EMBO/sd-character-level-ner huggingface dataset:
Figure 6KIC mice were treated in a survival study with rat IgG Mac48 (control) or 2G8. Median overall survival of rat IgG treatment was 72 days, while median overall survival of 2G8 treatment was 63 days (B). Tissues from (A) were stained for Ki67. Scale bars = 50 μm. Mutation profile of TGFBR2 in PDA patient samples from two independent sources (UTSW and TCGA). Overall survival of PDA patients from TCGA with and without TGFBR2 mutation. Loss-of-function mutation of Tgfbr2 was generated by CRISPR with two different gRNAs (mut1 and mut2) in the mouse PDA cell line BMFA3 derived from KPfC model. Control cell line (Tgfbr2wt) and two Tgfbr2-mutant cell lines (Tgfbr2mut1 and Tgfbr2mut2) were treated with TGFβ (30 ng/ml) for 5 hours. Cell lysates were harvested and western blotting for P-SMAD2, SMAD2, SMAD4, P-ERK1/2, ERK1/2, P-P38, P38, and actin was performed. Immunofluorescence was performed to study the expression and localization of E-cadherin and SMAD4 after TGFβ treatment (30 ng/ml for 24 hours) in control cell line and two Tgfbr2-mutant cell lines. Scale bars = 50 μm (fluorescent), scale bars = 100 μm (brightfield).
Figure 6PLTP is highly expressed and secreted from brown/beige fat and positively regulated by the PRDM16-PPARγ complex. Increased PLTP levels lead to reduced plasma levels of several sphingolipids and phospholipids lipid species, and enhanced cholesterol transport to the liver. In the liver, transported cholesterol is excluded by active synthesis and secretion of bile acids into circulation. In turn, secreted bile acids (e.g., cholic acid) in the circulation activates glucose uptake and thermogenesis in BAT. PLTP-mediated activation of BAT thermogenesis increases whole-body energy expenditure, prevents diet-induced body-weight gain, and improves systemic glucose and lipid homeostasis.
Interfering with the cardiolipin synthesis pathway can prevent mitochondrial fragmentation in Opa1 deficient fibroblasts (A) Schematic of cardiolipin (CL) biosynthesis pathway in mitochondria.Figure 5Schematic of cardiolipin (CL) biosynthesis pathway in mitochondria. Phosphatidic acid (PA) is transported to the inner membrane by PRELID1 where it is converted to CDP-diacylglycerol (CDP-DAG) and glycerol 3-phosphate (G3P) by TAMM41. Phosphatidylglycerol phosphate (PGP) is dephosphorylated to phosphatidylglycerol (PG) by PTPMT1. PG is either degraded to DAG or reacts with CDP-DAG to form CL in a reaction catalyzed by cardiolipin synthase (CLS1). Tafazzin (TAZ) catalyzes the remodeling of monolysocardiolipin (MLCL) to mature CL. CL is transported to the outer membrane and converted to PA by mitoPLD. PA is converted to DAG by Lipin1. PA can be supplied to the inner membrane from DAG conversion by Acylglycerol Kinase (AGK). Representative confocal micrographs of MEFs WT and Opa1Crispr MEFs treated with indicated siRNAs for 72 hours. Mitochondria (anti-TOM40, green) and nuclei (DAPI, blue). Scale bar=10μm. Supervised ML mitochondrial morphology quantification of (B) using WT MEFs with fragmented (Opa1 siRNA), normal (non-targetting NT siRNA), and hypertubular (Dnm1l siRNA) mitochondria. Data represent mean ± SD of three independent experiments, One-way ANOVA (726-4236 cells per cell line), (% fragmented); *** p < 0.001, ****p < 0.0001, ns; not significant. quantitative RT-PCR (qRT-PCR) measurement of Prelid1, Tamm41, Pgs1, Ptpmt1, and Cls1 expression in Opa1Crispr and WT MEFs. Fold change is indicated relative to WT control. Data represent mean ± SD of three independent experiments, One-way ANOVA. Whole cell phospholipidome of WT and Opa1Crispr MEFs treated with NT (non-targeting), Tamm41 or Pgs1 siRNAs. Data represent mean ± SD of five independent experiments; *p < 0.05, *** p < 0.001, ****p < 0.0001, ns; not significant. Representative confocal micrographs of MEFs WT, Pgs1Crispr and Dnm1lCrispr MEFs treated with indicated siRNAs for 72 hours. Mitochondria (anti-TOM40, green) and nuclei (DAPI, blue). Scale bar=10μm. Supervised ML mitochondrial morphology quantification of (G) using WT MEFs with fragmented (Opa1 siRNA), normal (non-targetting NT siRNA), and hypertubulated (Dnm1l siRNA) mitochondria. Data represent mean ± SD of >3 independent experiments, (3096-7238 cells per cell line), One-way ANOVA (% fragmented) ; *p < 0.05, ** p < 0.01, ****p < 0.0001, ns; not significant.
Figure 2A. MITOL degrades Parkin through the ubiquitin-proteasome system. HeLa cells stably expressing HA-Parkin were transfected with the indicated vectors and treated with DMSO or CCCP (10 µM) for 8 h alone or either with Bafilomycin A1 (10 µM) or Lactacystin (10 µM) for the final 5 h. Lysates of cells were subjected to an IB assay with the indicated antibodies. B. Overexpression of MITOL CS mutant fails to degrade Parkin. HeLa cells stably expressing HA-Parkin were transfected with the indicated vectors and treated with DMSO or CCCP (10 µM) for the indicated time. Lysates of cells were subjected to an IB assay with the indicated antibodies. MITOL CS mutant; lacking ubiquitin ligase activity. C. Parkin K220R mutant is not regulated by MITOL in mitophagy. HeLa cells were transfected with the indicated vectors and treated with DMSO or CCCP (10 μM) for the indicated time. Lysates of cells were subjected to an IB assay with the indicated antibodies. D. Endogenous MITOL attenuates Parkin in both CHX- and CCCP-treated conditions. WT or MITOL KO HeLa cells stably expressing HA-Parkin were treated with CCCP (10 µM) as indicated times. CHX (30 μM) was added 5 h after CCCP treatment. Lysates of cells were subjected to an IB assay with the indicated antibodies. E. MITOL regulates mitophagy. MITOL WT HeLa cells, MITOL KO HeLa cells and KO HeLa cells transfected with MITOL WT plasmids 0.5µg (+) and 5µg (++) stably expressing HA‐Parkin and mt‐Keima were treated with CCCP (10µM) for 8h. Then, mKeima was measured at 488 (pH 7) and 561 (pH 4) nm lasers using Flow Cytometer. Percentages of mitophagy were calculated from 30,000 cells in each independent experiment. Data represent the mean ± SD of three independent experiments. For statistical analysis, a one-way ANOVA with Tukey post-test was performed, **P < 0.01.
MAPK activity is linked to CRC cell differentiation states.Figure 4A Gene expression of LGR5 and EPHB2, along activity gradients of LGR5-ISC or MAPK target gene signatures. B Cell state distribution of SCN-aberrant CRC cells along gradients of LGR5-ISC or MAPK transcriptional signatures, as in A. C Cell state distribution of SCN-aberrant CRC tumor cells along MAPK signature activity, as in B, per tumor. Correlation between cell state distributions and MAPK target gene was calculated using Pearson´s r. For correlations and significances, see Table EV6. Color code as in Fig. 4B. D UMAP representations of single cell transcriptomes derived from P009T or P013T organoids, after MAPK blockade using MEK or combined MEK and EGFR inhibition. Color codes are treatment conditions or expression strength of signature, as indicated. Dashed line in P013T UMAP roughly separates control (DMSO) and MEK/EGFR inhibitor treated cells.
Figure 3SIRF detection of MRE11 at stalled forks in STN1-deficient U2OS cells. Representative SIRF images of MRE11 at normal or stalled replication forks are shown. Scale bars: 10 µm. ssDNA accumulation analysis in STN1-deficient U2OS cells with and without mirin treatment. Representative images of native BrdU staining are shown.
Figure 3a, Images show the localisation of endogeneous SEPT2 and SEPT7 in RPE1 ARLB13B-mRuby2 cells with basal body staining. The insets to the right show enlargements of the cilium. Scale bar: 4µm (large) and 2µm (small). b, STED microscopy images showing ciliated RPE1 cells expressing EGFP-SEPT2 or SEPT7-EGFP and stained for ARL13B. Scale bar: 1µm. c, Relative signal intensities were determined along the cilium for the indicated proteins at the ciliary tip (T), middle (M) and base (B) regions as specified in (b) (white lines). d, Representative images showing co-localisation of MKS3 or CEP290 in ciliated RPE1 ARLB13B-mRuby2 and GFP-SEPT2 cells. The white arrows point to the specific MKS3 or CEP290 signals. Scale bar: 1µm. e, Line scan measurements show the normalised signal intensities along the cilium for the indicated proteins as specified in (d) (dashed lines). f, Effect of nocodazole (Noco) treatment (3h) on the localisation of endogenous SEPT2 and SEPT7 at cilia in RPE1 cells. The insets show magnifications of the cilium. Scale bar: 5µm (large) and 2.5µm (small). g, Relative signal intensity of endogenous SEPT2 or SEPT7 along the cilium from (f). SEPT2 or SEPT7 signals at cilia were normalized to the total cilia length. Three biological replicates, n>90 cilia per sample and repetition. Data show in (g) includes mean ±s.d.; P values are calculated by two-tailed unpaired student t-test. See source data.
Figure 5A Plots of relative pre-mRNA abundance of tim (n=5) and Pdp1ε (n=3) determined by qRT-PCR in the whole head extracts of Nipped-A RNAi (timG4/+;Udcr2/UNipped-ARNAi-1) and control (timG4/+;Udcr2/+) flies during DD1. B ChIP assays to detect ub-H2B binding at E-box, TSS and gene body of tim, Pdp1ε, per and clk in Nipped-A RNAi (timG4/+;Udcr2/UNipped-ARNAi-1) and control (timG4/+;Udcr2/+) (n≥3). Data information: Student's t-test, *P < 0.05. Two-way ANOVA, significant effect between ub-H2B and IgG were found for all genomic regions tested (P<0.001). Significant effect of genotypes were found for tim E-box (P<0.05), Pdp1ε E-box (P<0.01), tim TSS (P<0.01) and Pdp1ε TSS (P<0.001).
a) [3H]thymidine incorporation by naive CD4+ lymphocytes stimulated for 72 h with agonistic mAb to CD3 and IFN-γ-deficient irradiated APCs, with IL-2 (10 U/ml) added 24 h after the initiation of culture.figf4(a) [3H]thymidine incorporation by naive CD4+ lymphocytes stimulated for 72 h with agonistic mAb to CD3 and IFN-γ-deficient irradiated APCs, with IL-2 (10 U/ml) added 24 h after the initiation of culture. (b) Accumulation of viable CD4+ T cells at 72 h in the presence of medium alone (Med) or IFN-γ (5 U/ml). (c) Flow cytometric analysis of the viability and division of CD4+ T cells labeled with CFSE and activated as described in a with T cell-depleted Ifng−/− APC samples in the presence or absence of IFN-γ (5 U/ml). Numbers in plots indicate percent CFSE+CD4+ T cell populations. (d-f) Death of Ifng−/− and Irgm1−/−Ifng−/− T cell populations activated with mAb to CD3 and expanded in IL-2, then treated with IFN-γ (d), agonistic mAb to CD3 (e) or Fas ligand (FasL; f), assessed by propidium iodide staining at 48 h after treatment. Open bars, Ifng−/−; filled bars, Irgm1−/−Ifng−/− (a,b,d-f). Data are representative of three independent experiments with similar results (mean and s.d. of triplicate cultures a,b,d-f).
Fis1K148R induces mitochondrial fission but does not cause muscle atrophy and preserves mitochondrial function.Figure 6(A)In vivo imaging of the mitochondrial network was performed in muscles expressing Fis1K148R. Adult muscles were transfected with plasmids encoding mitochondrially targeted yellow fluorescent protein (mtM13‐YFP) and Fis1K148R. Twelve days later muscles were observed in vivo with confocal microscopy. TMRM was injected into the muscles to monitor mitochondria, which retain membrane potential. Mitochondrial network was greatly altered but mitochondrial membrane potential was conserved. (B) FDB muscle fibres were transfected by electroporation in vivo. Eight days later adult fibres were isolated and placed in cell culture. Adult fibres were loaded with TMRM (5 nM) for 30 min at 37°C. TMRM accumulates in the mitochondria that maintain mitochondrial membrane potential. Oligomycin (Olm, 5 μM) and the protonophore FCCP (4 μM) were added at the indicated time points. TMRM staining were monitored in at least 10 fibres per construct (*P0.001). (C) hFisK148R‐transfected muscles were collected 12 and 21 days after transfection. Cross‐sectional area of transfected fibres, identified by anti‐myc immunofluorescence, was quantified (n=550). (D) Fis1‐dependent activation of MuRF‐1. MuRF‐1 promoter was co‐transfected into adult TA muscle with or without Fis1 or Fis1K148R. A renilla‐luciferase vector was co‐transfected to normalize for transfection efficiency. Eight days later, firefly and renilla‐luciferase activity was determined. Each condition represents the average of at least five independent experiments±s.e.m. (*P0.05).
Figure 4A, IgA1 and IgA2 subtypes specific for spike protein were measured in plasma and saliva at different time point (T0,T1, T2) of vaccinated naïve (plasma: n=64; saliva: n=63 (IgA1), n=55 (IgA2)) or SARS-CoV-2-Exp (plasma: n=21; saliva: n=18) subjects. Subjects received the first vaccine dose at day 0 (T0), and the 2nd dose at day 21 (T1) (indicated with an arrow). T2 corresponds to 7-10 days after the 2nd dose. For plasma samples the titers of antigen specific IgA1 and IgA2 are expressed in IU/ml and AU, respectively (see Methods). LoD is indicated by a dotted line: LoD (IgA1)=74.64; LoD (IgA2)=0.009. For saliva samples the titers of antigen specific Ig were normalized by dividing the values of SARS-CoV-2 specific Ig by total IgA concentration of each sample. The normalization was applied only to values higher than LoD. The adjusted values are expressed in AU. B, IgA1 and IgA2 subtypes specific for spike protein were measured in plasma and saliva of vaccinated naïve and SARS-CoV-2-Exp subjects 7-10 days after second dose (T2) and the antibody response was compared to that obtained in COVID-19 patients (plasma: n=28; saliva: n=26) and in non-vaccinated subjects (n=19, Control).
Figure 4Fluctuation analysis for the generation of Bu-1 loss variants. Top to bottom: wild type, timeless (clone 1), a timeless mutant (timeless ∆C) generated by CRISPR-Cas9 targeting exon 16 which truncates the protein removing the CTD containing both the DBD and the PARP binding domains. Then, complementation of timeless#1 with human Timeless (hTim), hTim∆816-1208 (lacking both the DBD and PBD), hTim∆816-965 (lacking the DBD) and hTim[1:1000], lacking the PBD. At least two independent fluctuation analyses were performed with 24-36 individual clones each cell line per repeat.
a) THPN induces the dissipation of ΔΨm in both time- and dose-dependent manners.figf4(a) THPN induces the dissipation of ΔΨm in both time- and dose-dependent manners. The compromised ΔΨm is shifted to the right upon THPN treatment in flow cytometry. '-' represents vehicle treatment only. (b) Effects of different plasmids on the THPN-induced dissipation of ΔΨm. Top, endogenous TR3 was knocked down by shRNA, and the siRNA-resistant version of TR3, TR3-NLS, TR3-ΔNLS or TAD (Supplementary Fig. 2c) were separately transfected into the cells as indicated. Bottom, similarly, the siRNA-resistant version of Nix or ΔTM were separately transfected into Nix knockdown cells. Transfected cells were treated with THPN (20 μM, 12 h), and their ΔΨm was assessed by flow cytometry. Ctrl, control. (c) ANT1 and VDAC1 are required for the THPN-induced autophagy. The ANT1 and VDAC1 shRNA-expressing plasmids, which also encoded EGFP for evaluating the transfection efficiency, were separately transfected into cells; mCherry-LC3 was cotransfected to display autophagy. Green cells are those transfected with ANT1 or VDAC1 shRNA, and the cells with red cytoplasmic puncta are autophagic cells (top; scale bars, 10 μm). (d) ANT1 and VDAC1 are required for the THPH-induced dissipation of ΔΨm and cell death. Endogenous ANT1 or VDAC1 was separately knocked down by shRNA, and the dissipation of ΔΨm was determined after THPN (20 μM, 12 h) treatment (left). ANT1 and VDAC1 knockdown abolishes the THPN-induced (20 μM, 48 h) cell death (right). All of the data are presented as the mean ± s.e.m. of three independent experiments. **P 0.01.
Endothelial Ang-2 upregulation correlates with WHO grading in human gliomas.Figure 1Figure 1: Endothelial Ang-2 upregulation correlates with WHO grading in human gliomas. ELISA displaying human Ang-2 (A) and Ang-1 (B) level in serum of healthy patients (Ang-2 n=4; Ang-1 n=21) or patients with low grade glioma (WHOII) (n=5), anaplastic astrocytoma (WHOIII) (n=7) or glioblastoma (WHOIV) (n=39) are shown. Figure 1: Endothelial Ang-2 upregulation correlates with WHO grading in human gliomas. ELISA displaying human Ang-2 (A) and Ang-1 (B) level in serum of healthy patients (Ang-2 n=4; Ang-1 n=21) or patients with low grade glioma (WHOII) (n=5), anaplastic astrocytoma (WHOIII) (n=7) or glioblastoma (WHOIV) (n=39) are shown. Figure 1: Endothelial Ang-2 upregulation correlates with WHO grading in human gliomas. ELISA displaying human Ang-2 (A) and Ang-1 (B) level in serum of healthy patients (Ang-2 n=4; Ang-1 n=21) or patients with low grade glioma (WHOII) (n=5), anaplastic astrocytoma (WHOIII) (n=7) or glioblastoma (WHOIV) (n=39) are shown. The TCGA database was accessed to obtain gene expression level for Ang-1, Ang-2, Tie2 and Tie1 in GBM in comparison to normal brain (C). Ang-2 expression and quantitative analysis of Ang-2 in healthy human brain (n=3), low grade diffuse glioma (n=14), anaplastic astrocytoma (n=12) or glioblastoma (n=11) is shown (D). Co-staining of Ang-2 and vWF (endothelial cells), αSMA (mural cells) and IBA1 (microglia) in different glioblastoma specimen. Normal brain tissue was used to assess Ang-2 staining specificity (E). Ang-2 predicts survival of glioblastoma patients (F). If not indicated differently, Kruskall-Wallis (Dunn´s post test) was applied, ** p < 0.05, *** p < 0.005; data are mean ± SEM
Deletion of Syk after Tam treatment of mb1‐CreERT2;Sykfl/fl miceSchematic representation of the targeted mb‐1 locus harboring the CreERT2 construct and the “floxed” Syk locus.Figure 1Schematic representation of the targeted mb‐1 locus harboring the CreERT2 construct and the "floxed" Syk locus. The construct is inserted between exons I and IV. The filled rectangles represent the exons of the mb‐1 allele. The open rectangle represents the CreERT2 construct followed by a poly‐adenylation (pA) site. The black circle represents the endogenous pA site of the mb‐1 allele. The "truncated" version of mb‐1 exon I shown here lacks the ATG start codon and is followed by the complete mb‐1 intron I with its splice donor and acceptor sites. Intron I was maintained to provide intron/exon splicing in the transcript and as a source of possible transcription regulatory sequences. The targeted Syk locus was targeted with 2 LoxP sites and has been described in Saijo et al (). RT‐PCR performed on cDNA from RNA isolated from splenic B cells derived from Tam‐treated mb1‐CreERT2 (left lane) or mb1‐CreERT2;Sykfl/flmice (right lane); the Syk‐ and Hprt‐specific amplification products were identified by agarose gel electrophoresis with Hprt used as an endogenous loading control. Immunoblot analysis of proteins isolated from B cells derived from spleens of mb1‐CreERT2 (left lane) or mb1‐CreERT2;Sykfl/fl (right lane) mice both treated with Tam as described; blots were probed with anti‐Syk and anti‐GAPDH Ab, GAPDH being used as a loading control. Intracellular flow cytometric analysis for Syk expression in B cells derived from the LN of Syk‐deficient and control mice. Genomic DNA analysis of Tam‐treated mb1‐CreERT2 or mb1‐CreERT2;Sykfl/flmice (as indicated). Upper row: amplification of floxed (fl) and wt (+) alleles. Middle row: amplification of the deleted (d) allele. Lower row: loading control (TC21, a gene unaffected by the deletion of Syk). Established control DNA was used to visualize the individual bands (as indicated).
Prazosin induces GIC apoptosis independently from α-AR.Figure 4A. Immunoblotting for pro-caspase-3 (pro-CASP3) and active caspase-3 (CASP3) in GICs treated with prazosin (PRZ) or vehicle (V) demonstrating that prazosin activates caspase-3. ZVAD, a caspase inhibitor, prevents prazosin-induced caspase-3 activation. kDa: kilodaltons. B. Immunoblotting for pro-caspase-9 (pro-CASP9) and active caspase-9 (CASP9) in GICs treated with prazosin (PRZ) or vehicle (V) demonstrating that prazosin does not activate caspase-9. C. Viability analysis of GICs treated with prazosin for 24h in the presence or absence of ZVAD, a caspase inhibitor. ZVAD counteracts prazosin-induced GIC death. *P=0.0286, n=4, two-sided Mann-Whitney U-test. D. Prazosin induces glioblastoma cell apoptosis in vivo. GFP+ and GFP- cells were analyzed from tumors in vehicle or prazosin-treated mice. Annexin V / DAPI staining was used to identify apoptotic cells by FACS. Tumors were initiated by GBM44 GICs implantation. E. Dose-response curve of prazosin on GIC survival (24h treatment). EC50 was determined with curve fit using nonlinear regression. F. Viability analysis of GICs treated with prazosin for 24h in the presence or absence of cirazoline, a subtype agonist of the α-ARs. Cirazoline did not alter GIC survival and counteracted only poorly prazosin-induced GIC death. *P<0.05, n=4, two-sided Mann-Whitney U-test. G. Membrane binding assays demonstrating the absence of prazosin binding sites in GIC membrane preparations. Positive control shows that prazosin binds to membrane preparations of yeast expressing 1-AR. PRZ: prazosin. CRZ: cirazoline. H. Immunoblotting for phosphorylated ERK1/2 (P-ERK1/2) and total ERK1/2 following prazosin treatment for 30 min. Prazosin induces ERK1/2 phosphorylation in GICs. I. SRE-luciferase reporter activity analysis of GICs treated with prazosin in absence or presence of U0126 (10 μM), an inhibitor of the ERK-activating kinase MEK. U0126 prevented prazosin-induced SRE-luciferase activation in GICs. J. Viability analysis of GICs treated with prazosin for 24 h in the presence or absence of U0126 (10 μM), an inhibitor of the ERK-activating kinase MEK. U0126 did not counteract prazosin-induced GIC death. Blockade of prazosin-induced ERK1/2 phosphorylation by U0126 was confirmed by immunoblotting (insert). PRZ: prazosin.
ISG screen identifies LY6E as a potent coronavirus restriction factor.Figure 1.factor.a, Duplicate screens depicting HCoV-229E infection (24 hours post-infection) in Huh7 cells ectopically expressing ISGs. b, HCoV-229E infection of Huh7 cells expressing LY6E or control vector (Empty). Blue: DAPI, green: HCoV-229E N protein, red: TagRFP encoded in SCRPSY vector. c-h, Effect of LY6E expression on diverse coronaviruses. Stably transduced cells were infected with HCoV-229E (c), HCoV-OC43 (d), MERS-CoV (e), SARS-CoV (f), SARS-CoV-2 (g), MHV-Gluc (h). Viral replication readouts included: titer determination by plaque assay (c), infectivity by nucleoprotein staining and quantitation by flow cytometry (d), limiting dilution assay (e,f,g), luciferase activity shown as relative light units (RLU) (h,j,l). i, Western blot of WT A549 or two LY6E KO clones (#3, #4). j, HCoV-229E infection of WT or LY6E KO A549. k, Western blot of WT or LY6E KO A549 reconstituted with CRISPR-resistant LY6E (CR LY6E) or control vector (Empty). l, HCoV-229E infection of CR LY6E-reconstituted WT or LY6E KO A549.