EMBO/sd-character-level-ner huggingface dataset:
Visualizing the various tumor cell fates in response to CTL attack in vitro(A) OVA-expressing lymphoma B cells are killed by OT-I CTL in a bulk cytotoxic assay.Figure 1(A) OVA-expressing lymphoma B cells are killed by OT-I CTL in a bulk cytotoxic assay. OT-I CTLs were cultured in the presence of OVA+ Eµ-myc B cells in either U-bottom (white) or flat-bottom (grey) plates. Tumor cell killing was evaluated by flow cytometry at the indicated time points. Data were pooled from three independent experiments with 3 distinct image acquisitions in each experiment. (***p<0.001, unpaired t‑test). (B) Scheme illustrating that perforin-mediated pore formation promotes the entry of calcium and propidium iodide (PI) in the cytosol of target cells. (C-D) Diverse outcome for tumor cells following interactions with CTLs. OT-I CTLs and OVA+ Eµ-myc B cells were labeled with the calcium-sensitive dye Indo-1, cultured together in the presence of propidium iodide (PI) and subjected to live imaging. Representative time-lapse images (C) and corresponding fluorescence quantification (D) showing the three distinct outcomes for CTL-tumor cell contacts (left, calcium response in T cells, and right, calcium and PI signal in targets): no detectable damage (no calcium signal in the target cells), reversible damage (transient calcium elevation and low uptake of PI), and irreversible damage (prolonged calcium elevation and bright PI staining). CTLs are outlined by white dashed lines. White arrowheads indicate tumor cells. Red arrowheads indicate tumor cells with internalized PI. Scale bar, 5 µm. (E) Pie chart representing the various fates of tumor cells following conjugation with a CTL. Data were pooled from three independent experiments with 3 distinct image acquisitions in each experiment.
Yip1A is involved in the formation of large vacuoles through the activation of IRE1, and Atg9 and WIPI1, but not DFCP1, are required for this process.Fig 4HeLa cells were transfected with each siRNA for 24 hr, and then treated with Tm for 5 hr to induce the UPR. (A, B) Representative confocal micrographs of control (left-hand panels), Yip1A-knockdown (middle panels), and IRE1-knockdown (right-had panels) cells after 0 hr or 5 hr of Tm treatment (A). The ER structure was visualized with an anti-calnexin antibody. Large vacuoles were observed in control cells (arrowheads), but not in Yip1A- or IRE1-knockdown cells. Scale bars are 10 μm. The percentage of cells with vacuoles was counted, and is shown in the bar graph (B). Data are means ± SD from three independent experiments (N = 100). **: p<0.01. (C, D) Representative confocal micrographs of control, Atg9-knockdown, WIPI1-knockdown, and DFCP1-knockdown cells after 0 hr or 5 hr of Tm treatment (C). The ER structure was visualized with an anti-calnexin antibody. Large vacuoles were observed in control and DFCP1-knockdown cells (arrowheads), but rarely seen in Atg9- or WIPI1-knockdown cells. Scale bars are 10 μm. The percentage of cells with vacuoles was counted, and is shown in the bar graph (D). Data are means ± SD from three independent experiments (N = 100). **: p<0.01.
Figure 7The control or CEP41 siRNA-transfected HUVECs were cultivated under hypoxia (1% O2) and their whole cell lysates were used for immunoblot assays of CEP41, phospho-AURKA, and AURKA. The resulting data were also compared to those of normoxic cells. Protein levels were normalized against β-ACTIN in the same blots. VEGFA (B) and VEGFR2 (C) mRNA levels were measured by qRT-PCR using cDNA from normoxic and hypoxic controls or CEP41-depleted cells. The expression of GAPDH was quantified for the normalization of those qRT-PCR results. Tg(kdrl:eGFP) zebrafish were injected with control- or cep41-MOs and then incubated under hypoxia at 28 hpf (a stage of high shear stress) for 2 h. They were subjected to immunostaining with phospho-AURKA-specific antibodies (red) and DAPI at 30 hpf. The insets indicate the representative areas from each immunostaining and red dots indicate activated aurka within ECs. Scale bars, 40 μm. Quantification of phospho-aurka-positive ECs (E) in equivalent fields of view for each MO-injected zebrafish in (D) are the result of three independent experiments with ≥ 20 embryos per condition. The top and bottom whiskers represent the maximum and minimum values, respectively. *P < 0.05 (Unpaired Student's t-test). Zebrafish vegfa mRNA levels were quantified by qRT-PCR in eGFP-positive ECs of control- or cep41-MO-injected Tg(kdrl:eGFP) zebrafish subjected to either normoxia or hypoxia at 30 hpf. The expression of zebrafish β-actin was quantified for the normalization of these qRT-PCR results. vegfr2 (G) mRNA levels were quantified by qRT-PCR in eGFP-positive ECs of control- or cep41-MO-injected Tg(kdrl:eGFP) zebrafish subjected to either normoxia or hypoxia at 30 hpf. The expression of zebrafish β-actin was quantified for the normalization of these qRT-PCR results. Control and CEP41-deficient HUVECs, transfected with expression vectors encoding nothing (MOCK), AURKA, AURKA-T288D, HIF1α, or HIF1α with CEP41 were cultivated under hypoxia and subjected to an in vitro angiogenesis assay for 18 h. Scale bars, 400 μm. Control and CEP41-deficient HUVECs, transfected with expression vectors encoding nothing (MOCK), AURKA, AURKA-T288D, HIF1α, or HIF1α with CEP41 were cultivated under hypoxia and subjected to an in vitro angiogenesis assay for 18 h. Scale bars, 400 μm. Quantification of tube node number in (I) and tube length in (J) from data examined within equivalent fields of view at each time point using the ImageJ angiogenesis analyzer. Data are shown as mean ± SD of five independent experiments with ≥ 5 tubulogenesis regions per condition.
METPlatform as a clinically-compatible "avatar".Figure 8A Schema of experimental design using GB-PDOC. B Correlation between response in GB-PDOC (left side of the graph) and its respective patient (right side of the graph). Response in GB-PDOCs was obtained by quantification of number of proliferative cancer cells found in Rx + TMZ treated normalized to DMSO treated (100%) PDOCs from the same patient. Representative GB-PDOC responding (R) or not (NR) to the standard of care that was provided ex vivo (Radiation (Rx): 2x 10 Gy + temozolomide (TMZ) 250 µM) are shown. Scale bar: 50 µm. Values are shown as mean ± s.e.m. (n=3-6 PDOCs per experimental condition per patient, each patient is represented in an individual bar. Fourteen patients included; each patient is an independent experiment). Time to progression of the patients after neurosurgery is represented in months. Response to Rx + TMZ was evaluated by volumetric measurement of the lesion (dashed line) based on MRI before and after the treatment. Representative patients responding (R) or not (NR) to the standard of care are shown. A white circle indicates progressive disease. Patients with ongoing response to Rx + TMZ (stable disease) are indicated with green bars. MGMT promoter methylation status is shown for each patient (met: methylated; unmet: unmethylated). N/A: not available. C Correlation between GB-PDOC and patient responses. GB-PDOC response is indicated by the mean value in percentage of proliferation post-treatment, where 40% represents the threshold. Patients are classified as responder (R) (green dots, 8 patients) or non-responder (NR) (white dots, 6 patients) according to the MRI evaluation. Values are shown in box-and-whisker plots where each dot is a patient and the corresponding GB-PDOC and the line in the box corresponds to the median. The boxes go from the upper to the lower quartiles and the whiskers go from the minimum to the maximum value. P value was calculated using two-tailed t-test. D Pie charts representing the percentage of patients where MGMT methylation status correlates with the expected therapeutic response in the patient and the same respect to the response of the GB-PCOC in METPlatform.
a) Localization of photoconverted KFERQ–PS–CFP2 (green) in LysoTracker-stained compartments (red) in mouse fibroblasts maintained in the presence or absence of serum.f3(a) Localization of photoconverted KFERQ-PS-CFP2 (green) in LysoTracker-stained compartments (red) in mouse fibroblasts maintained in the presence or absence of serum. (b) Percentage of co-localization of KFERQ-PS-CFP2 with the indicated proteins. Values are mean+s.e. of three different experiments with >50 cells counted per experiment. (c) Co-localization of photoconverted KFERQ-PS-CFP2 (green) with LAMP-1, LAMP-2 or LAMP-2A. (d) Immunofluorescence with antibodies that recognize PS-CFP2 (green) and hsc70 (red) in cells maintained in the absence of serum and fixed with methanol to eliminate the soluble cytosolic fraction. (e, f) Co-localization of photoconverted KFERQ-PS-CFP2 (green) with LC3, CD-M6PR, Rab5 (e) and ubiquitin (f) in mouse fibroblasts maintained in serum-free media. Scale bars, 5 μm.
Figure 1A) Schematic of the EMS mutagenesis screen carried out using the piRNA sensor and identification of a novel allele (mj261) of the rpb-9 gene. B) rpb-9 locus, in scale (top) and comparisons of RPB-9 protein domains to homologs in other species (bottom). The introduced mutation is highlighted in red. C) Expression patterns of rpb-9 in adult animals by RNA-sequencing. Quantification of piRNA sensor expression. Representative DIC and fluorescence microscopy images of piRNA sensor expression in wild type (mjIs144) (D) and rpb‑9 (mj261) animals (two images with varying GFP expression are shown, scale bar = 20 µm) (E) validation of transgene expression by RT-qPCR (n=3, error bars represent SD) (F) representative DIC and fluorescence microscopy images of piRNA sensor expression in the rpb-9 rescue (mj261; mjSi70) animals (two images with varying GFP expression are shown). Scale bar = 20 µm (G).
Fig.Figure 3A Experimental scheme. Groups of four to six-week-old C57BL/6 mice (n=5 per group) were immunized i.p. with 104-107 PFU of WNV-poly(A), PBS (negative control) or mock (untreated). On day 30 post-immunization, mice were challenged i.p. with 3× 107 PFU of WT WNV B-C Total anti-WNV IgG antibody (B) and neutralizing antibody titers (C) in mouse serum at the indicated days post immunization. (For B and C, the values represent mean ± sd in each group and the horizontal dotted line represents the limit of detection) Data information: For statistical analysis, one-way ANOVA analysis were used *p<0.5, **p<0.01, *** p<0.001,****p< 0.0001, ns represents not significant D-E Survival (D) and body-weight changes were monitored daily after challenge. (The values represent mean ± sd in each group) (E). Data information: For statistical analysis, two-way ANOVA analysis were used Log-rank test was performed *p<0.5, **p<0.01, *** p<0.001,****p< 0.0001, ns represents not significant F Viremia was quantified by plaque assay on day 2 post-challenge. (The values represent mean ± sd in each group, each symbol represents a mouse) Data information: For statistical analysis, two-way ANOVA analysis were used *p<0.5, **p<0.01, *** p<0.001,****p< 0.0001, ns represents not significant G-H T-cell responses in vaccinated mice were measured through ELISpot assay. Two groups of C57BL/6 mice (n = 4 / group) were i.p. inoculated with 107 of WNV-poly(A) or equal volume of PBS. ELISpot assay specific for IFN-γ in splenocytes was performed at 7 days post-immunization. Representative images were shown in (G) and the numbers of antibody secreting cells (ASCs) were counted (The values represent mean ± sd in each group, each symbol represents a mouse) (H). Data information: Mann-Whitney test was used for H. *p<0.5, **p<0.01, *** p<0.001,****p< 0.0001, ns represents not significant
Figure 1(A) Gene set enrichment analysis (GSEA) of differentially expressed genes in colorectal cancer from the GEO database (GDS4382). ES, enrichment score; NES, normalized enrichment score. n = 3 biological samples. (B) Kaplan-Meier survival curves for colon cancer patients with or without dysfunction of iron homeostasis in the GSE dataset (n = 590 samples, P = 0.016, Log-rank (Mantel-Cox) test). "Normal" represents the patients with unaltered expression of iron homoeostasis-related genes, including LTF, CP, FTH1, SLC30A1 and TFRC; "Dysfunctional iron homoeostasis" represents the patients with altered expression of iron homoeostasis-related genes. (C) Immunoblot analysis of protein levels of IREB2 in colorectal adenocarcinoma and its matched adjacent normal tissues (Left), the relative protein level of IREB2 was assessed by ImageJ software (Right) (n = 8 human samples, *P = 0.0114, two-tailed paired Student's t-test). (D) Endogenous IREB2 was immunoprecipitated from the lysates of primary colorectal adenocarcinoma and its matched adjacent normal tissue, and was subsequently analyzed by anti-ubiquitin antibody for the assessment of ubiquitination. (E) Mass spectrum analysis of IREB2-associated proteins. Mock or IREB2-FLAG was transfected into HEK293T cells and FLAG-tagged proteins were enriched by anti-FLAG M2 beads and incubated with lysates from normal mice colon tissues. Five matched peptides corresponding to OTUD1 were shown on the right panel. (F) In vivo ubiquitination assay of IREB2. HEK293T cells were transfected with indicated plasmids and subjected to immunoprecipitation with anti-FLAG antibody followed by western blot analysis. (G) Half-life analysis of IREB2 in wild-type (WT) and OTUD1─/─ NCM460 cells. Cells were treated with 100 μg/ml cycloheximide (CHX) and collected at the indicated times for western blot analysis (Up), the relative protein level of IREB2 was assessed by ImageJ software (Down) (n = 3 biological replicates, mean ± s.e.m., **P = 0.0049, ***P < 0.0001, two-tailed unpaired Student's t-test). (H) Flow cytometric analysis of TFRC expression in mock and OTUD1 expressing CT26 cells with AFC (50 μM) and Hemin (100 μM) treatment. MFI, mean fluorescence intensity. Control uses isotype-matched control antibody (n = 4 biological replicates, mean ± s.e.m., **P = 0.0010, ***P = 0.0008, two-tailed unpaired Student's t-test). (I) Flow cytometric analysis of TFRC expression in wild-type (WT) and OTUD1─/─ NCM460 cells with DFO (100 μM) treatment. MFI, mean fluorescence intensity. (n = 4 biological replicates, mean ± s.e.m., ***P = 0.0001, two-tailed unpaired Student's t-test).
Figure 1A. Ribbon representation of 10 energy-refined structure ensemble of FGF21core. B. Cartoon representation of a representative structure of FGF21core. β sheets for the barrel and triangular array regions were colored pink and cyan, respectively.
Figure 1(A) Quantification of free cholesterol levels by lipidomics analysis of total homogenates (TH), ER or MAM fractions isolated from WT and PS-DKO cells. Lipid units are represented as molar mass over total moles of lipids analyzed (mol %). Graphs represent fold change over controls. Unpaired t-test (n= 6 for TH, n=3 for MAM or ER; * p<0.05). (B) Quantification of free cholesterol levels in total homogenates of APP-DKO cells transiently expressing C99, C83 or AICD peptides, or treated with 5 µM Aβ42 oligomers for 16h. Cholesterol levels of WT cells are shown as a control. Lipid units are represented as molar mass over total moles of lipids analyzed (mol %). Graphs represent fold change over controls. One-way ANOVA (n = 4; * p<0.05). (C) Quantification of HMGCR enzymatic activity in PS-DKO and DAPT-treated WT cells. unpaired t-test (n=3; * p< 0.05). (D) Quantification of cholesterol uptake by incubating WT and PS-DKO cells, treated or untreated with BACE inhibitor IV (BI, 100 nM), with 2.5 µCi/mL 3H-cholesterol for the indicated times. Graph represents cholesterol radioactivity levels in total cell homogenates (TH). Two-way repeated measures ANOVA (Time, Group)(n = 3 independent experiments; * p< 0.05). (E-F) Quantification of 3H-cholesterol delivery to MAM (E) or bulk ER (F) after the indicated times. Control and BI-treated or untreated PS-DKO cells incubated with 2.5 µCi/mL 3H-cholesterol for the indicated times, before subcellular fractionation (Western blot of isolated MAM fractions shown in Fig. EV1F). Cholesterol delivery to MAM calculated by quantification of radioactivity levels in the isolated fractions. Two-way repeated measures ANOVA (Time, Group)( n=3 independent experiments; * p< 0.05) (G) Measurement of cholesterol uptake in the indicated cells by internalization of a fluorescent cholesterol analog [NBD-cholesterol (2 µM; upper panel, nuclei in blue)]. Endogenous free cholesterol was stained with filipin (lower panel). Graphs on the right represent fluorescence intensity (using ImageJ) and the percentage of cells with filipin punctae. Scale bar = 20 µm. One-way ANOVA (30-50 cells/condition from at least 3 independent experiments; * p<0.05, ** p<0.01, *** p<0.001).
Figure 7A) Double immunofluorescence staining for phosphorylated myosin light chain II (pMLC II) and VE-cadherin in mesenteric resistance artery endothelium from wild type, Tek-Cre::Lama5-/- and Lama4-/- mice (arrow heads). Scale bar is 10µm. B) Quantification of the pMLC II signal along VE-cadherin signal revealed significantly less overlap in Tek-Cre::Lama5-/- and more overlap in Lama4-/- endothelium, compared with wild type controls. The data are normalized to the skeletal length of the VE-cadherin signal. Values shown are means ± s.e.m resulting from quantification of 4 field of view per arteries, 4 mice /genotype. *P<0.05, **P<0.01, paired t-test.
Cdk4 deficiency in VMH SF1 neurons leads to increased heat production during cold exposure.Figure 5A Cdk4 mRNA expression in the dissected VMH of Cdk4flox/flox (n=5) and Cdk4flox/flox Sf1-Cre (n=6) mice B-C Rectal temperature was measured at 24°C (B), and rectal temperature was monitored during acute cold exposure at 4°C for 6 hours (C) in Cdk4flox/flox (n=15) and Cdk4flox/flox Sf1-Cre mice (n=15). D Hematoxylin-eosin (H&E) staining of iBAT sections (scale bar 100 μm) from Cdk4flox/flox and Cdk4flox/flox Sf1-Cre mice. E Expression of thermogenic genes and brown adipocyte markers expression in iBAT Cdk4flox/flox (n=9) and Cdk4flox/flox Sf1-Cre mice (n=9) mice as assessed by RT-PCR. F-G TH immunohistochemical (IHC) staining in iBAT sections (F) (scale bar 40 μm, arrows indicate TH parenchymal fibers) and corresponding quantification of the number of TH fibers relative to 100 adipocytes (G) (Cdk4flox/flox (n=5) and Cdk4flox/flox Sf1-Cre mice (n=5)). H-I Western blot analysis (H) and quantification of p-CREBS133 protein expression following 6 hours of cold exposure (4°C) (I) (n=3 biological replicates). HSP90 was used as a loading control.
The minimal active subdomain of TMEM59 directly binds ATG16L1 through the identified functional motif.Figure 6(A) Apposition events between GFP-ATG16L1 and endocytosed CD16:7-263-281. JAR cells were transfected with CD16:7-263-281 and GFP-ATG16L1 or GFP-BECLIN (as indicated), aggregated for 4 h and stained for the endocytosed chimera (red). Representative confocal microscopy pictures are shown. Two different examples are provided for GFP-ATG16L1. (B-D) 293T cells were transfected with the indicated constructs, lysed and subjected to GST immunoprecipitation using agarose beads coupled to glutathione (IP, immunoprecipitation; TL, total lysate). Shown are WBs against the indicated molecules. (B) AU-ATG16L1 co‐precipitates with wild‐type (WT) TMEM59-GST but not with a mutated version where the four essential amino acids were mutated to alanine (TMEM59-4M-GST). (C) Full‐length TMEM59 and TMEM59-Δ282 co‐precipitate with GST-ATG16L1, whereas the respective 4M versions do not. (D) AU-ATG16L1 co‐precipitates with a fusion protein between GST and the minimal active peptide of TMEM59 (GST-263-281), but not with a 4M version (GST-263-281-4M) or a GST fusion protein with the inactive portion of TMEM59-ID (GST-282-323). (E) HA-ATG16L1 expressed in bacteria co−precipitates with a GST-263-281 recombinant protein purified from bacterial cultures, but not with a 4M version of the same construct or GST-282-323. The indicated GST partners were expressed in bacteria and purified using agarose beads coupled to glutathione. The loaded beads were then used for HA-ATG16L1 pull−down from crude bacterial lysates. Shown are WBs against the indicated molecules (PD, pull−down). A Coomasie staining of a protein gel with the purified GST fusion proteins is shown. The right panel compares the amount of HA-ATG16L1 pulled down by GST-263-281 with the signal provided by direct anti−HA immunoprecipitation. This result shows that about 20-25% of the available HA-ATG16L1 protein is precipitated by GST-263-281. Asterisks indicate irrelevant bands in B and D.Source data for this figure is available on the online supplementary information page.
Figure 2A. Top: Cartoon representing the topology of the FUNDC1 protein in the mitochondrial membrane. The cytoplasmic domain that interacts with the ER protein Calnexin is shown. Bottom: Illustration of the different FUNDC1 truncation variants. B. HeLa cells were transfected with FUNDC1-MYC or empty vector for 24 h. Cell lysates were immunoprecipitated by anti-MYC antibody and immunoblotted with indicated antibodies. C. HeLa cells were transfected with FLAG-FUNDC1. About 24 h post-transfection, cell lysates were immunoprecipitated by anti-Calnexin and immunoblotted with anti-FLAG and anti-Calnexin antibodies. D and E. HeLa cells were transfected with the indicated FUNDC1-MYC constructs for 24 h. Cell lysates were immunoprecipitated using anti-Calnexin and immunoblotted using anti-MYC and anti-Calnexin antibodies. F. Immunoblots of subcellular fractions from HeLa cells transfected with scramble siRNA and exposed to hypoxia (1% O2) for 5 h. PNS: post-nuclear supernatant; CYTO: cytosol; ER: endoplasmic reticulum; MITO: mitochondria; MAM: mitochondrial-associated membrane. DRP1 (S) and DRP1 (L) indicate short and long exposures, respectively.G. Immunoblots of subcellular fractions from HeLa cells transfected with Calnexin siRNA (si-CNX) and exposed to hypoxia (1% O2) for 5 h. PNS: post nuclear supernatant; CYTO: cytosol; ER: endoplasmic reticulum; MITO: mitochondria; MAM: mitochondrial-associated membrane. DRP1 (S) and DRP1 (L) indicate short and long exposures, respectively.H. Quantification of the MAM:PNS ratio of FUNDC1 or DRP1 in HeLa cells treated with scramble siRNA or si-CNX and exposed to hypoxia (1% O2) for 5 h. Data are presented as mean ± s.e.m. from 3 independent experiments; *** P < 0.001.
Figure 1Cells were lysed and analyzed by SDS-PAGE and western blotting under nonreducing conditions (A) or reducing conditions (B). TMPRSS2 levels are expressed as percentages of TMPRSS levels in Calu-3 cells normalized to levels of EF2. *1 indicates TMPRSS2 (A) and pro-cathepsin L (B), and *2 shows cleaved TMPRSS2 (A) and cathepsin L (B). A549*, ACE2-expressing A549 cells; EF2, elongation factor 2. Vero and Caco-2 cells were infected with SARS-CoV-2 at MOIs of 0.2 for 8 h. Infected cells were then permeabilized and immunostained for the intracellular SARS-CoV-2 nucleoprotein (NP, red). Nuclei were stained with Hoechst (blue) before imaging by fluorescence confocal microscopy. Scale bars: 100 µm. Vero and Caco-2 cells were exposed to SARS-CoV-2 at MOIs of 0.3 and 0.2, respectively, and harvested 16 h later. After fixation and permeabilization, infected cells were stained with the primary mAb against NP. Infection was analyzed by flow cytometry. SSC-A = side scatter-area. Infection of Vero and Caco-2 cells was monitored over 24 h using the same flow cytometry-based assay used for the experiment shown in panel D. Infection is given as the total fluorescence associated with the NP protein-positive cells. MFI, mean fluorescence intensity. n = 3. SARS-CoV-2 mRNA levels were quantified by qRT-PCR in both Vero and Caco-2 cells infected at MOIs of 0.3 and 0.4, respectively, for up to 24 h. n = 3. Supernatants from infected cells were collected during the time course in F and assessed for the production of new infectious viral particles using a TCID50 assay on naïve Vero cells. n = 3-6.