EMBO/sd-character-level-ner huggingface dataset:
Figure 2(A-C) Mechanisms of three collective-survival circuits. The BlaM circuit consists of constitutive expression of BlaM to degrade carbenicillin upon cell lysis. BlaM works as an extracellular enzyme. The QS-CAT consists of QS-mediated expression of a chloramphenicol acetyl-transferase (CAT) that degrades chloramphenicol. The QS-BlaM circuit integrates QS control and extracellular carbenicillin degradation to establish collective survival.(D-F) Regulation modes for the three circuits. The BlaM circuit depends on antibiotic-mediated release of an enzyme into the environment to realize the collective survival. The QS-CAT circuit depends on QS regulation. The QS-BlaM circuit depends on both QS regulation and enzyme release (by lysis and export). G-I) Experimental validation of collective survival. All populations grew to high cell densities in the absence of antibiotics (Blue curves). In the presence of antibiotics, only populations with initial densities greater than NCT grew. NCT was increased with the concentration of antibiotics. Engineered bacteria with the QS-BlaM circuit exhibited a significantly higher NCT than those with the BlaM or the QS-CAT circuits. Carbenicillin was used for the BlaM and QS-BlaM circuits; chloramphenicol for the QS-CAT circuit. Each error bar represents the standard deviation from triplicate measurements. G-I) Experimental validation of collective survival. All populations grew to high cell densities in the absence of antibiotics (Blue curves). In the presence of antibiotics, only populations with initial densities greater than NCT grew. NCT was increased with the concentration of antibiotics. Engineered bacteria with the QS-BlaM circuit exhibited a significantly higher NCT than those with the BlaM or the QS-CAT circuits. Carbenicillin was used for the BlaM and QS-BlaM circuits; chloramphenicol for the QS-CAT circuit. Each error bar represents the standard deviation from triplicate measurements. G-I) Experimental validation of collective survival. All populations grew to high cell densities in the absence of antibiotics (Blue curves). In the presence of antibiotics, only populations with initial densities greater than NCT grew. NCT was increased with the concentration of antibiotics. Engineered bacteria with the QS-BlaM circuit exhibited a significantly higher NCT than those with the BlaM or the QS-CAT circuits. Carbenicillin was used for the BlaM and QS-BlaM circuits; chloramphenicol for the QS-CAT circuit. Each error bar represents the standard deviation from triplicate measurements. (G-I) Experimental validation of collective survival. All populations grew to high cell densities in the absence of antibiotics (Blue curves). In the presence of antibiotics, only populations with initial densities greater than NCT grew. NCT was increased with the concentration of antibiotics. Engineered bacteria with the QS-BlaM circuit exhibited a significantly higher NCT than those with the BlaM or the QS-CAT circuits. Carbenicillin was used for the BlaM and QS-BlaM circuits; chloramphenicol for the QS-CAT circuit. Each error bar represents the standard deviation from triplicate measurements.
Figure 2A. Amyloid staining (6E10) combined with miR-132FISH in ant-132- and scramble-injected mice at six months of age. Scale bar, 500 μm. B. Quantification of amyloid burden in hippocampus and cortex of ant-132-injected and control animals (see Materials and Methods). Sample size, n=4 per group. C, D. ELISA of soluble (TBS) and insoluble (Formic acid) Aβ40 and Aβ42 levels in the hippocampus of ant-132- (C) and miR-132-injected (D) animals at six and three months of age, respectively. Sample size, n=6 per group.
Figure 8A Representative whole-mount images of wounds from Ptger4flox/flox (Ptger4fl/fl) and Villin-Cre Ptger4flox/flox (VilCrePtger4fl/fl) mice four days post-biopsy injury. Scale bars, 750 µm. Wounds are outlined with a black dashed line. Asterisk indicates fibrin clot.B, C Quantification of percent healing (1 - [Day 4 wound area/original wound area] x 100) (B) and fibrin clot areas (C) ± s.e.m. (n = 7-8 mice per genotype with 3-4 wounds each, three independent experiments). *p=0.0270, **p=0.0057 by two-tailed unpaired t-test. D, E Representative images of serial wound tissue sections from the indicated genotypes. (D) Fluorescent images of sections stained for Claudin 4 (red). Nuclei are visualized with bisbenzimide (blue). Epithelial crypts adjacent to the wound bed are indicated by dashed yellow lines. Layer of adherent WAE cells indicated by white arrowheads. Non-adherent residual WAE cells indicated by orange arrows. D, E Representative images of serial wound tissue sections from the indicated genotypes. (E) Grayscale images of hematoxylin and eosin-stained sections. Insets of the boxed regions show a higher magnification of the region typically containing WAE cells (red arrowheads, dashed line indicates border with wound bed). Orange arrow indicates region of non-adherent residual WAE cells. Scale bars, 100 µm.
activity on the solubility of polyubiquitylated proteinsA.Figure 3A. Whole lysates of MCF7 and MCF7aTT cells exposed to heat shock and/or proteasome (MG132, 20 μM, 1h) and HSP70 (VER-155008, 50 μM, 1h) inhibitors were prepared as described in Materials and Methods. Total lysate, soluble fractions and insoluble fractions were stained by SYPRO Ruby (left) and quantified (right). (means +/- SD, n = 3, numbers above the bars indicate p values calculated with the two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli). B. The same total lysates as shown in (A) were assayed for the level of K48-linked polyubiquitin. Actin is shown as a reference. The levels of K48-linked polyubiquitin were quantified from multiple independent repeats. (means +/- SD, n = 5, numbers above the bars indicate p values calculated with the two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli). C. The soluble and insoluble fractions shown in (A) were assayed for the level of K48-linked polyubiquitin, and polyubiquitylated proteins were quantified (blue line graph, means +/- SEM, n = 4, numbers above the bars indicate p values). The data for bulk protein obtained in (A) were overlaid onto the graph for comparison (red line, means +/- SEM, n = 3).
m152 antagonizes STING translocation, but not its activation by cGAMP or its dimerization (A) Schematic representation of the key steps in the cGAS-STING signaling pathway.Figure 5(A) Schematic representation of the key steps in the cGAS-STING signaling pathway. (B) 293T cells were co-transfected with expression plasmids for IFNβ-Luc, pRL-TK and either ev or m152. Cells were further co-transfected with either Cherry-STING WT or the constitutively active Cherry-STING V154M (stimulated) or with IRES-GFP (unstimulated). Data is combined from three independent experiments and shown as mean ± SD. (C) iMEF stably expressing ev or m152-V5 were stimulated with 10 µg/ml ISD for the indicated times or left unstimulated (mock). Cell lysates were analyzed by SDS-PAGE under non-reducing conditions and subjected to IB with the specified antibodies. Data information: IB shown in (C) are representative of three and two independent experiments, respectively. Student's t-test (unpaired, two-tailed), n.s. not significant, *p<0.05, ****p<0.0001 (D) Two representative still images from live cell imaging experiments with iMEFgt/gt stably expressing Cherry-STING transfected with ISD (right panel) or left unstimulated (left panel). Red circles highlight representative translocated STING in ISD stimulated cells, which is used as an indicator of activation. (E) iMEFgt/gt stably expressing Cherry-STING and either ev or m152-V5 were stimulated with ISD. Live cell imaging was performed and STING translocation quantified 120 min and 180 min post stimulation. Data shown is one representative of two independent experiments. (F) iMEFgt/gt stably expressing Cherry-STING and V5-tagged m152 or corresponding ev were stimulated with 5 µg/ml ISD for the indicated time or left unstimulated (mock). Lysates were subjected to IB with the specified antibodies. Data information: IB shown in (C) and (F) are representative of three and two independent experiments, respectively. Student's t-test (unpaired, two-tailed), n.s. not significant, *p<0.05, ****p<0.0001
Figure 5A. Immunofluorescent staining for Rab11-FIP1 in TE1 and TE2 ESCC cell lines expressing a non-targeting control shRNA or shRNA against Rab11-FIP1. Scale bar= 50 μm B. Immunofluorescent staining for f-actin in TE1 and TE2 ESCC cell lines expressing a non-targeting control shRNA or shRNA against Rab11-FIP1. Scale bar= 50 μm C. Immunofluorescent staining for E-cadherin, in TE1 and TE2 ESCC cell lines expressing a non-targeting control shRNA or shRNA against Rab11-FIP1. Scale bar= 50 μm D. qPCR analysis of epithelial and mesenchymal gene expression in TE1 (left) and TE2 (right) cells expressing Rab11-FIP1 shRNA relative to control cells (dotted line). Graph error bars represent mean SEM (n=3 technical replicates from three different cell passages). n.s. TE2 SNAI2, *p<0.05,(Unpaired t-test). E. qPCR analysis of epithelial and mesenchymal gene expression following ZEB1 depletion in TE1 (left) and TE2 (right) cells expressing Rab11-FIP1 shRNA relative to control cells (dotted line). Graph error bars represent mean SEM (n=3 technical replicates from three different cell passages). **p<0.01, * p<0.05, n.s. TE1 CDH1, VIM. SNAI1, SNAI2, TE2 VIM, SNAI1 (Unpaired t-test).
Figure 1Physical appearance of wild type (WT), Wwox null injected either with AAV9-hSynI-GFP (the control virus) or AAV9-hSynI-mWwox-IRES-GFP virus at P17. Graph showing the body weight of WT, KO and KO+ AAV-mWwox at different days after birth (n= 4 per group). Kaplan-Meier survival graph indicates prolonged life span of Wwox knockout mice injected with AAV9-hSynI-mWwox [total n=18, spontaneously dead n=6, mice taken out for electrophysiology/electron microscopy/analysis, are shown in yellow, n=12] compared to mice injected with AAV9-hSynI-GFP (n=6) or the non-injected (n=8 (P <0.0001, Log-rank Mantel-Cox test). Immunofluorescence images showing the expression of WWOX (red) in brain tissue (cortex) at P17 from WT and Wwox null mice that were injected with AAV9-hSynI-mWwox (2x1010). Neurons are labelled with anti-NeuN antibody (green). The square white box shows the magnified area. White arrows show the transgene expression in NeuN positive cells. Graph showing the percentage of NeuN and WWOX double positive cells in different parts of the brain (Cortex, Hippocampus and Cerebellum) from KO mice injected with AAV-mWwox at P17. NeuN and WWOX double positive cells were calculated from 3 identical sagittal sections of the AAV-mWwox injected mice (n=3) brains. Expression of EGFP (green) is shown in brain tissue (cortex) of Wwox null injected with AAV9-hSynI-GFP (2x1010).
Figure 2A. Representative FACS plots of spleens from leukemic recipient mice that had received LSK, GMP or DN2 cell grafts immediately after retroviral Myc/Bcl2 transduction. Numbers indicate frequencies (%) of CD45.2 donor cells within the indicated gates summarizing the mean±SEM of at least 8 recipients per group from a total of 10 independent experiments. B. Cumulative survival of mice that received 2104 to 2105 freshly Myc/Bcl2-transduced LSK, GMP or DN2 cells; Graph shows n=11 Myc-Bcl2+LSK-, n=14 DN2-, and n=8 GMP-transplanted animals. C. Histological spleen sections of Myc-Bcl2+LSK-, GMP- or DN2-transplanted mice displaying myeloperoxidasestaining. Leukemic cell engraftment in spleens ranged from 75.1%-95.5%. D. Multiplex quantitative RT-PCR demonstrating strong expression of PU.1 in the myeloid (M3L: CD11b+Gr1+/CD3-CD4-CD8-) and biphenotypic T/myeloid (BL: CD11b+Gr1+/CD3+CD4+CD8+) fractions but not in the T-lymphoid (TL: CD11b-Gr1-/CD3+CD4+CD8+) fraction that were flow-sorted from diseased Myc-Bcl2+DN2-transplanted mice. Myeloid progeny of Myc/Bcl2+GMP-transplanted or T-lymphoid progeny of Myc/Bcl2+DN3-transplanted animals as well as healthy thymocytes (Thy) and BM cells from WT mice served as controls. Results are shown as fold over actin transcripts. E. D-J rearrangement PCR of flow-sorted T-lymphoid (TL), biphenotypic (BL) and myeloid (ML) fractions of donor splenocytes of a diseased Myc/Bcl2+DN2-transplanted mouse. All three fractions revealed a stable genomic reassembly of the Dβ1 TCR locus (Dβ1Jβ1.5) demonstrating clonality in DN2 leukemia. thy = thymus. F. FACS plot of diseased intrathymic grafts (CD45.2-gated), demonstrating that Myc/Bcl2+DN2 cells could produce multi-lineage leukemia within a T-cell supporting environment; Myc/Bcl2+GMPs remained myeloid restricted under these conditions. Numbers indicate cell frequencies within the gates (mean±SEM) representing six independent experiments. G. Intrathymic transplantation of mock-infected non-leukemic DN2 cells produced predominantly T-lymphoid but also myeloid progeny 23 days after injection. This differentiation outcome resembles that of freshly isolated DN2 cells (Bell and Bhandoola, 2008; Richie Ehrlich et al., 2011), and indicates that the short-term culture necessary for retroviral transduction did not interfere with the physiological lineage potential of DN2 cells.
Figure 6A-B. Inhibitor treatment scheme (A) and FACS plots (B) of 3C cultures treated with the Shh inhibitor cyclopamine or the BMP inhibitor dorsomorphin (mean ±SD; n=3). C. Quantification of experiments as in (B) shows enrichment of HFSCs upon cyclopamine (Cyclo) treatment, whereas dorsomorphin (Dorso) treatment leads to depletion of HFSCs (mean ±SD; n=3; *p ≤ 0.05, Kruskal-Wallis). D. Heatmap of significantly (padj value<0.05) differentially expressed genes from RNAseq analysis of 3C cultures treated with Cyclo or Dorso for 48h. HFSC identity genes (triangles) and genes implicated in HFSC lineage progression (circles) are indicated. E. RT-qPCR analysis of 3C cultures treated as in (D) confirms that Id 1,2 and 3 are significantly upregulated in Cyclo treated cells (mean ±SEM; n=4; *p ≤ 0.05, Mann-Whitney U test). F. RT-qPCR analysis of 3C cultures treated as in (A) shows upregulation of HFSC identity genes upon cyclopamine treatment whereas regulators of HFSC quiescence are downregulated upon dorsomorphin treatment (mean ±SEM; n=4; *p ≤ 0.05, ** p ≤ 0.01, Kruskal-Wallis). G. Inhibitor treatment scheme of 3C cultures treated from day 8 onward. H. Quantification of experiments as in E (mean ±SD; n=3-4; * p ≤ 0.05, ** p ≤ 0.01, Kruskal-Wallis). See also Appendix figure S6 and Dataset EV5.
Compartmentalization of DRiPs and polyUb proteins is catalyzed by the self-assembly of PML into nuclear bodies.Figure 4HeLa cells were lipofected with non-targeting siRNA control or a specific siRNA against PML. 72 h post-transfection, total proteins were extracted and expression levels of PML were analyzed by immunoblotting. TUBA4A was used as loading control. HeLa cells were lipofected with non-targeting siRNA control or a specific siRNA against PML. 72 h post-transfection, cells were treated with MG132 (10 µM) and OP-puro (25 µM) for 4 h. Left panel: automated quantitation of the number of PML-NBs/nucleus is reported. Statistical significance via 2‐tailed Student's t‐test. Cell number analyzed: 334 - 364; p < 10-10. Right panel: automated quantitation of the number of DRiP foci/nucleus. Cell number analyzed: 334 - 364; p < 10-10. Automated DRiP segmentation is based on azide signal. DRiPs and polyUb (FK1) protein distribution in cells treated as described in (A). Automated DRiP segmentation is based on azide signal. Quantitation of the number of DRiP foci enriched for polyUb (>2) is shown; n = 590 - 2114; statistical significance via 2‐tailed Student's t‐test; p < 10-10. Scale bars: 10 µm. Immunoblotting on protein extracts from HeLa cells lipofected with non-targeting siRNA control or a specific siRNA against Ubc9. A dilution curve of the control sample is shown. TUBA4A was used as loading control. Quantitation of the number of PML-NBs/nucleus from cells treated as described in (D). Statistical significance via 2‐tailed Student's t‐test. Cell number analyzed: 192 - 430; p < 10-10. Automated PML-NB segmentation is based on PML signal. Cells lipofected for 72 h with non-targeting siRNA control or a specific siRNA against Ubc9 were treated with MG132 (10 µM) and OP-puro (25 µM) for 4 h. Automated quantitation of the number of PML-NBs/nucleus is reported. Statistical significance via 2‐tailed Student's t‐test. Cell number analyzed: 151 - 225; p < 10-10 (see panel G). Quantitation of the number of PML-NBs/nucleus from cells treated as described in (D). Statistical significance via 2‐tailed Student's t‐test. Cell number analyzed: 192 - 430; p < 10-10. Automated PML-NB segmentation is based on PML signal. Representative pictures showing that Ubc9-depleted cells cannot efficiently induce the self-assembly of PML-NBs upon treatment with MG132 (10 µM) and OP-puro (25 µM) for 4 h. Scale bars: 10 µm.
USP22 knockdown in RIPK3-expressing HeLa cells induces resistance to TBZ-induced necroptotic cell death A HeLa TRex RIPK3 CRISPR/Cas9 control (ctr) and USP22 KO cells were treated with 1 µg/ml Dox overnight.Figure 3A HeLa TRex RIPK3 CRISPR/Cas9 control (ctr) and USP22 KO cells were treated with 1 µg/ml Dox overnight. Protein expression of induced Strep-RIPK3 was analyzed by Western blotting. GAPDH served as loading control. The asterisk marks an unspecific band. B HeLa TRex RIPK3 control and USP22 KO cells were incubated with 1 µg/ml Dox for 18 h before pre-treatment with 20 µM zVAD.fmk, 5 µM BV6 for 1 h. After pre-treatment, 10 ng/ml TNFα was added and cell death was measured after 4 and 5 h by analysis of PI-positive nuclei. C HeLa TRex RIPK3 control and USP22 KO cells were pre-treated with 20 µM zVAD.fmk, 5 µM BV6 for 1 h. After pre-treatment, 10 ng/ml TNFα were added for 1, 2, 3, 4 and 5 h. Protein expression of phosphorylated RIPK1, total RIPK1, total RIPK3, phosphorylated MLKL, total MLKL and USP22, without (left) or with (right) 1 µg/ml Dox treatment overnight, was monitored by Western blotting. GAPDH was used as a loading control. D HT-29 control cells and RIPK3 KO cells re-expressing PAM-mutated Dox-inducible RIPK3 WT were incubated overnight with 1 µg/ml Dox. Cells were pre-treated with 20 µM zVAD.fmk, 5 µM BV6 for 1 h. After pre-treatment, 10 ng/ml TNFα were added for 2 h, as indicated. Strep-RIPK3 was immunoprecipitated using anti‑Strep-beads and the indicated co-immunoprecipitated proteins were analyzed by Western blotting. β-Actin served as a loading control. E USP22 KO HT-29 cells and USP22 KO cells re-expressing PAM-mutated 3xFLAG-HA-USP22 were pre-treated with 20 µM zVAD.fmk, 5 µM BV6 for 1 h. After pre-treatment, 10 ng/ml TNFα was added for 2 h, as indicated. 3xFLAG-HA-USP22 was immunoprecipitated using anti-HA-beads and the indicated co-immunoprecipitated proteins were analyzed by Western blotting. β-Actin served as a loading control.
Figure 5The different PRKG2 promoter regions were inserted into the pGL4.1 luciferase report vector. The core promoter reporter vector of PRKG2 (-472/+131) was determined for transcription activity in cultured N2a cells, obtained from 3 independent experiments. One-way ANNOVA with Tukey's test; ***P<0.001. Data are mean ±SEM. The treatment of LAQ (5 or 10 μM) caused a marked inhibition on the transcription activity of the PRKG2 promoter detected by the luciferase assay from 3 independent experiments. One-way ANNOVA with Tukey's test. ***P<0.001. Data are mean ±SEM. The putative cis-elements (in box and sequences below) in the PRKG2 promoter were determined by TFSEARCH, ConTra and ALGGEN program analyses. Six AhR transcriptional factor binding sites were predicted according to the conserved sequence of "CACGC" or "GCGTG". The qPCR analysis of the transcripts of Cyp1a1 and Ugt1a6a, which were mediated by AhR, in the PLP-150Q mouse brain (corpus callosum). Note that Cyp1a1 and Ugt1a6a were significantly increased by LAQ (5 mg/kg). n=3 mice in each group. Student's t-test; Cyp1a1: ***P=0.00042; Ugt1a6a; ***P=0.00057. Data are mean ±SEM. The PRKG2 core promoter activity in N2a cells transfected with AhR siRNA or its scrambled siRNA control and then treated with 5 μM LAQ. The values of promoter activity via luciferase report assay were obtained from three independent experiments. One-way ANNOVA with Tukey's test. ***P< 0.001. Data are mean ±SEM. The semi-PCR detection of PRKG2 promoter DNAs associated with AhR that was immunoprecipitated by anti-AhR in ChIP assay. The N2a cells were treated with 10 μM LAQ or 10 nM 2,3,7,8-TCDD or DMSO for 12 hr. The quantification of PRKG2 promoter DNAs associated with AhR that was immunoprecipitated by anti-AhR in ChIP assay. The N2a cells were treated with 10 μM LAQ or 10 nM 2,3,7,8-TCDD or DMSO for 12 hr. The results were obtained from three independent experiments. One-way ANNOVA with Tukey's test. LAQ: ***P=0.00065; 2,3,7,8-TCDD: ***P=0.00014. Data are mean ±SEM
NSMCE2 is dispensable for DNA replication A WB illustrating the depletion of NSMCE2 in B cells isolated from 2‐month‐old CD19+/Cre animals of the indicated genotypes, 48 h after being stimulated in vitro with lipopolysaccharide (LPS).Figure 7A WB illustrating the depletion of NSMCE2 in B cells isolated from 2‐month‐old CD19+/Cre animals of the indicated genotypes, 48 h after being stimulated in vitro with lipopolysaccharide (LPS). Tubulin was used as a loading control. B Representative FACS analysis from the B‐cell cultures mentioned in (A). DNA content was measured with propidium iodide (PI). Note the presence of cells with > 4n DNA content in NSMCE2‐deleted cells. C, D Frequency of origin firing (C) and fork rate (D) were analyzed by DNA fiber analyses (see ) from fibers prepared from B‐cell cultures explained in (A). No significant differences were observed between Nsmce2+/+ and Nsmce2lox/loxB cells. Data are representative of two independent experiments. Error bars indicate SD. Between 200 and 300 tracks were measured to estimate fork rate and 300-500 for the calculation of origin firing frequencies. E Representative images of the DNA fibers used for the analysis explained in (C, D). CldU (red) and IdU (green) channels are shown.
Figure 7A Mean enrichment profiles performed with SeqMiner presenting the genomic distribution of H2Bac and H3K27ac reads obtained in ChIP-seq experiments in the different experimental groups, established along putative neuronal CBP enhancers (± 4Kb) (Kim et al., 2010). In the dorsal hippocampus, H2Bac levels are enriched at CBP enhancers in WT WEH (blue), decreased in tauopathic mice (TAU VEH, pink) and induced by CSP-TTK21 treatment (TAU MOL, green). B Mean profiles were separated in two sets of loci, including 17817 peaks highly enriched in H2Bac and showing a differential regulation in the experimental groups and 23331 peaks that were poorly enriched in H2Bac and not regulated Functional annotation of the 17817 peaks highly enriched in H2Bac performed with GREAT, GOTERM Cellular Component and Biological Process Mean profiles established with SeqMiner for all genes at TSS for H2Bac (Left) and H3K27ac (Right). H2Bac but not H3K24ac presents a differential regulation at TSS Mean H2Bac profiles established with SeqMiner on the differentially regulated genes obtained in transcriptomic experiments (RNA-seq), either decreased (left) or increased (right). The 'basal' H2Bac TSS enrichment state of these genes is differentially regulated whether the genes are decreased or increased in response to learning UCSC genome browser view of the c-fos genomic locus with H3K27ac ChIP-seq (blue), H2Bac ChIP-seq (green) and Input (black) signals. Blue shading indicates locations of the c-fos enhancers (Joo et al., 2016); gray shading indicates location of the promoter. Differential peaks were analyzed with SICER. This set of data shows that H2Bac levels were significantly decreased at e1 (FDR=1.95.10-08), e2 (FDR=0.0005) and e5 (FDR= 2.44.10-05) enhancers in THY-Tau22 compared to WT mice, and significantly enriched at e1 (FDR= 4.01.10-08) the regions encompassing e3, e4 and TSS (FDR=7.53.10-07) and e5 (FDR= 0.001) after CSP-TTK21 treatment. Expanded View Figure Legend