EMBO/sd-character-level-ner huggingface dataset:
linc-MYH is located in nuclei of proliferating and differentiated myocytes and interacts with the chromatin remodeler complex INO80 A RNA-seq.Figure 1A RNA-seq. of freshly isolated, proliferating and differentiating satellite cells reveals an increased expression of linc-MYH in proliferating and differentiating MuSCs, but no expression is found in in freshly isolated MuSCs. Expression of linc-MYH is confined to skeletal muscle in mouse (B) tissues. Expression of linc-MYH is confined to skeletal muscle in human (C) tissues. D Expression of linc-MYH is not detected in limb buds at E10.5 (n = 3), E13.5 (n = 3) and E15.5 (n = 3). Expression of linc-MYH in hindlimb muscle of E18.5, newborn and 7 day old animals (n = 3/3/3). Expression of linc-MYH in m. soleus (soleus), m. tibialis anterior (TA) and and m. extensor digitorum longus (EDL) muscle at 3 weeks (n = 3) and 8 weeks of age (n = 5). Biological replicates were used for all PCR experiments, data are mean ± S.E.M. E Murine linc-Myh is localized in nuclear extracts of C2C12 cells. Xist (nuclear) and Gapdh (mainly cytoplasmic) were used as controls (n = 2 nuclear / 3 cytoplasmic biological replicates.) Data are mean ± S.E.M. F-H RNA-FISH identifies linc-Myh in nuclei (DAPI, blue) of C2C12 myoblasts. (G) Adipor (cytoplasmic) and (H) Xist (nuclear) were used as controls. GO enrichment (I) analysis of proteins pulled down by in vitro transcribed RNA from nuclear extracts of C2C12 myoblasts. Linc-MYH RNA and three control RNAs were used in duplicate. (J) Heat map of proteins pulled down in the same experiments as in I, demonstrating that INO80 chromatin remodeler complex exclusively interacts with linc-MYH. Blue color indicates pull-down of the respective protein, white color indicates that the protein was not detected in the respective sample. RNA-IP experiments using an anti-INO80 antibody to isolate the INO80 complex from murine C2C12 cells (K) INO80 in L refer to replicate INO80 precipitation experiments. Precipitated RNAs were detected by RT-PCR. RNA-IP experiments using an anti-INO80 antibody to isolate the INO80 complex from human HSMM cells (L). INO80 II in L refer to replicate INO80 precipitation experiments. Precipitated RNAs were detected by RT-PCR.
Dynamics of intracellular nucleosides under starvation A Electron microscopic analyses to see autophagic bodies in the vacuoles under both growth and nitrogen starvation conditions.Figure 1A Electron microscopy analyses to see autophagic bodies in the vacuoles under both growth and nitrogen starvation conditions. Autophagic bodies typically contain cytosolic ribosomes. pep4∆prb1∆ and pep4∆prb1∆atg2∆ cells were grown in SD to mid‐log phase (OD600 = 1) and transferred to SD(‐N) for 5 h. The cells were examined by transmission electron microscopy as described in . Scale bar, 500 nm. B-D Time‐dependent changes in nucleoside contents under nitrogen starvation (B), rapamycin treatment (C), and carbon starvation (D) conditions. Wild‐type and atg2Δ cells were grown in SD and transferred to SD(‐N), SD medium with 0.2 μM rapamycin, or SD(‐C) at time 0. At the indicated time points, nucleosides were analyzed by LC/MS as described in . Results are presented as normalized intensities on the basis of peak height of each metabolite in wild‐type cells. All data are means of triplicate samples. The error bars represent the standard deviation.
The calcineurin inhibitor FK506 impairs innate immunity to Aspergillus fumigatus.Figure 1A C57BL/6 WT or Rag2−/− mice were immunosuppressed with hydrocortisone +/− FK506 and infected with 105 AF CEA10 conidia intranasally. Mortality was assessed using a 20% weight loss endpoint. Mortality was significantly increased in FK506 immunosuppressed mice reaching 100% by day 10 p.i. (P = 0.003 for HC versus HC+FK506 in Rag2−/− using log-rank test, n = 10 per group). B, C PAS-stained lung sections of Rag2−/− mice were scanned and inflammation quantified by pixel intensity threshold analysis in ImageJ. FK506-treated animals had increased inflammation at day 2 p.i. compared to control. P = 0.0126 (using Student's t-test), n = 3-5 per group. (C) Close-up of an infectious focus showing increased inflammation and fungal growth in the FK506-treated group. Scale bar: 400 μm. D, E Murine macrophages (J774A.1) were pre-treated with hydrocortisone, and the effects of FK506 on nuclear shuttling of the glucocorticoid receptor were assessed by confocal immunofluorescence microscopy. The nuclei are indicated in red, and the GR is indicated in green. Scale bar: 50 μm. FK506 did not cause increased translocation of the glucocorticoid receptor. Data represent mean + SEM calculated from 10 fields of vision. One out of 2 experiments is shown.
Generation of a zebrafish grk2 null mutation.Figure 1A Phylogenic tree showing the relationship of the various vertebrate Grk genes and the Drosophila gprk genes B Schematic representation of the exon 1 nucleotide sequence of zebrafish grk2 gene targeted by the ZFN. C Schematic representation of the zebrafish Grk2 protein domains and the truncated peptide predicted to be encoded by the grk2i283 mutant allele. D Zygotic grk2i283 mutant at 4dpf compared to wild type (N=80). Arrows indicate the position of swim bladder. Scale bar, 1mm
Lag time to division depends on the frequency of pulsed glucose for a subpopulation.Figure 2After 2 hour starvation, E. coli cultures were pulse fed 10 µM glucose at varying frequencies using the spin flask and plate reader systems, and optical density (OD) was measured over time (inset example figures). Grey dots are OD measurements and the black lines are an empirical fit (see Methods). For separate experiments (n = 18), the lag time is plotted against the frequency, represented as the time-integrated (TI) feedrate f (mmol glucose/g dry cell weight/hour). An empirical fit (grey solid line, see Methods) was used to separate the lag (non-dividing) and dividing phases Normalized absolute cell counts versus time show linear increases after lag time for exemplary feedrates. Data are mean ± standard error of technical replicates (n = 2-3). Lag predicted from the empirical fit is indicated by vertical dotted lines The average size of cells decreased after lag. Micrographs and cell length distributions (n > 400 per distribution) are shown for specific time points, with f = 0.6 mmol/g/h Immobilized cells in the microfluidic experiment divided after a lag time that decreased with increasing glucose pulse frequency. The labeled times indicate the period, time between pulses, for a given experiment Time course of the distribution of cellular DNA content. Sampled cells were stained with SYBR Green I and measured with flow cytometry over the course of a pulsing experiment (f = 0.6 mmol/g/h) The DNA content distribution over time is shown on the left side, and three specific time points are shown on the right. Within the first time point (t = 0), the highest distribution is taken to be high DNA content (2N), and the distribution at half of the 2N average was taken (1N) as medium DNA. DNA distributions were separated into medium (1N) and high DNA (2N). Distribution-specific estimated counts (see Methods) over time (f = 0.6 mmol/g/h) suggested that net division from high to medium DNA cells can explain the increase in cell counts and OD increase
a, HE-stained sections of the cerebral cortex, the gracile nucleus and cerebellum from control (Atg5flox/+; nestin-Cre) and Atg5flox/flox; nestin-Cre (Atg5flox/flox) mice at three months of age.figf2a, HE-stained sections of the cerebral cortex, the gracile nucleus and cerebellum from control (Atg5flox/+; nestin-Cre) and Atg5flox/flox; nestin-Cre (Atg5flox/flox) mice at three months of age. Purkinje cells are indicated with arrowheads. Arrows indicate eosinophilic spheroids, which represent axon swelling. Scale bar, 10 µm. b, Immunohistochemistry using an anti-calbindin antibody on cerebellum sections from control (Atg5flox/+; nestin-Cre) and Atg5flox/flox; nestin-Cre mice at six weeks of age. Scale bar, 25 µm. c, Loss of Purkinje cells in Atg5flox/flox; nestin-Cre mice. Purkinje cells were counted in comparable areas for each mouse, and three fields were counted in each area for each mouse. Data are normalized against values from control mice (Atg5flox/+; nestin-Cre). Values represent mean ± s.d. of three mice. Asterisk, P 0.01 (t-test). d, Apoptotic death of granular cells. Cerebellum sections from control (Atg5flox/+; nestin-Cre) and Atg5flox/flox; nestin-Cre mice at six weeks of age were subjected to TUNEL staining. Nuclei were stained with Hoechst 33258. Scale bar, 500 µm.
Figure 2Western blot analysis of ecto-Ronin expression Ronin del/del + TET-ON Ronin-FLAG ESC cultured in the presence of Dox or without Dox for 3 days. The endogenous levels of Ronin are compared to wild-type (WT) E14 ESC. Average growth rate per day (k) of ecto-Ronin (Ronin del/del TET-ON Ronin-FLAG) ESC cultured continuously in the presence of Dox (+ Dox); without Dox for 5 passages (-Dox) or grown without Dox for 3 passages and then switched to Dox-containing medium for another 2 passages (-Dox -> +Dox). Ordinary one-way Anova, error bars represent median with interquartile range, technical replicates (+ Dox, n = 13), (- Dox n = 13), (-Dox -> +Dox, n = 7) of three independent experiments. Tumour growth of Ronin fl/del, ecto-Ronin and Ronin del/del teratomas, n teratomas for each genotype = 3. Brightfield images of teratomas derived from Ronin fl/del and ecto-Ronin ESC. Haematoxylin/eosin staining of paraffin sections of Ronin fl/del and ecto-Ronin teratomas. In vitro differentiation of Ronin fl/del, ecto-Ronin (grown in the presence of Dox) and Ronin del/del ESC.
Figure 3A. Western blot for POT1 and DDR markers upon POT1 removal in clone 35 and overexpression of ectopic WT POT1. B. Telomere length and G-overhang length analysis for clone 35 upon POT1 removal and overexpression of ectopic WT POT1 (constant-field gel electrophoresis). C. Schematic of disease-associated mutations in POT1. CTCL - cutaneous T cell lymphoma, CMM - cutaneous malignant melanoma, CLL - chronic lymphatic leukemia, CP - Coats plus syndrome. D, E Telomere length and G-overhang length analysis for clone 35 upon POT1 removal and overexpression of ectopic POT1 variants carrying disease-associated mutations (constant-field gel electrophoresis).
Figure 3A, Cyclin E overexpression results in increased RMD when the DSB is close to one repeat. The U2OS (EGFP-SSA-3427-3) cell line carrying Tet-on-cyclin E plasmid was used, and RMD frequency was determined with or without induction of cyclin E expression by doxycycline (DOX, 0.5 µg/mL), which was added 24 hours before transfection of the indicated sgRNAs/Cas9. Cyclin E expression levels were validated by Western blot, using KU70 as the loading control. B, ATR is required for BIR/RMD upon cyclin E overexpression. RMD frequency was determined in U2OS (EGFP-SSA-3427-3) cells infected with lentivirus-expressed ATR shRNA or the control vector (Ctrl) with and without induction of cyclin E expression by DOX (0.5 µg/ml) after R29 sgRNA/Cas9 cleavage. ATR knockdown was shown by Western blot with KU70 as the loading control. C, A diagram showing the BIR/RMD model when a single-ended DSB is generated after replication fork collapse. D, RMD is stimulated when Cas9-D10A is used to create a nick near the repeat. The repair frequencies of the U2OS (MMEJ) reporter cell line (left) and U2OS (EGFP-SSA-3427-17) cell line (right) were determined after expression of the indicated sgRNAs/Cas9 and sgRNAs/Cas9-D10A. The distance between the sgRNA cleavage sites and the left and right repeat sequences is indicated (right bottom). E, RMD induced by Cas9-D10A is dependent on POLD3. U2OS (EGFP-SSA-3427-17) cells were infected with lentiviruses expressing shRNA for POLD3 or vector control and RMD was determined after cleavage by the indicated sgRNAs/Cas9-D10A. The distance between sgRNA/Cas9-D10A cleavage sites and the left and right repeat sequences is indicated at the bottom. The knockdown level of POLD3 was shown by Western blot, and KU70 was used as the loading control. F, p27 suppresses BIR/RMD and SSA/RMD. U2OS (EGFP-SSA-3427-3) cells carrying the Tet-on-cyclin E allele were infected with and without p27 lentiviral viruses. RMD was determined before and after induction of cyclin E overexpression by DOX (0.5 µg/mL) added 24 hours before transfection with the indicated sgRNAs/Cas9. Cyclin E expression levels and cell cycle profiles are shown in Appendix Fig S6.
Effects of RAR agonists on the growth of Luminal and Basal breast cancer cell lines.Figure 9Effects of RAR agonists on the growth of Luminal and Basal breast cancer cell linesThe indicated Luminal and Basal cell lines were challenged with increasing concentrations of ATRA, the RARα agonist, AM580, the RARβ agonist, UVI2003, and the RARγ agonist, BMS961, for 6 days. The complement of RAR-variant transcripts expressed in each cell line is shown in the left bar graphs (mean ± SD of two replicate measurements). The growth curves (sulforhodamine assay) of the cell lines are illustrated by the right linear plots. The results are expressed in % values relative to the corresponding control dishes treated with vehicle alone (right graphs). Each result is the mean ± SD of five replicate wells. ATRA sc = ATRA score.
A simplified model of PfPI4K-PfCDPK7 signaling in P.Figure 84'-phosphorylated phosphoinositides (4'-PIPs) generated by PfPI4K interact with the PH domain of PfCDPK7 (Kumar et al., 2014) and regulate the localization and thereby the function of PfCDPK7 (Fig. 5A), which in turn promotes the synthesis of phosphatidylcholine (PC) (Fig. 2A,D) by regulating enzymes (indicated in red) like ethanolamine kinase (PfEK) and phosphoethanolaminemethyltransferase (PfPMT) (Fig. 3,4) that are involved in PL metabolism (Fig. 2C). In addition, phosphoproteomics suggest that PfCDPK7 may contribute to vesicular trafficking via effectors (indicated in green) like PfRab11b but this pathway needs further investigation (Fig. 1A,C). Collectively, these events regulate intraerythrocytic development of the parasite.
Figure 7Western blot analyses of Phospho- or total JNK and c-Jun in human mo-DCs pre-treated with DMSO (1: 1000) or SP600125 (JNKi, 25 µM) for 1h and stimulated with R848 (10 µg/mL) for 15 min. B-C Human mo-DCs pre-treated with JNKi (SP600125, 25µM) (B) or AP-1 Inhibitor (T-5224, 20 µM) (C) for 1h were stimulated with R848 (10 µg/ml). CCL2 and IL-23 were analyzed by ELISA. (n = 5-11; 2-3 independent experiments). Data information: Data are shown as mean ± SEM. P‐values were calculated by paired, two-tailed t-test (B, C, Statistical significance: ns > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001. See Appendix Table S3 for exact P‐values. Representative flow cytometry plots (left) and histograms (right) of human mo-DCs stimulated with LL-37-RNA-40 for 24h are shown. Inhibitors (JNKi; 25 µM or AP-1 Inhibitor; 20 µM) were given 1h before stimulation. Plots shown are pre-gated on live, single, CD1a+ cells. E-F Human moDCs described in (D) were analyzed for mean fluorescence intensity of CD80 or CD86 by flow cytometry (E) and CCL2 or IL-23 protein by ELISA (F). (n = 7; 2 independent experiments). Data information: Data are shown as mean ± SEM. P‐values were calculated by paired, two-tailed t-test E and F). Statistical significance: ns > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001. See Appendix Table S3 for exact P‐values.
Figure 2(A) Heatmap showing the differential gene expression patterns between MDA-MB-231 PKM2 KO and parental cells (n=3). (B) Gene set enrichment analysis of the top enriched gene signatures associated with PKM2 knockout in (A). Hallmark 50 gene sets and KEGG 186 gene sets were used for this analysis. (C) Decreased cholesterol homeostasis signature in MDA-MB-231 PKM2 KO cells. Cholesterol homeostasis signature was generated by GSEA (v4.1.0) with adjusted p-value and normalized enrichment score (NES). (D) Heatmap showing the differentially expressed genes related to cholesterol homeostasis in PKM2 KO vs. parental MDA-MB-231 cells (n=3). (E) Volcano plot showing proteomic changes in PKM2 KO vs. parental MDA-MB-231 cells. Blue triangle symbol indicated PKM2 level; TMEM33, PKM1 and RNF5 were labeled in red dots. Horizonal dashed line indicated p-value 0.05 and vertical dashed lines indicated 2-fold cutoff. (n=3). (F) Correlation of PKM2 deficiency induced protein changes between MCF7 and MDA-MB-231 cells. Green highlights down-regulated lipid-related proteins. PKM1, TMEM33, OCIAD2 and TIMM8A were up-regulated and highlighted in red dots. (G) Western blot analysis of TMEM33, ABCA1, SREBP1, HMGCR, SCAP, FASN, LDLR, FDFT1, I DI1, RNF5, INSIG 1 and 2, and Calnexin in PKM2 KO MCF7 and MDA-MB-231 cells, and their respective parental cells. Arrow indicates nuclear SREBP1. P: precursor, N: nuclear.
Figure 4A Confocal immunofluorescence microscopy images of P1.HTR cells transfected with IDO1.WT or IDO1.Y149F and co-stained with antibodies recognizing IDO1 (green) and EEA1 (red). Nuclei were stained with DAPI (blue). The merge imagine is shown for all stainings or without DAPI (no DAPI). Scale bar, 10 µm. B Pearson's coefficient of IDO1 colocalization with EEA1 in P1.HTR cells transfected as in A, with each point representing colocalization within an image stack. Data represent mean ± SD (n > 24 cells from three independent experiments). Statistical analysis was performed using two-tailed unpaired Student's t-test. ***P < 0.001. C Distribution of IDO1.WT and the IDO1.Y149F mutants in homogenates of P1.HTR cells along the isopycnic sucrose gradient as in Fig 3D. Fractions were immunoblotted using antibodies specific for IDO1. D The distribution of IDO1.WT and IDO1.Y149F proteins along the isopycnic sucrose gradient is shown in term of densitometric analysis in each fraction (mean ± SD of three experiments).
Figure 6D. discoideum producing the LCV/PtdIns(4)P probe P4C-mCherry was infected (MOI 5, 48 h) with L. pneumophila wild-type strains (JR32, Corby), or phospholipase triple mutants (∆plcABC, ∆plaACD) harboring the Ptac-mCerulean-PflaA-gfp reporter construct, and fixed and treated with an anti‐ubiquitin antibody, LCV escape and cytosolic localization of the bacteria was assessed by confocal microscopy through quantification of amoeba containing intact, P4C-mCherry-positive LCVs or ubiquitin-decorated, cytosolic bacteria. (A) Representative images of amoebae harboring L. pneumophila confined within LCVs or released from the LCV to the cytosol are shown (scale bars: 5 μm). D. discoideum producing the LCV/PtdIns(4)P probe P4C-mCherry was infected (MOI 5, 48 h) with L. pneumophila wild-type strains (JR32, Corby), or phospholipase triple mutants (∆plcABC, ∆plaACD) harboring the Ptac-mCerulean-PflaA-gfp reporter construct, and (A-C) fixed and treated with an anti‐ubiquitin antibody, or (D) lysed, fixed and analyzed by flow cytometry. LCV escape and cytosolic localization of the bacteria was assessed by confocal microscopy through quantification of amoeba containing intact, P4C-mCherry-positive LCVs or ubiquitin-decorated, cytosolic bacteria. (B) The percentage of D. discoideum with intact LCVs was assessed for the following number of cells: JR32, n = 132; JR32 ∆plcABC, n = 606; Corby, n = 265; Corby ∆plaACD, n = 357. (C) The percentage of D. discoideum harboring ubiquitinated L. pneumophila was assessed for the following number of cells: JR32, n = 100; JR32 ∆plcABC, n = 602; Corby, n = 281; Corby ∆plaACD, n = 389. (D) Quantification of GFP-positive bacteria of the L. pneumophila strains by flow cytometry and FlowJo software. Data shown are means and SEM from (B, C) three biological replicates and (D) a technical triplicate (two-tailed Student's t test; *P < 0.05, and **P < 0.01).
Figure 1(A) WT, Irgm1-/-, Irgm2-/- and Irgm3-/- mice (n = 4 mice/genotype) were injected i.p. with LPS (8 mg/kg). Serum was collected 4 hours post injection (hpi) and concentration of various cytokines determined via a preconfigured Luminex multiplex panel. Relative concentration (fold change relative mean of WT) is shown for the indicated cytokines (absolute cytokine concentrations of same experiment are shown in Fig EV1). (B) WT (n = 9), Irgm1-/- (n = 7), Irgm2-/- (n = 9) and Casp1-/-Casp11-/- (n = 7) mice were injected i.p. with LPS (8 mg/kg). Serum was collected 4 hpi and concentration of IL-1β, IL-18, and TNFα was measured via ELISA.
a) Above: percentages of cells that accumulate MBds (>1) in a range of different cell types, as indicated.figf4(a) Above: percentages of cells that accumulate MBds (>1) in a range of different cell types, as indicated. Below: doubling times of representative cell lines aligned with MBd-accumulation data. Data are presented as mean±s.d.; cell lines are examined in triplicate (MCF-10A, DLD-1, MCF-10AT, MCF-7, H1 and H9) or quadruplicate (e.v. B6 MEFs, HeLa, SAOS-2 and MCF-10CA1a), except hRPE-1 (n=6), U2OS (n=7) and NCC-IT (n=8). Horizontal line, cell lines with different MBd accumulation potentials (14-fold) but similar doubling times. (b) Cells pulse-chased with EdU show a decrease in EdU intensity (x axis) over time (y axis), reflecting dilution of dye after cell divisions. (c,d) After a 96 h chase period, EdU levels were compared between cells with MBd numbers of >1, 1 and 0 (y axis) in HeLa (c) and SAOS-2 cells (d). In both cases, no significant differences were noted (c, P=0.2101; d, P=0.5609, one-way analysis of variance, with at least 800 cells analysed for each experiment, n=3), indicating similar cycling rates among different subpopulations of cells. (b-d) Each graph is a representative experiment. Cells analysed are shown by green points, the medians are depicted by vertical red lines, and horizontal red lines with ticks illustrate the interquartile range.
Figure 4A. Most significantly altered transcriptomic pathways in PEO muscle (n=4) and in control subjects muscle after mAD (n=8). Blue color represents down-regulated and red up-regulated pathways. B. Genes, the expression levels of which formed the pathway results in A.