EMBO/sd-character-level-ner huggingface dataset:
Figure 3A, B. Carfilzomib and proteasome inhibitor MG132 activate Il-1β luciferase in macrophages. (A) BMDMs were treated with IL-4 (20 ng/mL) for 24 hours and then stimulated by DMSO, Carfilzomib (1 μM), or MG132 (5 μM). (B) BMDMs were stimulated by DMSO, Carfilzomib (1 μM) or MG132 (5 μM)) for 1 hour, then induced with IL-4 (20 ng/mL). Luciferase assays were monitored 12 hours after stimulation. The data are means ± SD of three independent experiments. ***p < 0.001 (student's t-test). C. MG132 promotes the expression of M1 macrophage markers and inhibits the expression of M2 macrophage markers. BMDMs were pretreated with IL-4 (20 ng/mL) for 24 hours and then stimulated by DMSO or MG132 (5 μM). RNA was extracted from BMDMs 6 hours after stimulation and the expression of M1 (Il-1β / Il-6 / Inos) or M2 (Cd206 / Arg1) macrophage markers were quantified through RT-qPCR. The data are means ± SD of three independent experiments. *p < 0.05, ***p < 0.001 (student's t-test). D. Knockdown of β5 subunit of proteasome in macrophages promotes the expression of proinflammatory factors. RNA was extracted from Raw264.7 (shGFP or shPsmb5) and the expression of Il-1β and Il-6 were quantified through RT-qPCR. The data are means ± SD of three independent experiments. *p < 0.05, ***p < 0.001 (student's t-test). E. Carfilzomib, Bortezomib, MLN9708 and MG132 induce ER stress response. BMDMs were pretreated with IL-4 (20 ng/mL) for 24 hours and then stimulated by DMSO or Carfilzomib (1 μM), Bortezomib (1 μM), MLN9708 (2 μM), MG132 (5 μM). RNA was extracted from cells 3 hours or 6 hours after stimulation and the expression of ER stress related genes (Bip, Chop) were detected through RT-qPCR. The data are means ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 (student's t-test). F. ER stress agonists, TUN and TG, promote the expression of M1 macrophage markers, as well as reduce the expression of M2 macrophage markers. BMDMs were pretreated with IL-4 (20 ng/mL) for 24 hours and then stimulated by DMSO, TUN (500 nM) or TG (500 nM). RNA was extracted from cells 6 hours after stimulation and indicated genes were quantified through RT-qPCR. The data are means ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 (student's t-test). G. Inhibition of ER stress impairs the ability of Carfilzomib, Bortezomib, MLN9708 and MG132 to reprogram M2 towards M1-like macrophages. BMDMs were induced by IL-4 (20 ng/mL) for 24 hours, then pretreated with 4-PBA (5 mM) for 1 hour and stimulated by Carfilzomib (1μM), Bortezomib (1μM), MLN9708 (2 μM), MG132 (5 μM). RNA was extracted from cells 6 hours after stimulation and the expression of Il-1β, Il-6 and Inos were detected through RT-qPCR. The data are means ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 (student's t-test).
Figure 6A) Immunofluorescence characterization of reconstructed TA against vW (green) and Dys (red). B) Enlarged view of dashed area in (A): white arrows indicate new vessels. Inset: dashed area higher magnification (arrows indicating vWF positive capillaries) C) Superimposed image in (B) over LacZ stained image. D, E) Vessel density assessment by means of fluorescence, statistical significance was analyzed by Anova test (P < 0.05 was considered significant: *=0,0019).
Figure 3A Schematic diagram showing the domain structure of human Ykt6 protein. Ykt6 has two cysteine residues, Cys194 and Cys195, at the fifth and fourth positions from the C-terminus. B Alignment of the C-terminal amino acid sequences of Ykt6 from various species. The conserved tandem cysteines are highlighted in gray. C Farnesylation of recombinant Ykt6 by FTase. Recombinant Ykt6 (5 μM) was incubated with FTase (0 or 50 nM) and 3H-FPP (1 μM; ~3,500 dpm/pmol) at 37ºC. Reactions were stopped at the indicated time points, and the amount of 3H-farnesyl transferred to Ykt6 was quantified by scintillation counting (mean ± SEM, n = 3). D Geranylgeranylation of recombinant Ykt6 by GGTase-III. Recombinant Ykt6 (5 μM) was incubated with increasing concentrations of GGTase-III (0-100 nM) and 3H-GGPP (1 μM; ~3,000 dpm/pmol) for the indicated times, and the amount of 3H-geranylgeranyl transferred to Ykt6 was quantified (mean ± SEM, n = 3). E Geranylgeranylation of Cys to Ser mutants of Ykt6 by GGTase-III. WT and mutant Ykt6 proteins (5 μM each) were incubated with GGTase-III (100 nM) and 3H-GGPP (1 μM) for the indicated times, and the amount of 3H-geranylgeranyl transferred to Ykt6 was quantified (mean ± SEM, n = 3). F Sequential prenylation process of Ykt6. SAM, S-adenosylmethionine; SAH, S-adenosylhomocysteine; PPi, inorganic pyrophosphate. G Mass spectra of prenylated Ykt6 peptides. Recombinant Ykt6 was farnesylated by FTase (FTase), further processed by RCE1 and ICMT (RCE1/ICMT), and finally geranylgeranylated by GGTase-III (GGTase-III). After purification to homogeneity, the prenylated Ykt6 proteins were digested with chymotrypsin (left) or V8 protease (right) and analyzed by MALDI-TOF mass spectrometry. The calculated m/z values for the C-terminal peptide ions are shown in parentheses. The peaks labeled with an asterisk correspond to amino acids 14-38 of Ykt6 (AKVVLLKAAYDVSSFSFFQRSSVQE; m/z 2806.48). Effects of C-terminal processing and Cys195 farnesylation on Cys194 geranylgeranylation by GGTase-III. Purified recombinant unprenyl Ykt6, Cys195-farnesyl Ykt6, and Cys195-farnesyl Ykt6ΔAIM (5 μM each) were incubated with GGTase-III (100 nM) and 3H-GGPP (1 μM) for the indicated times, and the amount of 3H-geranylgeranyl transferred to Ykt6 was quantified. (mean ± SEM, n = 3). I Gel filtration chromatography of purified unprenyl Ykt6, Cys195-farnesyl Ykt6ΔAIM, and Cys194/195-diprenyl Ykt6ΔAIM (135 μg each) on a Superdex 200 column. Dotted lines indicate the elution profile of unprenyl Ykt6 for comparison. The prenylated Ykt6 proteins were eluted slightly later than the unprenylated Ykt6, suggesting more compact and stable folds.
Fig.Figure 7A) Wildtype (BY4741) cells labeled with FM4-64 show extensive contact zones between vacuoles. Scale bar: 2 µm. B) Schematic view of FM4-64 diffusion through a small fusion pore located centrally (left panel) or laterally (right panel). The laterally positioned fusion pore leads to diffusion paths of unequal length for fluorescence recovery on both sides of the bleached vacuole. The red line indicates the intersections with the membrane where fluorescence recovery is measured. C) FRAP analysis of FM4-64-labeled wildtype cells was performed by acquiring pictures in continuous acquisition mode after bleaching, for a total period of 5 s. Three representative frames after photobleaching are shown. The bars in the magnified parts of frames 2 and 3 illustrate the positions at which the recovery of FM4-64 fluorescence was quantified. Scale bar: 2 µm. D) Quantification of recovery experiments in (C). A delay of >50 ms between the appearance of the signal on both sides of the photobleached vacuole was considered as asymmetric recovery. The histogram shows means and s.d. from 3 independent experiments evaluating 50 stained vacuoles each. A Schematic view of the various possible positions of a fusion pore and its consequences for our ability to detect asymmetric FM4-64 recovery. The contact zone between the two vacuoles is tilted by 90° in the middle panel, which gives four examples for the positioning of the pore (shown as a black dot). Asymmetric recovery could only be detected if the pore is sufficiently far from the center of the contact zone and close to the optical plane that is imaged (the cases marked in red).
Trf1-deficient carcinomas present high amount of telomeric DNA damage and apoptosis.Figure 3Percentage of cells showing γH2AX foci in carcinomas of the indicated genotypes (left panel). Representative images of γH2AX immunohistochemistry (right panel). Percentage of cells showing 3 or more γH2AX and RAP1 colocalizing foci (TIFs) (left panel). Representative images of γH2AX and RAP1 double immunofluorescence (right panel). Yellow arrowheads: colocalization of γH2AX and RAP1. Percentage of active caspase-3 (AC3)-positive cells (left panel). Representative images of AC3 immunohistochemistry (right panel).
Translational readthrough in heterologous context (A) AGO1 ISR can drive translational readthrough in a heterologous context of RHOA.Figure 4AGO1 ISR can drive translational readthrough in a heterologous context of RHOA. AGO1 coding sequence in AGO1-TGA-ISR-FLuc construct was replaced by RHOA coding sequence (582 nucleotides) and the readthrough assay was done as described above in HeLa cells. 3′UTRRho, 159 nucleotides in the proximal 3′UTR of RHOA (NM_001664); ISRAgo1, Inter-stop codon region of AGO1. ***, P < 0.0001. AGO1 ISR can drive translational readthrough in a heterologous context of renilla luciferase (RLuc). AGO1 coding sequence in AGO1-TGA-ISR-FLuc construct was replaced by RLuc coding sequence and the readthrough assay was done as described above in HeLa cells (left) and in vitro using rabbit reticulocyte lysate system (right). **, P = 0.006; ***, P = 0.0001. Tethering of N-peptide-tagged Ago1 or Ago1x can induce readthrough of AGO1. Two BoxB elements were cloned downstream of the AGO1 and upstream of the in-frame FLuc as shown in the schematic. This construct was transfected in HeLa cells expressing N-peptide-tagged or FLAG-tagged Ago proteins as indicated. Readthrough was measured as described above. *, P = 0.035 (with Welch correction); **, P = 0.016.
Figure 1Cross-link connectivity map of SVgrG-Tle1H complex. The two proteins are represented as rectangles in which the residues number and known domains are indicated. Straight lines represent inter-molecular cross-links, while dotted lines represent intra-molecular cross-links. Only the cross-links identified in two independent experiments were considered. Raw and cured data are provided in Dataset EV1. Schematic representation of an alignment of Tle1 orthologs using ClustalW and draw alignment tool in KEGG(Kanehisa et al, 2017). Red box, bit score ≥ 200; pink box, bit score ≥ 80-200. Ordered locus names of the identified genes are indicated on the right. Domains are represented in grey boxes (Hcp, PF05638; PAAR (P), PF05488; VgrG, PF05954, COG3501; DUF2345, PF10106). The N-terminal segment found in EAEC Tle1 protein and absent in the others is represented by a blue box. Antibacterial competition assay. E. coli K12 recipient cells (W3110 gfp+, kanR) were mixed with the indicated EAEC attacker cells (1:4 ratio): WT, ∆T6SS-1 and ∆tle1-tli1 carrying pBAD18 and pBAD33 vectors, or producing the indicated proteins, and spotted on SIM 0.02 % arabinose agar plates for 4-h at 37°C. The image of a representative bacterial spot is shown. The number of recovered prey cells is indicated in the upper graph (in log of colony-forming units (cfu)). The black, dark grey and light grey circles indicate values obtained from three different spots, and the average is indicated by the bar. Western blot analysis of the production of Tle1V and Tle1∆1-26 V is shown in the inset. The experiment was performed in triplicate and a representative result is shown. Co-immunoprecipitation assay. Lysates from E. coli K-12 W3110 cells producing VSVG-tagged Tle1 (Tle1V) or VSVG-tagged Tle1∆1-26 truncated variant (Tle1∆1-26 V) were mixed with lysates from W3110 cells producing FLAG-tagged VgrG (VgrGFL) or not (-, empty vector) and subjected to immunoprecipitation on anti-FLAG-coupled beads. The mixed soluble lysates (Input) and the immunoprecipitated material (IP) were subjected to 12.5% SDS-PAGE and immunodetected with anti-FLAG (upper panel) and anti-VSVG (lower panel) monoclonal antibodies. Molecular weight markers (in kDa) are indicated on the left. This experiment was performed in triplicate and a representative result is shown.
Fig.Figure 1Representative flow cytometry dot plots showing the myeloid cell landscape in eWAT of 16-week-old mice fed on a high-fat diet (HFD) (right) and the corresponding lean controls (left). The upper panel shows the F4/80 versus CD11b staining profile gated by CD45+CD24-Ly6G- cells. The middle panels show the MHCII versus CD11c staining profile of F4/80hi macrophages and Ly6C versus MHCII staining profile of F4/80int cells, respectively. The lower panel shows the Ly6G versus CD11b staining profile gated by total CD45+cells (n = 8 mice). Representative phenotype profiling of distinct myeloid subpopulations in lean or obese (HFD 16 weeks) eWAT: CD206, CD301, CD9, CD163, Cx3CR1, CCR2, and Trem2 expression is shown in the respective colored histograms specific for each cell type (n = 3-5 mice per group). The y axis shows relative cell count for each population (normalized to mode). Pearson correlation coefficients between eWAT weight and cell numbers of MHCIIlow, MHCIIhi, CD11c+ ATMs, Ly6ChiMHCII-, Ly6ChiMHCII+, Ly6C-MHCII+, and Ly6G+ neutrophils. Significance was determined using the Pearson correlation test. High-dimensional UMAP analysis shows a dynamic distribution of distinct myeloid clusters in eWAT during aging and the development of obesity. Three timepoints were analysed (8, 12, and 16 weeks) with annotated, colored clusters based on their expression of 13 different markers (n = 3 for each timepoint). Illustration of global topological relationships between clusters of different myeloid cell populations in the eWAT based on the result in D.
Senescence entry by human cells is mostly transcriptionally driven.Figure 1Proliferating and senescent IMR90 and HUVECs assayed for β-galactosidase activity (left), cell cycle profiling via FACS (middle) and proliferation via MTT assays (right). Heatmaps showing changes in gene expression levels upon senescence (log2FC) of genes encoding selected chromatin-associated factors. For each gene shown, statistically significant expression changes were recorded in at least one cell type. Western blots showing changing protein levels on the path to senescence in IMR90 and HUVECs. Passage 6 cells represent the proliferating state, and passages 21 and 34 the senescent state for HUVECs and IMR90, respectively. Super-resolution (gSTED) imaging of HMGB1 distribution in proliferating HUVEC nuclei counterstained with DAPI. Bar: 2μm. Representative immunofluorescence images of IMR90 and HUVECs (left) showing reduced HMGB1 levels in senescent nuclei; bean plots quantify this reduction (right; N is the number of cells analyzed per each condition/cell type). Bars: 5 μm. *: P<0.01; Wilcoxon-Mann-Whitney test. Scatter plots showing correlation between RNA-seq (transcription) and Ribo-seq (translation; log2) in proliferating and senescent IMR90. Pearson's correlation values (ρ) and the number of genes in each plot (N) are also shown.   As in panel F, but correlating RNA-seq and whole-cell proteome changes.   Heatmap showing GO terms/pathways associated with the gene subgroups from panel G (colour-coded the same way). The number of genes in each subgroup (N) are indicated.
Impact of tumor mutational burden on overall survival across treatment groupsOverall survival stratified by tumor mutational burden (TMB) across treatment groups in 3,217 patients.Figure 7Overall survival stratified by tumor mutational burden (TMB) across treatment groups in 3,217 patients. Samples were classified as TMB-high if the amount of non-synonymous mutations per expressed MB (rnaMB) was ≥ the median number of non-synonymous mutations per rnaMB across the whole SCAN-B cohort (0.082 mutations per rnaMB) and TMB-low otherwise. In each Kaplan-Meier plot, TMB-low cases are plotted in blue, TMB-high cases are plotted in red, the log-rank P value is given, and the hazard ratio (HR) for TMB-high is given with a 95% CI and after uni-variable and multi-variable (MV) Cox regression adjustment. Covariables included in the MV analysis were age at diagnosis, lymph node status, tumor size, and the variables denoted by the following symbols: ¶, ER, PgR, HER2, and NHG; ¤, ER, PgR, and NHG; $, HER2 and NHG; #, NHG. Abbreviations: TMB, tumor mutational burden; ER, estrogen receptor; PgR, progesterone receptor; HER2, human epidermal growth factor receptor 2; NHG, Nottingham histological grade; HoR, hormone receptor; TNBC, triple-negative breast cancer.
Figure 6(A&B) Non-biased proteomics of TGFβ-treated naïve (WT) and COUP-TFII overexpressing (COUP-TFII-OE) cells revealed that COUP-TFII promotes the expression of proteins enriched in metabolic process critical for myofibroblasts, in particular, suppression of FAO and enhancement of glycolysis. Top pathways ranked by NES (gene set enrichment with FDR < 0.05) altered in COUP-TFII-OE compared to naïve C3H10T1/2 cells (n=2). (C) TGFβ1 significantly increased more lactate production by COUP-TFII-OE (OE) cells when compared to WT or COUP-TFII-KO (KO) cells treated with TGFβ1. Interestingly, overexpression of COUP-TFII alone (without TGFβ1) significantly increased lactate production compared to WT (n=3). **p<0.01, ****p<0.0001 by one-way ANOVA, mean ± SD. (D) WT, COUP-TFII overexpression (OE) and COUP-TFII knockout (KO) cells were seeded in Seahorse XF-24 cell culture microplates. The cells were rendered quiescent in 0.5% FBS DMEM overnight and then treated with or without 10 ng/ml TGFβ1 for 24 hours. All the cells were incubated in the glycolysis stress test medium without glucose and pyruvate, followed by sequential treatments with glucose (10mM), oligomycin (5μg/ml) and 2-DG (50mM). Real-time extracellular acidification rate (ECAR) was recorded as the baseline (before glucose), the rate of glycolysis (after glucose), glycolytic capacity (after oligomycin), and glycolytic reserve (after 2-DG) (n = 10). (E) COUP-TFII OE significantly increased, while COUP-TFII KO decreased, in the baseline, rate of glycolysis, glycolytic capacity, and glycolytic reserve when compared to WT cells under TGFβ1 stimulation. Without TGFβ1 treatment, there is no difference on the baseline and glycolysis among WT, OE, and KO cells. COUP-TFII KO significantly decreased glycolytic capacity and reserve compared to WT, even without TGFβ1 stimulation (n=10), *p<0.05; **p<0.01; ****p<0.0001 by one-way ANOVA, mean ± SD. (F) 2-deoxyglucose (2-DG) treatment inhibits TGFβ1-induced glycolysis (left) and fibrosis (right) in COUP-TFII-OE cells by qRT-PCR (n=6). **p<0.01; ****p<0.0001 by one-way ANOVA, mean ± SD. We confirmed that 2-DG suppressed TGFβ1-induced proteins of αSMA and ColA1 in COUP-TFII OE cells without change the protein of COUP-TFII by western blot (n=3). **p<0.01 by one-way ANOVA.
Figure 6A 15NO3- -uptake (δ15N‰) in X. laevis oocytes in the presence of equal concentrations of Cl- or gluconate at ~pH 5.5. The oocytes were exposed to 10 mM Na15NO3 for 2h. Biological replicates: n=4 (2 oocytes in each sample). Data information: Asterisks denote significant difference: **P<0.01, *P<0.05 (Student's t-test). In the boxplots, horizontal black bars represent the median, horizontal red bars represent the mean. Boxes represent the middle 50% of the distribution, and whiskers represent the entire spread of the data. Means were compared using Student's t-test, α= 0.05. B Oocytes were exposed to 1.17 mM 36Cl- for 2h in Cl free condition. Biological replicates: n=6 (4 oocytes in each sample). Data information: Asterisks denote significant difference: **P<0.01, *P<0.05 (Student's t-test). In the boxplots, horizontal black bars represent the median, horizontal red bars represent the mean. Boxes represent the middle 50% of the distribution, and whiskers represent the entire spread of the data. Means were compared using Student's t-test, α= 0.05. C Oocytes were exposed to 1.17 mM 36Cl- for 2h with 1 and 10 mM NO3-. Biological replicates: n=6 (4 oocytes in each sample). D The same data as in C is shown without AtNPF6.3 to allow for better comparison between MtNPFs. Biological replicates: n=6 (4 oocytes in each sample). Data information: Asterisks denote significant difference: **P<0.01, *P<0.05 (Student's t-test). In the boxplots, horizontal black bars represent the median, and black cross represent the mean. Boxes represent the middle 50% of the distribution, and whiskers represent the entire spread of the data. Means were compared using Student's t-test, α= 0.05.
Figure 2Pathway enrichment analysis on genes co-evolving with SHLD2 in Mammalians and Vertebrates using a phylogenetic profiling approach followed by an Enrichr-based analysis. Quantification of the Neutral Comet assay. U2OS cells stably expressing shCtrl, shREV7 or shSHLD2 were exposed to IR (10 Gy) and run in low melting agarose under neutral conditions. Immunofluorescence against DNA stained with SYBR Gold was performed to measure the tail moment. U2OS cells were transfected with the indicated siRNA. 48h post-transfection the cells were treated with NCS to induce DNA damage and the cells were harvested at 0, 1, 2, 4 and 24 hr post NCS treatment. Flow Cytometry analysis of phosphorylated-H2AX signal was used to measure γ-H2AX endogenous signal. Sensitivity to IR monitored by colony formation assay. MCF-7 cells stably expressing the the indicated shRNAs were exposed to increasing doses of IR. 24 h post-irradiation, the cells were re-seeded to allow colony formation, fixed and stained with 0.4% crystal violet. Shown is the quantification of colonies per condition which possessed more than 50 colonies. MCF-7 cells stably expressing the the indicated shRNAs 24 h post-irradiation, the cells were re-seeded to allow colony formation, fixed and stained with 0.4% crystal violet. Shown is the quantification of colonies per condition which possessed more than 50 colonies. cells were exposed to increasing doses of UV radiation.
"TIL-matrix-release-and-reactivate" strategy through targeting TNC and CXCR4TNC contributes to an immune suppressive TME in breast cancer by retaining CD8 TIL in the stroma.Figure 9TNC contributes to an immune suppressive TME in breast cancer by retaining CD8 TIL in the stroma. TNC induces CXCL12 in a TLR4 dependent manner. The TNC protein binds CXCL12 generating a substratum that tethers CD8 TIL in the stroma thus preventing infiltration of CD8 TIL into the tumor nests and subsequent tumor cell killing. This mechanism could be relevant in human breast cancer as patients with tumors of high TNC expression show high CD8 TIL in the (TNC-rich) stroma which correlates with shorter MFS. (Lower left) Upon removal of TNC anti-tumor immunity is activated and characterized by lower CXCL12 expression, higher CD8 TIL nest infiltration and, a reduced metastasis rate as previously shown (Sun et al., 2019). In human breast cancer low TNC and high CD8 TIL correlate with longest MFS. (Lower right) TNC impacts CD8 TIL through CXCL12/CXCR4. Inhibition of CXCR4 led to increased CD8 TIL infiltration, higher tumor cell apoptosis, and reduced tumor growth and subsequent lung metastasis. Also macrophages, loosing their M2 phenotype, infiltrate the tumor nests more numerously contributing to the observed activated CD8 TIL phenotype (e.g. less PD-L1, CTLA4). Targeting TNC and CXCL12 could direct a novel "TIL-matrix-release-and-reactivate" strategy to improve ICT in breast cancer.
Figure 8(A) The studied patient harbored compound heterozygous mutations in COX6A2 gene (Inoue et al, 2019). (B) Scheme depicting the main functions analyzed by Wechsler Adult Intelligence Scale-III, the test used to evaluate the intelligence and cognitive abilities of the patient. (C) Values obtained by the patient. Orange indicates lower than average values.
Tamoxifen treatment decreases proliferation and increases apoptosis in epithelial cells of PDAC tissues.Figure 6(A) Immunofluorescence images of PDAC tissues from KPC mice treated with vehicle control, and 2mg of tamoxifen, scale bar is 100 μm. Upper panels: Ki67 staining is used as a surrogate of proliferation. White arrows indicate Ki67 positive nuclei in epithelial cells. Lower panels: Cc3 staining shows the cells undergoing caspase-mediated apoptosis. Tamoxifen panels show higher levels of yellow staining, which indicates higher percentage of apoptotic epithelial cells. (B) Quantification of staining in panel A (n=4 animals per condition, and n≥ 5 sections per animal, two experimental repetitions). Histogram bars represent mean ± s.e.m.; ***P<0.001, t-test