EMBO/sd-character-level-ner huggingface dataset:
LUZP1 binding affinity for myosin light chain (MLC) depends on the phosphorylation status of MLC.Figure 3Representative confocal micrographs of wild-type (WT) Eph4 cells treated with 100 μM Y27632 for 30 min. Artificial reductions in di-phosphorylated myosin light chain (ppMLC) levels with Y27632 inhibited LUZP1 junctional localization. Scale bar, 10 μm. Bar plots with dot density plots showing that LUZP1 mean intensities within circumferential rings (CRs) were significantly reduced after Y27632 treatment [39.75 ± 6.74 arbitrary units (a.u.) (control) vs. 27.7 ± 4.21 a.u. (Y27632)]. n = 3. **p < 0.01 (Mann-Whitney U test). Bars and error bars represent the mean ± standard deviation (SD). Representative confocal micrographs of WT Eph4 cells treated with 100 nM Calyculin A for 30 min. Artificial upregulations in ppMLC levels with calyculin A promoted LUZP1 junctional localization. Scale bar, 10 μm. Bar plots with dot density plots showing that LUZP1 mean intensities within CRs were significantly upregulated after calyculin A treatment [39.19 ± 8.32 a.u. (control) vs. 42.24 ± 5.40 a.u. (calyculin A)]. n = 3. **p < 0.01 (Mann-Whitney U test). Bars and error bars represent the mean ± SD. A schematic drawing of the positive feedback loop between ppMLC and LUZP1 at tight junction (TJ)-associated CRs to promote apical constriction. Representative confocal micrographs of Venus-LUZP1-expressing LUZP1 KO (REV) Eph4 cells in the apical plane. Scale bar, 10 μm. Magnified micrographs of Fig 3F. Binary images on the right show the colocalization of ppMLC and LUZP1. Scale bar, 1 μm. Schematics of wild-type MLC (WT-MLC), di-dephosphomimetic MLC (AA-MLC, with T18A and S19A mutations), and di-phosphomimetic MLC (DD-MLC, with T18D and S19D mutations). In vitro direct binding assay between FLAG-LUZP1 and GST-MLC. Representative immunoblots are shown. S, supernatant; P, pellet.   A representative plot in which the amount of bound GST-WT-, AA-, or DD-MLC/FLAG-LUZP1 was plotted with the Michaelis-Menten fitted curve.   Bar plots with dot density plots showing that the dissociation constant (Kd) of LUZP1 from DD-MLC was significantly lower than that from WT-MLC and AA-MLC [4.04 ± 1.05 μM (WT-MLC) vs. 4.88 ± 1.79 μM (AA-MLC) vs. 0.85 ± 0.39 μM (DD-MLC)]. n = 5 (WT-MLC and AA-MLC) and n = 4 (DD-MLC). *p < 0.05 [Kruskal-Wallis test followed by Steel-Dwass test]. Bars and error bars represent the mean ± SD.
Figure 1(A, B) C57BL/6 mice were left untreated (NT) or were intraperitoneally treated with indicated amounts of AG for 3 d. Lung sections stained with Haematoxylin and eosin(H&E) (A) and quantification of lung lesion burden from H&E-stained sections (B). Data information: Data are means + SD of indicated numbers of mice from 1 of n=3 independent experiments with similar results and each symbol represents 1 mouse. Data are representative of n=3 independent experiments. One-way ANOVA followed by Dunnett's post hoc test were used for statistical analysis, respectively. ns, not significant; *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001. Scale bar, 200 μm. (C) Sequence and secondary structure of identified aptamers against AG as predicted with DNAMAN version 6.0. (D-G) H&E staining of lung sections from mice 4 weeks after intranasal infection with M. bovis BCG (D and E) or Mtb H37Rv (F and G) in the absence or presence of intranasally administrated AG aptamers (1 μg) once at a 1 week-interval. Quantification of lung inflamed regions shown in (E and G). Data information: Data in are means + SD of indicated numbers of mice from 1 of n=3 independent experiments with similar results and each symbol represents 1 mouse. Data are representative of n=3 independent experiments. One-way ANOVA followed by Dunnett's post hoc test and Student's t test were used for statistical analysis, respectively. ns, not significant; *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001. Scale bar, 200 μm.
IL-6 concentration correlated with α-SMA-expressing CAFs abundance in human and mouse pancreatic tumours and plasma.Figure 7A-C Immunohistofluorescence using an anti-α-SMA or anti-IL-6 antibody in paraffin-embedded sections from MIA PaCa-2-Gluc and CAF cells orthotopic co-xenografted tumours from Fig5A (A) or from human tumour subcutaneously xenografted from Fig5D (B) in nude mice treated or not with gemcitabine + SOM230 (representative of five different tumours). Representative co-localization of α-SMA with IL-6 in human PDAC samples (C) (n = 15). Scale bar = 100 μm. D, E Plasma was collected from MIA PaCa-2GLuc and CAFs (*P = 0.034, **P = 0.007) (D), or human pancreatic tumour (*P = 0.028, **P = 0.004) (E), xenografts in nude mice and human IL-6 plasmatic concentrations (mean ± SD) were quantified by ELISA (n = 5 mice / group). * treated versus untreated mice.
Figure 3A. Strategy for quantification of TNF-RSC-associated proteins. Cells were treated with FLAG-TNF-α for 5 and 15 minutes, and in control FLAG-TNF-α was added directly in the lysates. The TNF-RSCs were affinity-purified using anti-FLAG antibody, proteins were mixed, proteolysed and the relative abundance of proteins quantified with LC-MS/MS. B. Validation of the TNF-RSC interacting proteins. A549 cells were stimulated with FLAG-TNF-α for the indicated time and the TNF-RSCs were isolated as described in Figure 3A. TNF-α-stimulation-dependent interaction of the indicated proteins was confirmed by isolating the TNF-RSC and blotting with the indicated antibodies. The amount of the analyzed proteins in the input material used for the TNF-RSC isolation is also shown. C. Quantification of ubiquitin linkage peptides in the TNF-RSC. The box plot shows the relative abundance of K48, K63 and linear ubiquitin linkage-specific peptides in the TNF-RSCs isolated from TNF-α-stimulated (15 min) and control cells quantified by SILAC-based MS. D. Strategy for identification of SPATA2 interacting proteins. SILAC-labeled A549 cells were transfected with the GFP-SPATA2 or GFP alone, and one of GFP-SPATA2 transfected cell population was left unstimulated and the other one was stimulated with TNF-α for 15 minutes. Proteins were affinity-purified using GFP-Trap agarose and analyzed by LC-MS/MS. E. The list of proteins enriched in SPATA2pull-down. The table shows proteins that were enriched in GFP-SPATA2pull-down. The columns under "Unstimulated" shows SILAC ratios of the indicated proteins in SPATA2 pull-downs from unstimulated cells, whereas the columns under "Stimulated (TNF-α 15 min)" shows SILAC ratios of the indicated proteins in SPATA2 pull-downs from cells stimulated with TNF-α for 15 minutes. F. Validation of TNF-α-stimulation-independent association of SPATA2 with CYLD. GFP-SPATA2 was immunoprecipitated from unstimulated and TNF-α-stimulated cells, and probed with CYLD antibody. G. HOIP interacts with SPATA2. FLAG-HOIP expressing A20.2J cells were SILAC labeled and FLAG-HOIP was immunoprecipitated from unstimulated cells and associated proteins were quantified using LC-MS/MS. The table contains proteins that were enriched in HOIPpull-down compared to control pull-down. H. Validation of SPATA2 interaction with HOIP. GFP-tagged RNF31 was purified from unstimulated or TNF-a-stimulated (15 min) HCT116 cells and the immunoprecipitates were blotted with SPATA2, CYLD and GFP (HOIP) antibodies. The lower panels show expression of the indicated proteins in the input. The asterisk indicates unspecific band.
Knockdown of beclin 1 suppresses the Spns1 deficiency in zebrafish.Figure 2(A) Schematic representation of the zebrafish beclin 1 (zbeclin 1) gene, its mRNA and protein products. A splice-blocking beclin 1 MO was designed to overlap the intron-exon boundary at the 5′-splice junction of exon 4 in the zebrafish beclin 1 gene. To detect aberrantly spliced RNA products, two forward primers were designed for exon 3 (EX3 primer) and exon 4 (EX4 primer), and one reverse primer was designed for exon 7 (EX7 primer) within the beclin 1 gene. The zebrafish beclin 1 gene has a total of 11 exons having three unique domains [BH3 domain, coiled-coil (CCD) domain, and evolutionarily conserved (ECD) domain], and the beclin 1 MO was anticipated to disrupt the BH3 domain encoded by exon 4 and exon 5. (B) Splicing detection of zbeclin 1 mRNA by RT-PCR. Amplified PCR fragments show the intact sizes of the two amplicons for EX3-EX7 and EX4-EX7 following control (water) injection or only spns1 MO injection. Either beclin 1 MO (12 ng/embryo) injection or coinjection of spns1 MO (4 ng/embryo) and beclin 1 MO (12 ng/embryo) generated a skipping of exon 4 (beclin 1Δexon4). This was detected by the presence of an altered EX3-EX7 amplicon and undetectable EX4-EX7 product. The deletion of exon 4 was confirmed by sequencing. Injected embryos were harvested for total RNA isolation at 54 hpf. (C and D) Rescue of the spns1 morphant by beclin 1 knockdown. (C) The yolk opaqueness phenotype appearance in control-injected (water), spns1 MO-injected, and spns1 and beclin 1 MOs-coinjected embryos was followed through 72 hpf. At 24 hpf, opaqueness commenced from the yolk extension region, which had almost disappeared or was severely damaged (more than 95% of spns1 MO-injected animals) with an extension of opacity to the entire yolk at 48 hpf. By 72 hpf, yolk opaqueness became highly dense throughout most of the spns1 MO-injected embryos, which usually died within another 24 h. Scale bar, 250 µm. (D) Clarification of the yolk opaqueness phenotype in spns1 morphants at 72 hpf. As described in (C), more than 95% of the spns1 MO-injected embryos showed a 'mostly opaque' yolk at 48 hpf, and such embryos subsequently died. Animals showing a 'partially opaque' yolk could sometimes be recovered and subsequently survived 96 h and beyond. beclin 1 MO coinjection dramatically increased (more than 10 times) the animal numbers with the partial yolk opaque phenotype. (E) Survival curve for spns1 morphant and spns1;beclin 1-double morphant larvae (log rank test: χ2 = 162.5 on one degree of freedom; p<0.0001).
Figure 5A. The graph shows the number of differentially expressed (DE) genes 24h following SNA or DCA compared to their respective controls. B. RNAseq read counts show differential occupancy of H3K9ac on specific gene regions, including an increased occupancy close to the transcriptional start site (TSS) after SNA only, when compared to corresponding controls. C. Bar graph shows the number of genes that are upregulated or downregulated following SNA or DCA (RNAseq) with an increased or decreased H3K9ac occupancy (ChIPseq), respectively. D. Odds ratio analysis of the enrichment/depletion of genes with differential H3K9ac occupancy across each upregulated and downregulated gene cluster in (C); the numbers in red represent the p value given by Fisher's exact test (ns= not significant). E. Out of the 321 upregulated genes associated with increased H3K9ac upon SNA, the majority does not show a change in expression following DCA (grey), while some of them are downregulated (white). F. Cytoscape visualization of the protein network resulting from HDAC3 interactome and H3K9ac ChIPseq and RNAseq upon SNA. The signalling network was created by combining the protein interactome of HDAC3 and H3K9ac dependent genes with increased gene expression upon SNA. HDAC3 is in red at the center of the network. The line thickness is proportional to the interaction score. Highlighted in orange circles are the validated pathways and targets via IHC (Figure 6) or ChIP-qPCR (EV Figure 4), regeneration associated genes (RAG) are highlighted in orange triangles.
Fibroblastic FAK inactivation reduces M2 macrophage polarization, migration and correlates with M2 macrophage number in human PDAC samples (A-B) Relative frequencies of tumour-infiltrating M1 macrophages and M2 macrophages analyzed by flow cytometry at 21 days (A) and 38 days (B) after grafting.Figure 4(A-B) Relative frequencies of tumour-infiltrating M1 macrophages and M2 macrophages analyzed by flow cytometry at 21 days (A) and 38 days (B) after grafting. (C) Schematic of BMDM isolation and macrophage polarization assay. (D) Impact of conditioned media (CM) secreted by activated mouse fibroblasts expressing FAK-WT or FAK-KD on mouse BMDM-derived macrophage polarization. Relative frequencies of macrophages expressing CD80, CD86, Nos2, CD206, CMH2, Dectin, and CD163 markers acquired by flow cytometry and processed using FlowJo software. (E) Impact of soluble factors secreted by CAFs on macrophage migration. Left: schematic of BMDM derived M1 or M2-like macrophages. Right: Representative pictures and quantification of M1 and M2 macrophage migration at 24 and 48h towards CM secreted by CAFs (previously treated or not with FAK inhibitor for 48h). Cells were stained using Cristal Violet and percentage of cristal violet positive area quantified using Cell Observer videomicroscope (Zeiss) and analyzed with ImageJ. (F) Quantitative real-time PCR conducted with gene-specific primers (CCL2, CCL5 and M-CSF) on four different FAK-I-treated (16h) and non-treated CAFs for gene expression. The normalized fold change ± SEM was calculated using the delta-delta Ct method with RSP16 as a control gene; values are mean +/- SEM. Each individual patient-derived activated fibroblasts was assigned to a specific symbol (G) Elisa quantification of CCL2 (MCP1) concentration (µg/ml) in conditioned medium from hCAFs treated or not with FAK-I during 72h. * p <0.05, by paired t-test; values are mean +/- SEM. Each individual patient-derived activated fibroblasts was assigned to a specific symbol (H) Left: Representative immunohistochemistry pictures of phosphorylated FAK on tyrosine (Y) 397 and of CD206 in serial sections of human PDAC tissues. Scale bar: 200µm Middle: Correlation plot between fibroblastic pY397 FAK score and CD45 or CD206+ cell number within human PDAC samples. Fibroblastic pY397 FAK score and percentage of CD206 positive cell quantifications were performed on a 47 patient cohort based on IHC staining. Spearman r = 0.4145, p=0.0019. Right: Percentage of CD206 positive cells in PDAC patient expressing either low or high fibroblastic FAK activity score.
Figure 7A. Western blot of LC3B in kidney homogenates from leupeptin-treated (40mg/kg, 6h) fasted Ofd1fl/y;creKsp and control mice. (Right) LC3B-II protein levels normalized to Actin (loading control) are expressed as fold increase versus control mice, n=4 mice/group. B. Representative images of GFP-LC3 puncta (autophagosomes, green) in aquaporin2-positive (AQP2, red) renal distal tubules from P8 Ofd1fl/y;creKsp;GFP-LC3 and Ofd1fl/y;GFP-LC3 mice. Scale bar=10μm. Hoechst labels nuclei. Bar graphs on the left show quantitative analysis of GFP-LC3 puncta; n=4 mice/group for a total of 300 AQP2 positive nuclei/group analyzed. C. Western blot of LC3B in kidney homogenates from leupeptin-treated (40mg/kg, 6h) fasted Ofd1-IND and control mice. (Right) LC3B-II protein levels relative to βTubulin (loading control) are expressed as fold increase compared with control mice, n=5 mutant/8 control mice . D. Western blot of Atg13 in Ofd1fl/y;creKsp and control kidneys at pre-cystic (P8, left) and cystic (P22, right) stages. Gapdh was used as loading control. Atg13 protein levels are expressed as fold change compared with control mice; n=7 mutant/5 control mice at P8, n= 4mice/group at P22. E. Haematoxylin & eosin staining of kidney sections from Ofd1fl/y;Atg7+/+;creKsp and Ofd1fl/y;Atg7fl/fl;creKsp mice. (Right) Graph shows fold change of the cysts number observed in Ofd1fl/y;Atg7fl/fl;creKsp animals compared with Ofd1fl/y;Atg7+/+;creKsp; n=12 sections/animal were analyzed; n=6 mice/group. Scale bars=1mm. F. Blood Urea Nitrogen was quantified on blood withdrawn from Ofd1fl/y;Atg7fl/fl;creKsp, Ofd1fl/y;Atg7+/+;creKsp and control mice at P45. n=8 mice per group.
PVM decoration with autophagy adaptors correlates with p62 but not ubiquitination A) HFFs were stimulated with IFNγ (10 U/mL for 24 h) or left unstimulated and subsequently treated with PYR41 (1 µM) for 2 h prior to infection.Figure 3A) HFFs were stimulated with IFNγ (10 U/mL for 24 h) or left unstimulated and subsequently treated with PYR41 (1 µM) for 2 h prior to infection. Cells were washed and subsequently infected with Pru parasites (MOI=3) for 3 h. The percentage of vacuoles that stained positive for total ubiquitin, p62 or LC3B was determined (n=3). For all the co-staining experiments IFNγ-stimulated HFFs infected for 3 h with the Pru strain were used. For each staining at least 50 vacuoles were scored (n=3). On the right-hand side, a representative fluorescent image is shown for the Toxoplasma Pru strain. Scale bar is 20 µm. The yellow box inside each representative image is shown as inset pictures with higher magnification. The total number of coated vacuoles was set at 100% and the percentage of vacuoles positive for Ub, p62 was calculated. For all the co-staining experiments IFNγ-stimulated HFFs infected for 3 h with the Pru strain were used. For each staining at least 50 vacuoles were scored (n=3). On the right-hand side, a representative fluorescent image is shown for the Toxoplasma Pru strain. Scale bar is 20 µm. The yellow box inside each representative image is shown as inset pictures with higher magnification. The total number of coated vacuoles was set at 100% and the percentage of vacuoles positive for Ub, LC3B was calculated. For all the co-staining experiments IFNγ-stimulated HFFs infected for 3 h with the Pru strain were used. For each staining at least 50 vacuoles were scored (n=3). On the right-hand side, a representative fluorescent image is shown for the Toxoplasma Pru strain. Scale bar is 20 µm. The yellow box inside each representative image is shown as inset pictures with higher magnification. The total number of coated vacuoles was set at 100% and the percentage of vacuoles positive for Ub, GABARAP was calculated. For all the co-staining experiments IFNγ-stimulated HFFs infected for 3 h with the Pru strain were used. For each staining at least 50 vacuoles were scored (n=3). On the right-hand side, a representative fluorescent image is shown for the Toxoplasma Pru strain. Scale bar is 20 µm. The yellow box inside each representative image is shown as inset pictures with higher magnification. The total number of coated vacuoles was set at 100% and the percentage of vacuoles positive for p62 LC3B was calculated. For all the co-staining experiments IFNγ-stimulated HFFs infected for 3 h with the Pru strain were used. For each staining at least 50 vacuoles were scored (n=3). On the right-hand side, a representative fluorescent image is shown for the Toxoplasma Pru strain. Scale bar is 20 µm. The yellow box inside each representative image is shown as inset pictures with higher magnification. The total number of coated vacuoles was set at 100% and the percentage of vacuoles positive for p62 GABARAP was calculated.
Crystal structure of Ser65‐phosphoUbStructure of phosphoUb in space group P1 with 8 molecules in the asymmetric unit (Table ).Figure 3A Structure of phosphoUb in space group P1 with 8 molecules in the asymmetric unit (Table ). Ub molecules are colored differently, and atoms for pSer65 are shown as spheres with red oxygen and purple phosphorous atoms. B Six of eight phosphoUb molecules form similar dimer interactions, where phosphoUb molecules interact via their Ile44 patch. This resembles the crystal structure of Lys48‐linked diUb [pdb‐id 1aar (Cook et al, )]. Atoms for pSer65 are shown as in (A), and the Ile44 side chain is shown as blue sticks. C Representative 2|Fo|‐|Fc| electron density, contoured at 1 σ, covering the phosphorylation site. pSer65 is shown in ball‐and‐stick representation, and neighboring side chains are shown in stick representation with red oxygen and blue nitrogen atoms. D In wt Ub (left), Ser65 forms a hydrogen bond with the backbone amide of Gln62. Upon phosphorylation (right), an oxygen atom from the phosphate forms the same backbone hydrogen bond. E Cartoon representation and electrostatic surface potential of Ub (left) and phosphoUb (right) in identical orientations. Electrostatics were calculated using CHARMM (www.charmm-gui.org) and are colored from red (negative potential) to blue (positive potential).
Figure 1Quantitative expression of Lif mRNA in the colon of DSS-challenged and control mice (n=6 per group). LIF measured by ELISA in mouse colon explant supernatants from DSS-challenged and control mice (n=4 per group). Quantitative expression of Lif mRNA in LPLs and IECs from DSS-challenged and control mice (n=4 per group). Quantitative expression of Lif mRNA in the colon of SPF mice treated with or without antibiotics (Abx) at the indicated time points during colitis induction (n=3 per group). Quantitative expression of Lif mRNA in LPLs and IECs from mice treated with or without Abx at the indicated time points during colitis induction (n=3 per group). mRNA expression of the indicated genes in IECs stimulated by LPS (2.5μg/ml), PGN (3μg/ml) or LTA (2μg/ml) for 2 hours. Serum concentrations of LIF in IEC supernatants untreated (G) followed by LPS (2.5μg/ml) or LTA (2μg/ml) for 12hours, as determined by ELISA. Serum concentrations of LIF in IEC supernatants pretreated with monensin (H) followed by LPS (2.5μg/ml) or LTA (2μg/ml) for 12hours, as determined by ELISA. Immunoblot analysis of the expression and modification of the indicated proteins in the total cell lysate of CD4+ T cells (I) and IECs (J) treated with LIF (20 ng/ml) at different time points. The lower panel shows the intensity analysis of the bands from three independent experiments. Stat4 mRNA expression in LPLs and IECs (n=6). The proliferation rate of CD4+ T cells (L) and DLD-1 cells (M) was determined in the presence or absence of LIF.
Figure 7 A Blood glucose during glucose tolerance test (GTT) on female mice after 11 weeks of HFD and 3 weeks of HFD+Rosi (n=8). ***P=0.001, in repeated measures 2x2 ANOVA with Holm-Sidak posttests (Cited4-/- vs. Cited4+/+)   B Fasting serum insulin in female mice after 10 weeks of HFD ("Before Rosi") or 11 weeks of HFD and 2.5 weeks of HFD+Rosi ("After Rosi") (n=8). ***P=0.0009 in repeated measures 2x2 ANOVA with Holm-Sidak posttests (After Rosi vs. Before Rosi); P=0.725 (Cited4-/- vs. Cited4+/+ Before Rosi)   C Blood glucose during GTT on male mice as in (A) (n=5/8). Repeated measures 2x2 ANOVA with Holm-Sidak posttests (Cited4-/- vs. Cited4+/+)   D Fasting serum insulin in male mice as in (C) (n=7/7/8/8). ***P=0.001 (Cited4+/+), ***P=0.0008 (Cited4-/-) in repeated measures 2x2 ANOVA with Holm-Sidak posttests (After Rosi vs. Before Rosi)   E, F Blood glucose during insulin tolerance test (ITT) with 1 U insulin per kg body mass on female mice after 12 weeks of HFD (n=8) (E) or 16 weeks of HFD and 4 weeks of HFD+Rosi (n=7) (F), expressed as % of the 0-time point value. **P=0.003 in repeated measures 2x2 ANOVA with Holm-Sidak posttests (Cited4-/- vs. Cited4+/+). G Area under the curve (AUC) of blood glucose during the ITT (0-120 min) in (E,F). 2x2 ANOVA with Holm-Sidak posttests (After Rosi vs. Before Rosi)   H, I Blood glucose during insulin tolerance test (ITT) on male mice as in (E,F), n=8 (H), n=7/8 (I). Repeated measures 2x2 ANOVA with Holm-Sidak posttests (Cited4-/- vs. Cited4+/+). J Area under the curve (AUC) of blood glucose during the ITT in (H,J). **P=0.001, ***P=0.00006 in 2x2 ANOVA with Holm-Sidak posttests (After Rosi vs. Before Rosi)   K, L Fasting serum non-esterified fatty acids (NEFA) in female (K) or male (L) mice after 10 weeks of HFD ("Before Rosi") or 11 weeks of HFD and 2.5 weeks of HFD+Rosi ("After Rosi"), n=7/8/8/8 (K), n=6/6/6/8 (L). **P=0.001 in 2x2 ANOVA with Holm-Sidak posttests (After Rosi vs. Before Rosi)