from control (NaCl-injected, left panels) or Tie2-GFP mice 25 and 35 days (center and right panels, respectively) post-intrasplenic injection of 5x103 CT26. Large metastatic foci in the liver of mice that received CT26 cells are indicated (arrowheads). The dashed line in the H&amp;amp;E panels identifies the metastasis margin; ✱=CRC metastatic area; scale bars=100μm. D. Liver RNA was extracted from mice transplanted and injected as described in B (NaCl n=4, day 25 n=7, day 35 n=4). The average GFP expression value of NaCl injected mice estimated by quantitative real-time PCR was set to 1 and utilized as reference to calculate the fold increase values of CT26 injected mice; data pooled from three independent experiments; mean values are shown; error bars=S.E.M.; p-values were calculated by Mann-Whitney test. E. Confocal immunofluorescence images of representative liver sections from Tie2-GFP mice that were injected intrasplenically with either NaCl (upper panels) or 5x103 CT26 (bottom panels, 25 days post-injection). Note that TEMs (identified as GFP+, macrophage mannose receptor [MMR]+ and F4/80+ cells) gather in the proximity of CRC metastatic foci (identified by the dashed lines); ✱=CRC metastatic area; scale bars=100μm.
Figure 1A. Diagram of the calyx of Held synapse. B. Strategy for selective deletion of all neurexins at the Calyx of Held by crossing PV-Cre driver mice with neurexin-1/2/3 (Nrxn123) triple conditional knockout (TKO) mice (Zhang et al., 2016; Chen et al., 2017). C. Representative images of calyx synapses from littermate control and Nrxn123 TKO mice. Brainstem sections were labeled with antibodies to vGluT1 (red) and Syt2 (green). Scale bar, 10 μm. D. Summary graphs of the synaptic vGluT1 and Syt2 immunostaining intensity (normalized to control). E-H. Representative traces of spontaneous EPSCs (sEPSCs)(E), and summary graphs of the sEPSC frequency (F), amplitude (G), and kinetics (H) recorded from littermate control and Nrxn123 TKO mice.
A possible mechanism of Ub-mediated autophagic sequestration of invading bacteria and transfection reagent–coated latex beads.Figure 7.A possible mechanism of Ub-mediated autophagic sequestration of invading bacteria and transfection reagent-coated latex beads. After internalization and endosomal rupture, Ub is conjugated to host cellular proteins in endosomes that contain Salmonella or transfection reagent-coated latex beads (top). Ub would be recognized by three pivotal components of the autophagic machinery, notably the Atg16L1 complex, the ULK1 complex, and Atg9L1 (bottom right). Atg16L1 localizes to the ubiquitinated target through three mechanisms, the interaction of coiled-coil domain (CCD) with FIP200, the interaction of WD repeats (WDR) with Ub, and the 194-195 region-mediated mechanism (bottom left).
Figure 6evaluation of S100A9+ neutrophil and CD8+ T cell infiltration in histologic sections of human CRC tumors. , Anti-S100A9 immunostaining on a representative human CRC section. Arrowheads indicate neutrophil infiltration at the tumor boarder. Co-immunostaining for CD8 (brown) and S100A9 (gray-blue) in the same CRC sample as shown in A. evaluation of S100A9+ neutrophil and CD8+ T cell infiltration in histologic sections of human CRC tumors. Quantification of S100A9+ cells (upper panel) and CD8+ T cells in different areas on CRC tumor sections containing tumor border with adjacent benign mucosa as determined by a specialized pathologist: "Tumor center" was tissue within tumor >200μm from tumor border, "Tumor border" was tissue within tumor <100μm from tumor border and "Adjacent benign mucosa" was mucosa tissue outside tumor <100μm from tumor border. Per section, all identifiable tumor border and adjacent benign areas, as well as three arbitrary tumor center areas were scored. Sections were derived from 10 individual patients. Gene expression scores were generated as described in material and methods. Boxes are lower and upper quartiles with median as solid lines; horizontal lines define minimum and maximum; dots define outliers. , Neutrophil scores in tumor samples categorized according to CMS subtypes. Gene expression scores were generated as described in material and methods. Boxes are lower and upper quartiles with median as solid lines; horizontal lines define minimum and maximum; dots define outliers. Comparison of T cell gene expression scores in CMS4 tumors within two publicly available CRC gene expression datasets (median, lower and upper quartiles; horizontal lines define minimum and maximum; dots define outliers). Samples are categorized according to the medians (high = >median; low = neutrophil and TGFβ gene expression scores. Upper panel: TCGA dataset. Lower panel GSE39582 dataset. Proliferation of activated blood T cells in vitro in co-culture with increasing ratios of CRC patient-derived autologous blood neutrophils. CD3+ T cells and CD66b+ neutrophils were isolated and cultured individually for each patient. Bar graphs display relative numbers of proliferated T cells after three days of co-culture. Each dot per condition represents a neutrophil sample from an individual patient (n=13). Bars represent mean + SD. Proliferation of activated blood T cells in vitro in co-culture with autologous blood neutrophils. Cells were derived from both CRC patients (n=2) and healthy volunteers (n=5). T cells and neutrophils were cultured at a 1:1 ratio and treated with vehicle (T + Ne) or 10µm Galunisertib (T + Ne + TGFBRi). Each line represents a neutrophil sample from an individual healthy volunteer.
Figure 2A A schematic illustration of the protein structure of Drosophila Efa6, and various truncated Efa6 proteins. Live confocal images of efa612/GX6w- mutant ddaC neurons at WP stage and 16 h APF. Expression of Efa6FL (C) rescued the pruning defects in efa612/GX6w- mutant ddaC neurons (B). Red arrowheads point to the ddaC somas. Live confocal images of efa612/GX6w- mutant ddaC neurons at WP stage and 16 h APF. Expression of Efa6Nterm (D), Efa6ΔPH (F) and Efa6Nterm+CAAX (H), but not Efa6Cterm (E) or Efa6ΔMTED (G), rescued the pruning defects in efa612/GX6w- mutant ddaC neurons Red arrowheads point to the ddaC somas. I-J Quantification of dendrite severing defects and unpruned dendrite lengths at 16 h APF.
Figure 2(A) Induction scheme for generating the Gata6 iKO. Mice were induced during either telogen (PD21) or anagen (PD30) for up to 10 days (n 3 mice/time point/genopye). (B) Representative images of hematoxylin staining of skin sections from the telogen induced mice sacrificed at 2, 4, and 10 days (d) post TM induction. (C) Images of hematoxylin staining of skin section from the anagen induced mice. Quantification shown in Figure EV2D. Scale bars: 30 m.
Figure 2(A) Human IPF lung fibroblasts were transiently transfected with control or WT1 siRNA for 72hrs and AURKB transcripts were quantified. ****P < 0.00005, unpaired t-test, (n=4). (B) Lung-resident fibroblasts from TGFα mice on Dox for 4 wks were transiently transfected with control or WT1 siRNA for 72hrs and AURKB transcripts were quantified. ****P < 0.00005, unpaired t-test, (n=4). (C) Immunoblot analysis of AURKB and WT1 in the lysates of non-IPF fibroblasts transduced with control or WT1-adenoviral particles for 72hrs. **P < 0.005, unpaired t-test, (n=3). (D) Quantification of AURKB transcripts in primary fibroblasts from IPF lung treated with either control or WT1 siRNA and stimulated with media or TGFα (100ng/mL) for 16hrs. ****P < 0.00005, unpaired t-test, (n=4). (E) Primary lung-resident fibroblasts were isolated from stromal cultures of TGFα mice placed on Dox for 8 wks. Cell lysates were prepared, and the ChIP assay was performed with anti-WT1 antibody or normal rabbit IgG as a negative control using AURKB gene promoter-specific PCR primers. Non-immunoprecipitated DNA is represented as input DNA (product size, 104 bp). ***P < 0.0005, unpaired t-test, (n=2). (F) AURKB-promoter luciferase activity was measured in HEK293 cells transfected with control or WT1 overexpressing (OE) vector. ****P < 0.00005, one-way-ANOVA, (n=6).
Figure 2A Following transfection of HeLa cells with control or TIAR siRNAs for 72 hours, mitotic cells were analyzed by IF microscopy and scored for chromatin (mean ± SE, n = 3). B HeLa cells were analyzed as in (A) and scored for spindle defects (mean ± SE, n = 3). Data information: statistical significance was determined by unpaired Student's t-test; *, p < 0.05; **, p < 0.01; ***, p < 0.001. C HeLa cells were transfected with control or TIAR siRNAs for 4 days, and subjected to colcemid for 2 hours prior to preparation of metaphase spreads. Chromosomes were stained with Hoechst (red) and anti-CENP-A antibody (green); the yellow arrowhead marks a chromosome break. D Metaphase spreads were prepared as in (C), and the frequency of cells with chromosomal breaks was quantified (mean ± SD, n = 3 independent experiments, approximately 30 metaphase spreads were assessed per experiment and condition). E Metaphase spreads were prepared as in (C), and the frequency of cells with scattered chromosomes was quantified (mean ± SD, n = 3). Data information statistical significance was determined by unpaired Student's t-test; *, p < 0.05; **, p < 0.01; ***, p < 0.001. F HeLa cells were transfected with control or TIAR si-RNAs, alone or together with Cdc25B siRNA, and 4 days later subjected to colcemid for 2 hours prior to preparation of metaphase spreads. The frequency of cells with scattered chromatids was quantified (mean ± SD, n = 3 independent experiments, approximately 30 metaphase spreads were assessed per experiment and condition). G Metaphase spreads were prepared as in (F), and the frequency of cells with chromosomal breaks was quantified (mean ± SD, n = 3). Data information , statistical significance was determined by unpaired Student's t-test; *, p < 0.05; **, p < 0.01; ***, p < 0.001.