EMBO/sd-character-level-ner huggingface dataset:
Miro negatively regulates the recruitment of Drp1 to peroxisomes and mitochondria.Figure 6A) Representative images of proximity-ligation assay (PLA) of Fis1 and Drp1 in WT and DKO MEFs. Scale bar is 10 μm. Red dots indicate interaction of Fis1 and Drp1. Blue is DAPI. B) Quantification of fluorescent dots from PLA of Fis1 and Drp1 in WT and DKO MEFs. Dotted line indicates the sum of the Fis1 and Drp1 single antibody controls (n=3 experiments). Data information: For B) statistical significance was quantified by a two-tailed Student's t test. *, ** and *** denote p < 0.05, p < 0.01 and p < 0.001, respectively. Data are represented as mean ± SEM. C) Representative images of a Fis1-Drp1 PLA in DKO MEFs expressing either GFP, GFPv1 (GFP-tagged variant 1 of Miro1) or GFPv4 (GFP-tagged variant 4 of Miro1) n=30 cells per condition over three independent experiments). Scale bar is 10 μm. Red dots indicate interaction of Fis1 and Drp1. Blue is DAPI. D) Quantification of PLA fluorescent dots per cells between GFP, GFPv1 or GFPv4 transfected DKO MEFs (n=30 cells over three independent experiments). Data information: In D) a one-way ANOVA with a Newman-Keuls post-hoc test was used to calculate statistical significance. *, ** and *** denote p < 0.05, p < 0.01 and p < 0.001, respectively. Data are represented as mean ± SEM. E) Western blot of Drp1, Fis1 and actin from WT and DKO whole cell lysates. F) Quantification of normalised band intensity of Drp1 and Fis1 in WT and DKO MEFs (n=3). Data information F), statistical significance was quantified by a two-tailed Student's t test. G) Zooms of endogenous Drp1, mitochondria (Mitotracker) and peroxisomes (PMP70) in WT and DKO MEFs. Scale bar is 5 μm. H) Integrated density of Drp1 signal on Mitotracker-positive and PMP70-negative structures in WT and DKO MEFs. I) Integrated density of Drp1 signal on PMP70-positive and Mitotracker-negative structures in WT and DKO MEFs. Data information G-I n=42 cells per condition over three independent experiments. For , H) statistical significance was quantified by a two-tailed Student's t test. *, ** and *** denote p < 0.05, p < 0.01 and p < 0.001, respectively. Data are represented as mean ± SEM.
Experimental and computational analysis Scheme of this study Nine tissues/cells from the F1 hybrid of the two mouse inbred strains C57BL/6J and SPRET/EiJ were subject to the 3'mRNA-seq.Figure 1Nine tissues/cells from the F1 hybrid of the two mouse inbred strains C57BL/6J and SPRET/EiJ were subject to the 3'mRNA-seq. Sequencing reads were mapped to the reference genomes of the parental strains respectively and classified as allele-specific reads or common reads. Only reads uniquely mapped in PASs annotated in the PolyA_DB were used for PAS usage calculation. Total PAS usage (calculated by the sum of allelic and common reads) in different tissues was utilized in within-tissue comparisons and analysis of APA patterns across tissues. The allelic reads were utilized to study allele-specific APA patterns and cis-regulatory divergence.
Figure 6Total intracellular glutathione (GSH + GSSG per 106 cells), as determined by GR re-cycling assay after 24h MK-571 treatment in six different cell lines (n ≥ 3, except HCT116 WT n = 2). *p = 0.014, Paired t test. Each dot represents one cell line, see Fig. EV6A for individual cell lines and n. Intracellular oxidized glutathione (GSSG per 106 cells) as shown by LC-MS in OVCAR-3 (R248Q TP53) cells at 24h treatment with APR-246 +/- MK-571 (n = 3). Intracellular reduced glutathione (GSH per 106 cells) as shown by LC-MS in OVCAR-3 cells at 24h treatment with APR-246 +/- MK-571 (n = 3). Western blot analysis of MRP1 (Cell Signaling), xCT and GAPDH of OVCAR-3 cells treated with APR-246 +/- MK-571 for 24h. Western blot analysis of MRP1 (Cell Signaling), xCT and GAPDH of HCT116 WT and R248W cells treated with APR-246 +/- MK-571 for 24h. Intracellular cystine (CySS per 106 cells) as shown by LC-MS in OVCAR-3 cells at 24h treatment with APR-246 +/- MK-571 (n = 3). Intracellular cysteine (Cys per 106 cells) as shown by LC-MS in OVCAR-3 cells at 24h treatment with APR-246 +/- MK-571 (n = 3).
Cdk4 deficiency promotes thermogenesis and mitochondrial biogenesis in iBAT.Figure 2A Gross morphology of iBAT from Cdk4+/+ and Cdk4-/- mice. C Hematoxylin-eosin staining and UCP1 immunohistochemical (IHC) staining of iBAT sections (scale bar 100 μm) from Cdk4+/+ and Cdk4-/- mice. D-E Western blot analysis of UCP1 and CDK4 protein expression (D) and quantified protein levels of UCP1 (E) (n=4 biological replicates) in iBAT. HSP90 was used as loading control. F Expression of thermogenic genes and brown adipocyte markers in iBAT of Cdk4+/+ (n=6) and Cdk4-/- (n=6) mice as assessed by RT-PCR. G-H Transmission electron microscopy (TEM) images of iBAT (G) (scale bar 5 μm (upper panel) and 1 μm (lower panel, LD indicates lipid droplet and M mitochondria)) and quantification of lipid droplet number (LD) and mitochondrial volume (Mito) (H) (Cdk4+/+ (n=4) and Cdk4-/- (n=4)). I Respirometry analysis of iBAT as measured by the Oroboros system (Cdk4+/+ (n=6) and Cdk4-/- (n=6)).
Loss of THEM6 impairs PCa tumour growth following ADT.Figure 1A, Volcano plot of the differentially modulated proteins in 22rv1 (CRPC) versus CWR22res (HN) tumours. Red and blue dots represent the proteins that are significantly up- and down-regulated, respectively (p-value < 0.05, FC = 1.5). Data information: Data reproducibility: n = 3 tumours per group. B, Western blot analysis of THEM6 expression in CRPC (22rv1 and LNCaP AI) and HN (CWR22res and LNCaP) prostate orthografts. VCL was used as a sample loading control. Data information: Data reproducibility: : n = 1 gel loaded with three prostate orthografts per condition. C, IHC staining of THEM6 expression in CWR22res and 22rv1 orthografts. Scale bar represents 100 µm. Data information: Data reproducibility: representative image from 3 independent biological experiments. D, Western blot analysis of THEM6 expression in CTL and THEM6 KO CRPC cells. HSC70 was used as a sample loading control. Data information: Data reproducibility: : representative image from 3 independent biological experiments. E, Proliferation of CTL and THEM6 KO CRPC cells after 72 hours. Data are expressed as a percentage of CTL cells. Data information: Data are presented as mean values +/- SD. Panel Statistical analysis: 1-way ANOVA with a Dunnett's multiple comparisons test. Data reproducibility: n = 3 independent biological experiments. F, Cell stiffness (Young's modulus) of CTL and THEM6 KO CRPC cells measured by atomic force microscopy. Data information: : Centre line corresponds to median of data, top and bottom lines correspond to maximal and minimal values. Statistical analysis: 1-way ANOVA with a Dunnett's multiple comparisons test. Data reproducibility: (up): n = 46 (CTL); 47 (KO1); 49 (KO2) cells measured. (down): n = 31 (CTL); 43 (KO1); 50 (KO2) cells measured. G, Tumour volume (measured by ultrasound) of CTL and THEM6 KO 22rv1-derived orthografts developed in surgically castrated mice. H, Tumour volume of CTL and THEM6 KO LNCaP AI-derived xenografts developed in surgically castrated mice. I, Tumour volume (measured by ultrasound) of CTL and THEM6 KO CWR22res-derived orthografts. Orchidectomy was performed 3-weeks after cell implantation. Data information: Data are presented as mean values +/- SD. Statistical analysis: G, H: two-tailed Mann-Whitney U test. I: Kruskal-Wallis test. Data reproducibility: G, I: n = 5 mice per group. Panel H: n = 7 mice per group.
Analysis of centrosome- and GA-MT nucleation A Confocal images of WT, akap KO, c5rap2 KO, pcnt KO, pc-ak-2KO and pc-c5-2KO cells stained for EB1 (green) and the centrosomal protein CAP350 (red).Figure 2Confocal images of WT, akap KO, c5rap2 KO, pcnt KO, pc-ak-2KO and pc-c5-2KO cells stained for EB1 (green) and the centrosomal protein CAP350 (red). High magnification single-channel images of selected areas are shown at right. Scatter plot showing quantification of EB1 comets from three independent experiments as that shown in A. A region of interest of 3 μm radius around the CAP350 signal was used to count the number of comets. Horizontal black lines represent the mean. MT regrowth experiments of WT cells mixed with either WT (as a control), akap KO, c5rap2 KO, pcnt KO, pc-ak-2KO or pc-c5-2KO cells. Samples were fixed 3 min after NZ washout and stained for EB1 (green) and the indicated protein in the red channel. Quantification of EB1 signal intensity around the centrosome WT and pc-ak-2KO cells were transfected with a combination of three siRNAs against CEP192, fixed and triple labelled for EB1, CEP192 and CAP350, as a centrosomal marker. Scramble siRNA was used as a control. Confocal images of WT, akap KO, c5rap2 KO, pcnt KO, pc-ak-2KO and pc-c5-2KO cells stained for EB1 (green) EB1 comets around the centrosome Three min MT regrowth experiments in WT and pc-ak-2KO cells transfected with either scramble or CEP192 siRNAs. Quantification of the EB1 signal intensity around the centrosome MT regrowth experiments in WT and c5rap2 KO or pcnt KO cells. Cells were stained as indicated. Arrows point the centrosome. Quantification of EB1 signal intensity at Golgi membranes.
cAMP-dependent pathway mediates resistance to CX-5461-everolimus co-treatment(A) Enrichment analysis by GeneGO MetaCore® of the polysomal RNAseq data comparing CX-5461+EV-resistant (CMB) and drug-naïve cells (CTRL) (n=3; false discovery rate (FDR) ≤ 0.Figure 3(A) Enrichment analysis by GeneGO MetaCore® of the polysomal RNAseq data comparing CX-5461+EV-resistant (CMB) and drug-naïve cells (CTRL) (n=3; false discovery rate (FDR) ≤ 0.05; fold change (FC) ≥ 1.5 or ≤-1.5). (B) Abundance of intracellular 3'5'-cyclic AMP as measured by a liquid chromatography (LC)-mass spectrometry (MS) analysis. Graphs represent mean ± SEM of n=3 (5-6 technical replicates each). (C) Western analysis of EPAC1 and EPAC2 abundance, as well as active GTP-bound RAP1 levels in the indicated early-passage cells during their log-phase growth period (n=3). Actin and total RAP1 were used as loading controls. (D) Propidium iodide (PI) exclusion assays of CMB cells treated with CX-5461 and everolimus as indicated in the presence or absence of a selective PKA inhibitor H89 for 48 hours. (E) PI exclusion analysis of early-passage drug-naïve (CTRL) lymphoma cells treated with CX-5461 and everolimus in the presence or absence of selective PKA activator 6-Bnz-cAMP for 48 hours. (F) PI exclusion analysis of the CMB cells treated with CX-5461 and everolimus as indicated in the presence or absence of EPAC1 inhibitor CE3F4 or EPAC2 inhibitor ESI-05 for 48 hours. (G) PI exclusion analysis of early-passage drug-naïve (CTRL) cells treated with CX-5461 and everolimus in the presence or absence of the selective EPAC activator 8-pCPT-2-O-Me-cAMP for 48 hours.
Figure 4A Autophosphorylation as measured in a ELISA assay is shown for WT and interaction mutants in presence of control vesicles (PC; phosphatidylcholine) or PI(4,5) P2 containing vesicles. Plotted are mean values from two independent autophosphorylation reactions, each determined in duplicates (n=4). Mutant nomenclature is as shown in Table 2. B Catalytic turnover activity as measured in a coupled kinase assay is shown. Plotted are mean values from at least three independent experiments. C To determine whether autophosphorylation occurs in cis or trans, autophosphorylation was measured for WT or a kinase dead form of FAK (KD, contains K454R mutation) in presence or absence of a substrate deficient FAK mutant (Y397F) and/or PI(4,5) P2 vesicles. PI(4,5) P2 negative experiments contain control PC vesicles instead. Note that the scales of the two plots are different, i.e. autophosphorylation is increased in presence of PI(4,5) P2. Plotted are mean values from two independent phosphorylation reactions, each determined in duplicates (n=4). Left: Schematic of possible cis- and trans-autophosphorylation (red arrows) in mixtures of FAK-WT/FAK-Y397F and FAK-KD/FAK-Y397F. D Src phosphorylation of Y577 in FAK in presence of PC or PI(4,5) P2 vesicles as determined by an ELISA method. The plot on the left is obtained in presence Src containing only the kinase domain (Src254-536) and the plot on the right with Src containing additionally the SH3 and SH2 domains (Src84-536). Plotted are mean values from two independent phosphorylation reactions, each determined in duplicates (n=4).
InlK is a virulence factor of L.Figure 1A. The inlK gene locus in L. monocytogenes compared with the same genomic region in the related non-pathogenic species L. innocua. The stem and circle represent transcription terminators. B. Kaplan-Meier curve represents the survival of BALB/c mice over time. Four BALB/c mice in each experimental group were infected i.v with 104 L. monocytogenes wild-type (EGD-e) or ΔinlK mutant. C. The L. monocytogenes EGD-e wild-type strain (WT), the ΔinlK mutant (ΔinlK) and the complemented strain (ΔinlK+pPL2 inlK) 104 CFU were inoculated i.v into BALB/c mice. Animals were euthanized 24 h, 48 h, 72 h or 96 h after infection and organs were recovered, homogenized, and homogenates serially plated on BHI. The number of bacteria able to colonize liver (left panel) and spleen (right panel) is expressed as log10 CFU. Four animals per bacterial strain, per time points and per experiment were used. Statistical analyses were performed on the results of 3 independent experiments using the Student t test. P values of <0.05 were considered statistically different and are labeled here as *.
Figure 2(A) Gene ontology (GO) enrichment analysis of gene transcripts up-regulated in MCK-miR-499muscle identified a number of terms related to mitochondrial function and muscle contraction. Gene expression array data generated from the gastrocnemiusmuscle of 8-week-old male MCK-miR-499mice were compared to littermate controls (NTG). (B) Expression of the nuclear-encoded and mitochondrial-encoded genes (RT-qPCR) in the gastrocnemius muscle from the indicated genotypes (n = 5 mice per group). *p < 0.05. (C) Representative western blot analysis performed with gastrocnemius muscle total protein extracts prepared from the indicated mice using myoglobin, cytochrome c, and β-actin (control) antibodies (n = 8 mice per group). (D) Expression of genes (RT-qPCR) involved in muscle glucose and fatty acids metabolism in the gastrocnemius muscle from the indicated genotypes (n = 5 mice per group). *p < 0.05. (E) LDH isoenzymes were separated by polyacrylamide gel electrophoresis using whole cell extracts from NTG heart (Ht, control) and gastrocnemius muscle from the indicated mice. A representative gel showing 3-4 independent mice per group is shown.(F) Quantification of LDH isoenzyme activity gel electrophoresis in E. Values represent the mean % (+/- SEM) total LDH activity. *p < 0.05. (G) Cross-section of the gastrocnemius muscle from a 3-month-old male NTG and MCK-miR-499 stained for myosin I ATPase activity (Top), SDH (Middle), and α-GPDH (Bottom). Representative images were shown. Scale bar: 500 μm.
Neuronal survival after exposure to SIV-infected microglia supernatant in the presence of different autophagy related drugs.Figure 6(A). Neuronal survival tested by MTT. There are significant decreases of neuronal survival after exposure to SIV-infected microglia supernatant for 3 or 24 hr, which is significantly abrogated in the presence of rapamycin. Rapamycin alone has no effect. MK-801 (100 µM) also prevented the neurotoxicity induced by SIV-infected microglia supernatant. *** P<0.001, for n = 6 experiments. (B) Neuronal survival was examined with MTT in cultures exposed to SIV-infected microglia supernatant for 24 hr and in the presence or absence of different drugs such as 3-MA (1 mM) or Baf (100 nM). There is no significant difference between cells exposed to SIV-infected microglia supernatant and a pretreatment with 3-MA followed by exposure to SIV-infected microglia supernatant. However, there is a significant decrease of neuronal survival exposed to SIV-infected microglia supernatant when cells are pretreated with Baf. The SIV-infected microglia supernatant from no infected monkey (no Sup) doesn't affect neuronal survival and there is no significant difference with control samples. All values are mean±SEM.
Fig.Figure 4(A) Localization of c-Myc in uninfected Caco-2 cells (first row), Caco-2 cells infected with V. parahaemolyticus vopZ'ΔvscN1 (wild type VgpA, second row), Caco-2 cells infected with vopZ'ΔvscN1vgpA' (third row), Caco-2 cells infected with vopZ'ΔvscN1vgpA':pvgpA (fourth row) and Caco-2 cells infected with vopZ'ΔvscN1vgpA':pvgpAL10A (fifth row). Fixed cells were stained with DAPI (blue), and anti-EBP2 (red), and anti c-Myc (green) antibodies. White arrows are examples indicating the co-localization of EBP2 and c-Myc. (B Detection of c-Myc or EBP2 in nuclear extracts from uninfected Caco-2 cells or after infection with indicated strains. Following infection, nuclear extracts were obtained and loaded on protein A/G beads cross-linked with anti-EBP2 (B, IP: anti-EBP2) Input and eluates were blotted with anti-EBP2, anti-c-Myc or anti-VgpA antibodies, respectively. Quantified value (mean ± standard deviation) as measured by band intensity (normalized to uninfected) from three independent experiments are presented under the signals of the blot of IP samples. n.d., not determined. C) Detection of c-Myc or EBP2 in nuclear extracts from uninfected Caco-2 cells or after infection with indicated strains. Following infection, nuclear extracts were obtained and loaded on protein A/G beads cross-linked anti-C-Myc (C, IP: anti-c-Myc) antibodies, respectively. Input and eluates were blotted with anti-EBP2, anti-c-Myc or anti-VgpA antibodies, respectively. Quantified value (mean ± standard deviation) as measured by band intensity (normalized to uninfected) from three independent experiments are presented under the signals of the blot of IP samples. n.d., not determined. (D) Purified proteins EBP2, c-Myc and GST-VgpA or GST-VgpAL10A were mixed and then pulled down with protein A/G beads cross-linked with anti-EBP2. Input and eluates were blotted with anti-EBP2, anti-c-Myc or anti-VgpA antibodies, respectively. Quantified value (mean ± standard deviation) as measured by band intensity (normalized to EBP2+c-Myc sample without GST-VgpA and GST-VgpAL10A) from three independent experiments are presented under the signals of the blot of IP samples. n.d., not determined. (E) Caco-2 cells were infected with the indicated strains and treated with cycloheximide (CHX) for the indicated time. Whole cells lysates were analyzed by western blot using anti-c-Myc and anti-Actin antibody.
Figure 2A Three-dimensional reconstruction of serial H&E frontal sections of miniature pig mandibles at embryonic day 60 (E60) and E90; deciduous canine (DC) in purple, permanent canine (PC) in yellow, and alveolar socket in green. The red, blue, and green arrows indicate the width of the labial alveolar socket, lingual alveolar socket, and DC, respectively. n = 3. B DC and labial and lingual alveolar socket widths in the horizontal plane at the bottom of the PC during E60 and E90. n = 3. Data information: Data represent the means ± SEM. Unpaired t-tests, *P < .05, **P < .01, ***P < .001. C The proportion of DC width relative to the total alveolar socket width during E60 and E90. n = 3. Data information: Data represent the means ± SEM. Unpaired t-tests, *P < .05, **P < .01, ***P < .001. D Micro-CT imaging of the whole mandible at E60; boxed region is magnified in D' (top panel). (D') Mandible slice isolated with Geomagic software. White dashed lines mark DC. E 3-D color map (left) after alignment of mandible slices before and after (sham) surgery showing a comparison of the surface points. Coronal sections through cusp tips (transparent squares, left) were selected for 2-D comparisons (middle); right panels are magnifications of yellow-boxed regions. The solid purple contour and dotted black contour indicate the pre- and post-surgery shapes, respectively. The distance between the two contours are the colored line segments showing the distance and direction of the movement. The colored ball in the 2-D comparison marks the position of the maximum displacement. The PC position is indicated in pink. n = 3. F Dissected mandible slice with a "U" shape. G In the cup model, the mandible slice was simplified as a cup according to its dimensions (unit, mm). Red arrows indicate the uniform force (stress) exerted on the inner wall of the mandible. The bottom of the cup was fixed to avoid rigid body motions. H 3-D color map shows the extent of deformation based on the cup model established with ANSYS software. I Probable range of mechanical stress inside the mandible evaluated by multiple simulation tests in which serial stress and Young's modulus were inputted into the cup model; the cup was set with the outer surface fixed (left, dr = 0 denotes that radial deformation of the outer surface equals 0) or the outer surface free (right). J Deformation of the mandible walls under a series of stress levels with different Young's moduli. Data were obtained from multiple tests as in I. Gray horizontal lines indicate upper and lower limit values of mean mandible wall displacement (79.74 and 36.48 µm, respectively); dashed colored lines indicate results of the free outer surface; solid colored lines indicate results of the fixed outer surface (with consideration of good linearity of the simulation results, the corresponding result points of the simulation series were omitted and replaced by solid or dashed colored lines for clarity); the actual stress value should be between the two extreme boundary conditions. Results show that the probable stress level ranged from 3-20 kPa. n = 3.
Figure.Figure 5A. RT-qPCR analysis of KDM6A/B in SCNT embryos injected with siRNA-6A or siRNA-6B. The SCNT embryos were subject to injection of 10 μM or 20 μM  siRNA as indicated. Data are mean expression relative to Gapdh with siRNA-control normalized to 1. Error bars, s.e.m., n = 3. **P < 0.01, ***P < 0.001 according to two-tailed Student"s t-test. n.s., not significant. B. The bar chart shows the KDM6A/B transcript levels in SCNT embryo with 20 μM siRNA-6A/B. All data are mean expression relative to Gapdh with control siRNA injected SCNT embryos normalized to 1.  Error bars, s.e.m., n = 3, **P < 0.01 by Student&quo C. Immunofluorescence staining results showing H3K27me3 of 2-stage SCNT embryos. Embryo was injected with siRNA as indicated. The H3K27me3 levels between control and double injected with siRNA-6A-6B cannot observe any difference, but a marked decrease was observed when injected with either siRNA-6A or siRNA-6B. Scale bar, 20 μm D. Western blot showing the expression of KDM6A and KDM6B in SCNT embryos after injection of different siRNA as indicated. Protein lysates from 1,500 embryos were loaded in each lane. The results of one representative of two independent experiments are presented E. Schematic illustration of siRNA injection into oocytes and SCNT F. Representative images of SCNT embryos at 115 h after injection of different siRNA as indicated. These images are representative examples of the quantification shown in Fig 5G. n ≥ 3. Scale bar, 50 μm. G. Injection of siRNA-6B improved the preimplantation development rate of SCNT embryos. Both cumulus cells, Sertoli cells, and C57 MEF cells were used as donor cells. The bovine intraspecies SCNT embryos derived from bovine ear fibroblast cells. Shown is the percentage of embryos that reached the indicated stages. ♂ and ♀ indicated the male and female, respectively. Error bars, s.d., n ≥ 3. H RT-qPCR analysis for select ZGA genes in ICSI and SCNT embryos. The SCNT embryo was injected with siRNA as indicated. Results were normalized based on the geometric mean of the expression levels of two reference genes (Ywhaz and Gapdh). Error bars, s.e.m., n = 3. *P < 0.05, **P < 0.01, ***P < 0.001 according to two-tailed Student"s t-test. n.s., not signif