EMBO/sd-character-level-ner huggingface dataset:
mTORC1 phosphorylates TFEB at serine 142 (S142).Figure 3(A) Torin 1 induces S142 dephosphorylation. HeLa cells were treated as indicated and total and nuclear extracts were probed with a TFEB p‐S142 phosphoantibody and with anti‐FLAG antibody. Disappearance of TFEB S142 phosphorylation upon starvation or Torin 1 treatment correlates with accumulation of TFEB in the nuclear fraction. (B) mTORC1 in‐vitro kinase assays. Highly purified FLAG-S6K1, TFEB-3 × FLAG, or TFEBS142A-3 × FLAG were incubated with radiolabelled ATP without kinase, with purified mTORC1 or with mTORC1+Torin 1, and analysed by autoradiography. The lower panel shows a FLAG immunoblot of the substrates. (C) Schematic representation of TFEB protein structure with the predicted mTORC1 phosphorylation sites and their conservation among vertebrates (for mTORC1 phophosite prediction see Material and methods). Numbering is according to human isoform 1. (D) Sequence conservation scores of the phosphosites and quantitative agreement between mTOR consensus motif and the sequence around the phosphosites of TFEB. (E) S142 and S211 regulate TFEB localization. FLAGimmunostaining (red) of HeLa cells expressing serine‐to‐alanine mutated versions of TFEB-3 × FLAG. Nuclei were stained with DAPI (blue). Values are means of five fields containing at least 50 transfected cells. Student's t‐test (unpaired) ***P0.001. Scale bars represent 30 μm.
(A) 293T cells were cultured in complete medium made of dialyzed serum for 24 h which was then replaced with medium in which one amino acid was eliminated and cultured for 8 h.Figure 1(A) 293T cells were cultured in complete medium made of dialyzed serum for 24 h which was then replaced with medium in which one amino acid was eliminated and cultured for 8 h. Then, WB was used to measure p-AMPK, p-ACC, p-Raptor and total AMPK, ACC, Raptor protein expression. "Nor" = normal medium. Actin served as the loading control. The red text "-Cys" indicates that cystine deficiency most significantly activates AMPK and its substrates. 293T cells were treated with cystine-deficient medium for 0, 0.5, 1, 2, 4, or 8 h (B) or treated with medium containing 200 µM, 100 µM, 50 µM, 25 µM or 0 µM cystine for 8 h (C); RCC4, SK-MES, SK-hep-1 or Hep3B cells were treated with cystine-deficient medium for indicated time (D). Protein levels of p-AMPK, p-ACC, p-Raptor and total AMPK, ACC, Raptor were measured by WB, and actin served as the loading control. (E) A schematic diagram of the hypothetical mechanism showing how cystine deprivation induces AMPK activation. (F) WB analysis of p-AMPK, p-ACC, p-Raptor and total AMPK, ACC, Raptor protein in 293T cells transfected with shRNAs targeting LKB1, CaMKK2, TAK1 or a non-targeting control (NTC) that were treated with cystine-deficient medium for 8 h. Actin served as the loading control. (G) WB analysis of p-AMPK, p-ACC, p-Raptor and total AMPK, ACC, Raptor protein in 293T cells and RCC4 cells treated with 1 µg/ml STO-609 or DMSO for 8 h during cystine deprivation. Actin served as the loading control. (H) 293T cells were transfected with HA-AMPKα1 alone or together with Flag-CaMKK2 for 48 h and then cultured with cystine-deficient medium or complete medium for 8 h. Cell lysates were immunoprecipitated with anti-Flag, and then, WB analysis was performed. (I) 293T cells cultured with cystine-deficient medium or complete medium for 8 h were harvested and subjected to immunoprecipitation with anti-AMPKα, followed by WB analysis with anti-AMPKα and anti-CaMKK2.
Figure 1A Scheme of the breast cancer patient's clinical status and therapies. Course of disease progression (blue) and treatment scheme (green) of the patient giving rise to CTC-ITB-01 are indicated. Timeline of progression and treatment indicated in years and months (mo). Drugs were administered at standard dosage according to indicated pattern. The red star represents the time point of blood sample collection. B Representative pictures of different CTCs from the initial CellSearch® analysis of the metastatic breast cancer patient who gave rise to the breast CTC line. The detected tumor cells display clear keratin and DAPI staining, CD45-negativity as well as lack of, or very weak (4, 8), ERBB2 expression. Cells of small (about 5 µm in diameter, 1, 2) and large size (larger than 10µm in diameter, 2,3) were detected. While some CTCs displayed dot-like perinuclear keratin signals (1, 2), the majority showed diffuse keratin staining. Additionally, CTC clusters of 4-8 cells were present (5, 6). Some CTCs showed multiple/lobed nuclei (4, 7, 8). C Bright field images of CTC-ITB-01 cells growing adherently. D Bright field images of CTC-ITB-01 cells growing non-adherently (relation between adherent and non-adherent cells: 80/20 %).
Figure 3A . MC3T3E1 cells were transfected with pTOPFLASH together with pCMV-β-gal. After 24 h, the cells were treated with DMSO, KY-02061 in DMSO, or 5 µM PolyR-DBM for 2 days. The luciferase activities of whole cell lysates were measured and normalized with β-galactosidase activities. [n=3] B . Primary calvaria cells were isolated from the calvariae of 4-day-old mice (Manton et al, 2007), and treated with DMSO or KY-02061 in DMSO for 4 days. ALP activity levels were visualized by ALP staining. C. Primary calvaria cells were treated with DMSO or 5 µM KY-02061 in DMSO for 14 days. The cells were subjected to immunofluorescence analyses to visualize Runx2 (green) and β-catenin (red). The cell nuclei were counterstained with DAPI (blue). D, E. Calvariae from 4-day-old mice were cultured ex vivo for 7 days with KY-02061 in DMSO (D). The calvaria thicknesses were measured from the stained sections using Image Pro software (E). [n=3]
Figure 4Measurement of cytokines present in cell culture supernatants of T cells co-cultured with either tumor-derived neutrophils (T + Ne; n=3) or with tumor-derived monocytes (T + Mo; n=3). Cytokines with a log2 fold change >0.6 or <-0.4 were considered as differentially present in supernatants. Shown is a heatmap of scaled cytokines levels (z-score) differentially present in T + Ne compared to T + Mo. Adjusted p-values were calculated by moderated t-test (limma) and Benjamini post test and were <0.05 only for MMP9 and RELMβ. Supernatants of T cells alone (T only; n=2) served as internal control. T cells cultured with monocytes did not have differential presence of cytokines compared to T cells alone. Co-immunostaining for MMP9 (green) and S100A9 (red) on a representative tumor section derived from an Apcfl/fl-Cdx2CreERT2 mouse. Right panel shows a higher magnification of indicated area of the left panel. mRNA expression levels of Mmp9 (C) or Tgfb1, Tgfb2 and Tgfb3 (D) in indicated FACS sorted cell types isolated from mouse colon tumors (n=4). Data are derived from RNA-sequencing and are displayed as reads per kilo base per million mapped reads (rpkm). Neutrophil - T cell co-cultures treated with vehicle (T + Ne), 10µm Galunisertib (T + Ne + TGFBRi), T cell proliferation after three days of indicated co-culture conditions. T cell proliferation index is numbers of proliferated T cells under indicated condition relative to the number of proliferated T cells when cultured alone. Neutrophil - T cell co-cultures treated with vehicle (T + Ne), 10µm of the MMP inhibitor GM6001 (T + Ne + MMPi) Protein levels of free active TGFβ1 in cell culture supernatants of indicated conditions determined by ELISA. Neutrophil - T cell co-cultures treated with vehicle (T + Ne), 10µm of the MMP inhibitor GM6001 (T + Ne + MMPi) T cell proliferation after three days of indicated co-culture conditions. T cell proliferation index is numbers of proliferated T cells under indicated condition relative to the number of proliferated T cells when cultured alone. Neutrophil - T cell co-cultures treated with vehicle (T + Ne), 10µm Galunisertib (T + Ne + TGFBRi), 10µm of the MMP inhibitor GM6001 (T + Ne + MMPi) or with 10µm Galunisertib plus 10µm GM6001 (T + Ne + TGFBRi + MMPi). T cell proliferation after three days of indicated co-culture conditions. T cell proliferation index is numbers of proliferated T cells under indicated condition relative to the number of proliferated T cells when cultured alone.
Figure 7(A) Metaplot of CG, CHG, and CHH methylation of genes and TEs in the genomes of wild type, epcr1/2, and arid2/3/4 plants. H indicates C, A, or T. (B) Metaplot of CG, CHG, and CHH methylation on chromosome 3 of Arabidopsis in wild type, epcr1/2, and arid2/3/4 plants. (C) Distribution of arid2/3/4 and epcr1/2 hyper-DMRs of CG, CHG, and CHH sites in pericentromeric regions and two chromosome arms. A pericentromeric region refers to 6 million base pairs of a centromere-flanking region on each chromosome. (D) Boxplot of DNA methylation in the wild type, nrpe1, epcr1/2, and arid2/3/4 mutants. The hypo-DMRs identified in the nrpe1 mutant was defined as RdDM target loci. Asterisks indicate that DNA methylation is significantly (P<0.001) decreased in the mutants as determined by paired student t-test. Horizontal lines represent the median, and the bottom and top of the box represent the 25th and 75th percentile. The whiskers represent data range within 1.5× of the interquantile range. (E) Heat maps of CG, CHG, and CHH methylation at RdDM target loci. The hypo-DMRs identified in the nrpe1 mutant were defined as RdDM target loci and analyzed in the wild type, nrpe1, eprc1/2, and arid2/3/4 mutants. Black and light yellow indicate low methylation and high methylation, respectively.
Figure 5A Graphs show the quantification of lamin B1 intensity and circularity. N = 3. An average of 12 nuclei was examined in each culture. Data are expressed as a percentage of controls. Bars represent the mean ± S.E.M. *P < 0.05 and **P < 0.01 as compared to mApple-C1 control neurons (two-tailed unpaired Student's t test). Exact P values are reported in Appendix Table S3.
Caspase-2 has no impact on the degree of aneuploidy in HCC.Figure 4Copy number variations were determined using whole genome sequencing of 30 nuclei "mini-bulks" isolated from Casp2-/- or wt tumors (n=11 per genotype). Diploid and tetraploid pools of tumor cell nuclei were analyzed separately. A, B (A) Representative dot plots show the degree of aneuploidy and heterogeneity and are quantified in (B, empty symbols). Data are represented as median, statistical significance determined by one-way ANOVA with multiplicity correction (Sidak-Holm); * p≤0.05, ** p≤0.01. N-numbers refer to biological replicates.
Figure 1(A) 12-lead ECG of patient 1. (B) 12-lead ECG of patient 2. (C) ECG of patient 2 during epinephrine test (A- represents baseline ECG; B- ECG at 17 min and 47 s of the beginning of epinephrine infusion (0.2 μg/kg of epinephrine) and C- ECG after the end of epinephrine infusion). (D) Pedigree of a family diagnosed with arrhythmias; Arrow indicates the proband, solid symbols represent family members affected by arrhythmias who are also homozygous for mutations identified by exome sequencing. Crossed symbols indicate deceased individuals. Half-filled symbols correspond to individuals heterozygous for mutations identified by exome sequencing. (E) ECG of patient 3 (subject IV:13) showing mild QTc prolongation. (F) ICD interrogation of patient 3 (subject IV:13) reveals an episode of VT.
Figure 2Quantitative fluorescent microscopy imaging shows a dose-dependent increase in the number of C9 DPR reporter positive cells with increasing concentrations of an apoptotic stimulator, staurosporine (SSP). The C9 DPR reporter in the GP frame was utilized for these experiments. n = 4. Filter trap binding assays using DPR-specific antibodies show all DPR levels are increased for the sense-coding C9 DPR reporter when NSC34 cells are challenged with stress-inducing compounds. These stress-inducing compounds broadly cover distinct or overlapping cellular pathways (see Table EV2) Antibodies specific for the C9orf72 NRE DPRs, poly-GA, -GP, or -GR, were used to quantify DPR levels normalized to GAPDH 24 h post transient transfection of the C9 DPR reporter constructs. n = 4. Quantitative fluorescent microscopy imaging shows that NSC34, rat primary cortical neurons have increased relative DPR fluorescent intensity levels following compound induced cellular stress. In NES-mIFP cotransfection positive NSC34 and rat primary cortical neurons there is a significant increase in C9 DPR reporter fluorescent intensity levels 24 post treatment with most stress-inducing compounds. Fluorescent intensity was not normalized to NES-mIFP fluorescent levels. NSC34: n = 10 with m > 500 cells analyzed per n. Cortical neuron: n = 6 with m > 40 cells per n. Endogenously expression of DPRs in iPS spinal motor neurons (iPS sMN) derived from C9orf72 NRE patients increases following induction of cellular stress. DPR fluorescent intensity in iPS sMN was measured using the DPR-specific antibodies described above. IPS sMN: n = 4 with m > 20 cells per n.
Figure 1(A-C) Western blot analysis of THP-1 cells infected with (A) HSV-1 at MOI 5 for 3 h, 6 h, 12 h, 18 h, and 24 h (B) CHIKV at MOI 5 for 3 h, 6 h, 12 h, 18 h, and 24 h (C) JEV at MOI 5 for 3 h, 6 h, 12 h, and 24 h. (D-H) Western blot analysis of THP-1 cells untreated or treated with (D) 5'pppRNA ( 1 μg/ml) for 3 h, 6 h, 12 h, 18 h and 24 h (E) Poly I:C (1 μg/ml) for 3 h, 6 h, 9 h, 12 h and 24 h (F) Poly dA:dT (1 μg/ml) for 3 h, 6 h, 9 h,12 h and 24 h (G) cGAMP (1 μg/ml) for 6 h, 12 h, 18 h, and 24 h (H) dsDNA (1 μg/ml) for 3 h, 6 h, and 12 h and probed with antibodies as indicated. (I-L) Control and IRGM knockdown THP-1 IFN reporter cells were treated with (I) Poly I:C (1 μg/ml) for 8 h, 12 h and 24 h or (J) Poly dA:dT (1 μg/ml) for 12 h and 24 h or (K) IFN-β (500 ng/ml) for 8 h and 12 h or (L) cGAMP (1 μg/ml) for 12 h and 24 h and the supernatant was subjected to luciferase reporter assay using QuantiLuc reagent (M-P) Control and IRGM knockdown THP-1 cells were untreated and treated with Poly I:C (1 μg/ml) for 8 h and the total RNA was subjected to qRT PCR with primers of (M) MX2 (N) ISG15 (O) OAS1 and (P) IFN-β. (Q-T) Control and IRGM knockdown THP-1 cells were untreated and treated with Poly dA:dT (1 μg/ml) for 8 h and the total RNA was subjected to qRT PCR with primers of (Q) MX2 (R) ISG15 (S) OAS1 and (T) IFN-β.
SIRT7 regulates KCC4 activity during a decrease in external pH.Figure 3A KCC4 acetylation at the indicated pH in HEK293 transfected with KCC4. After lysis, KCC4 was immunoprecipitated using an anti-FLAG antibody, and then analyzed by SDS-PAGE/Western blotting using an anti-acetylated-lysine (AcK) or anti-KCC4 antibody. Non-transfected cells (UT) were used as a control. B HEK293 cells were transfected with KCC4 together with SIRT7 with a scramble shRNA (-) or a shRNA against SIRT7 (+), and KCC4 activity and protein level were determined at the indicated pH. SIRT7 expression in these conditions is presented in C. C Nuclear and cytoplasmic SIRT7 levels at different pH values in HEK293 cells. At a lower pH, SIRT7 appears to translocate to the cytoplasm.
Figure 7A, B Immunoblot analysis of HeLa WT and HeLa CALCOCO1 KO cells. The cells were starved for 6 hours (HBSS) as indicated and treated with Baf A1 as indicated. Numbers below the blots in A represent relative intensity of the bands in the shown blots normalized against GAPDH loading control. In A, the panels are collected from more than one western blot experiment but for clarity, only a single GAPDH loading control is shown. In B, the bars represent the mean±sd of band intensities of three independent experiments as quantified using ImageJ. Statistical comparison was analyzed by one-way ANOVA followed by Tukey multiple comparison test and significance displayed as **p ˂ 0.005, *p ˂ 0.01; ns is not significant. C, D Immunoblot analysis of HeLa CALCOCO1 KO cell lines reconstituted with EGFP-CALCOCO1. Expression of EGFP-CALCOCO1 was induced or not with tetracycline and the cells were treated with MG132 or Baf A1 as indicated. Numbers below the blots in C represent relative intensity of the bands in the shown blots normalized against actin loading control. In C, the panels are collected from more than one western blot experiment but only a single actin loading control is shown. In D, the bars represent the mean±sd of band intensities of three independent experiments as quantified using ImageJ. Statistical comparison was analyzed as in B and significance displayed as **p ˂ 0.005, *p ˂ 0.01; ns is not significant. E HeLa CALCOCO1 KO cell lines reconstituted with EGFP-CALCOCO1 were treated with tetracycline or not to induce expression of EGFP-CALCOCO1. Abundance of the ER was quantified from widefield fluorescence images of endogenous RTN3 staining (see Materials and methods). Data are presented as mean ± sd of three independent experiments. Statistical comparison was analyzed as in B and significance displayed as ***p ˂ 0.001, **p ˂ 0.005, *p ˂ 0.01; ns is not significant. F, G Immunoblot analysis of cells analyzed in C. The bars in G represent the mean±sd of band intensities relative to the loading control from three independent experiments quantified using ImageJ. Statistical comparison was analyzed by one-way ANOVA followed by Tukey multiple comparison test. Significance is displayed as **p ˂ 0.005, *p ˂ 0.01; ns is not significant. H, I Immunoblot analysis of HeLa CALCOCO1 KO cells stably expressing EGFP-CALCOCO1 in fed or starved conditions and transfected with the indicated VAPA/B siRNAs.
Figure 8A Representative live-cell STED super-resolution images (t=0 s) showing WT Hela cells stained with TMRM. Box marks selection shown as a zoom in panel (B) (red hot LUT). Scale bar 500 nm. B Time-lapse image series of two mitochondria stained with TMRM (1.5 s/frame). Green asterisks show merging events and redistribution of TMRM intensity from a high-intensity cristae to a low-intensity cristae (two such instances are shown at 3 and 7.4 s). Green arrows pointing inward connected by solid line at 1.5 and 5.9 s indicate imminent merging events. Scale bar 500 nm. C Representative live-cell STED super-resolution images (t=0 s) showing WT Hela cells stained with TMRM. Box marks selection shown as a zoom in panel (D) (red hot LUT). Scale bar 500 nm. D Time-lapse image series of two mitochondria stained with TMRM (1.5 s/frame). Green and magenta asterisks show cristae merging and splitting cycles (shown at 5.9 s and 7.4 s). Green arrows pointing inward connected by solid line and magenta arrows pointing outward connected by dotted line show sites of imminent merging and splitting events, respectively. Scale bar 500 nm. E Representative time-lapse images of WT Hela cells stained with NAO, and imaged using Airyscan microscope, indicate cristae merging and splitting dynamics. Yellow and magenta asterisks show merging and splitting events of cristae respectively. Yellow arrows pointing inward connected by solid line and magenta arrows pointing outward connected by dotted line show sites of imminent merging and splitting events, respectively. Scale bar 500 nm.
Figure 7A) HEK293A were treated with VPS34-IN1 (1μM) alone or in the presence of MRT68921 (1μM) for the indicated time. Untreated VPS15 KOs were included as a positive control and lysates analysed by Western blot. Identical samples were loaded in parallel for VPS34, ATG13, PRKAB2 analysis, indicated by dashed line. B) HEK293A and VPS15 KOs were fixed and the labelled for p62 (green) and FIP200 (red). C) HEK293A and VPS15 KOs were fixed and the labelled for ubiquitin (green), Ferritin (red) and p62 (blue). D) HEK293A and VPS15 KOs were stained for p62 (green), ATG9 (red) and LAMP1 (blue). E) HEK293A and VPS15 KO cells were fed, or starved for 2 hrs before fixation, and labelling with ULK1 (green), ATG9A (red) and GM130 (blue). Dashed boxes show magnified regions of interest.
Figure 3A The partner-binding regions of CD53 and CD81. Their EC2 sequences are aligned. Identical residues are shaded in dark grey, similar residues in light grey, and partner-binding regions identified by HDX-MS in orange. Secondary structures are indicated above. Representative HDX-MS curves showing the protected regions in CD53 and CD2. The HDX experiments were performed (manually or with a LEAP automation system) in triplicate. Error bars show the standard deviation. C Left, CD53-CD2 docking model. The HDX-MS identified regions are shown as a red rod in CD53 and as a blue arrow in CD2. The black box indicates the CD2 transmembrane region and the dashed line indicates the linker to Ig domains. Right, CD53-CD2 docking model with CD53 modeled in the closed conformation. The modeling was based on the CD81 structure, in which EC2 reorients to interact with the TM domain. Docking of CD19 Ig to CD53 EC2 is in the same manner as Left. D Representative HDX-MS curves showing the protected regions in CD81 and CD19. The HDX experiments were manually performed in triplicate. Error bars show the standard deviation. E Left, CD81-CD19 docking model in the closed conformation. The HDX-MS identified binding regions are colored as in C. Right, Modeled open conformation of CD81 and docking with CD19 (in the same manner as Left). MD simulation (1 µs) of this open conformation is shown in Fig 4 and Movie EV1.
Figure 3(A) Subcellular localization of MAP4K4-GFP in Arabidopsis mesophyll protoplasts. MAP4K4-GFP (green) partially co-localized with the plasma membrane dye FM4-64 (red). 14 hours after transformation, photographs were taken when the plasma membrane of mesophyll protoplasts was stained by FM4-64 (< 3 min). Scale bars = 10 µm. (B) Auto-phosphorylation of recombinant HisMBP-MAP4K4 wild-type, no auto-phosphorylation in the kinase-inactive HisMBP-MAP4K4-D139A protein. The phosphorylation level was determined by Pro-Q Diamond phosphoprotein gel stain (upper panel), protein loading amounts were visualized by Coomassie G-250 staining (lower panel). The predicted molecular weight of HisMBP-MAP4K4 is 118.4 kDa. (C) BiFC visualization of interaction between MAP4K4 and BIK1 in Arabidopsis mesophyll protoplasts. BIK1-nYFP and MAP4K4-cYFP were co-expressed in protoplasts to show fluorescence of complemented YFP signal (green), 14 h after transformation, the results were photographed by confocal laser scanning microscopy, scale bars = 10 µm. Chl indicates Chloroplast, BF indicate Bright field. nYFP and cYFP constructs were used as empty controls. MAP3K5-K375M and MKK4 were used as control. (D) MAP4K4 associates with BIK1 in vivo by Co-IP. BIK1-HA and MAP4K4-GFP were co-expressed in plants by crossing of 35S::BIK1-HA and 35S::MAP4K4-GFP transgenic lines. 14 days old plants were treated (+) with 1 µM flg22 or H2O (-) for 10 min. GFP trap beads were used for immunoprecipitation, and target proteins were detected by western blots using anti-GFP or anti-HA antibodies. The predicted molecular weight of BIK1-HA, MAP4K4-GFP is 49.3 kDa and 103.8 kDa respectively.
Fig.Figure 3A The rationale of footprinting analysis as illustrated by actual ATAC-Seq peaks, HINT-ATAC footprint hits, and example TF motif instances near RAG1 gene in DN2 population of thymus donor 6. B Heatmap of ln(odds ratio) of the 50 TFs showing the most differential abundance of binding motifs in the open chromatin regions. TFs with an SD of the motif counts <10 are excluded.
The amplitude and frequency of miniature EPSCs of Venus-positive cells are inversely correlated with the fluorescence intensity A.Figure 5A. Scatter plots showing Venus-fluorescent intensities and the median amplitude of miniature EPSCs (mEPSC) in the LA of the AV-KO mice and their littermates (AV-WT). B. Scatter plots showing Venus-fluorescent intensities and the mean frequency of miniature EPSCs in the LA of the AV-KO and their control littermates (AV-WT). Three typical pictures of Venus-positive cells with various fluorescent intensities are shown. Lines in each panel are regression lines showing inverse correlation of the fluorescent intensity with the mEPSC amplitude (A) or the mEPSC frequency (B). The black dotted lines are regression lines for AV-WT mice, the red dotted lines are those for AV-KO mice, and black solid lines are those for all cells.