EMBO/sd-character-level-ner huggingface dataset:
Cells accumulate ROS under starvation conditions.Figure 1(A) CHO cells were grown in a control medium (see Materials and methods) or starved for 3 or 13 h, after which they were incubated in 50 μM DHE and visualized as detailed in Materials and methods. (B) CHO cells were grown as in (A), treated with 5 nM MitoTracker Red for 30 min at 37°C, washed, treated with 30 μM DCFDA and visualized as detailed in Materials and methods. (C) HeLa cells grown in 96‐well plates were deprived of serum (αMEM), completely starved (EBSS) or maintained in a control medium (α-MEM, 10% FCS) for 2 h, after which they were treated with DCFDA as in (B) and subsequently analyzed in a fluorimeter, as explained in Materials and methods. (D) Data collected from the fluorometric measurements were analyzed as detailed in Materials and methods.
Antisense inhibition of miR-210 specifically in vascular endothelium improves existing manifestations of PH in vivo.Figure 6A Following the schema of Fig5A in the presence of hypoxia but the absence of SU5416, intravenous delivery of anti-miR-210 ameliorated the elevation of RVSP compared with anti-miR-Control (N = 6/group, ***P = 0.001). B Schema of the strategy to determine whether delivery of anti-miR-210 specifically to the vascular endothelium via packaging with 7C1 nanoparticles improves hypoxia-induced PH ("7C1 reversal protocol"). C, D Consistent with specific delivery to the vascular endothelium after intravenous administration (Dahlman et al, 2014), 7C1-mediated delivery of anti-miR-210 (N = 6/group) decreased miR-210 expression in PECAM+ pulmonary vascular endothelial cells, **P = 0.0013, (D) but not PECAM-negative pulmonary cells, NS P = 0.3775 (C). E Correspondingly, endothelial-specific anti-miR-210 (N = 6/group) increased ISCU1/2 expression in PECAM+ cells even in the presence of chronic hypoxia, ***P = 0.0003. F-I Delivery of anti-miR-210 (N = 5/group) decreased endothelin-1 in hypoxic mouse lung tissue, *P = 0.0198 (F). As a result, in contrast to anti-miR-Control (N = 8), endothelial-specific anti-miR-210 ameliorated the elevation of RVSP (N = 6), **P = 0.0024 (G), and pulmonary vascular remodeling (N = 6/group), **P = 0.0021, *P = 0.0117 (H-I).
In Vitro and In Vivo Lytic Replication of the WT and Recombinant γHV68 Viruses.Figure 6(A) Single-step (right) and multiple-step (left) growth curves of the WT and recombinant γHV68 viruses in BHK21 cells. (B) Acute replication of the WT and mutant vBcl-2 γHV68 viruses in the lungs of BALB/c mice at 5 dpi (up) and 7 dpi (down) after intranasal infection determined by viral titers in the lungs of the infected mice (left) or by real-time PCR of the viral genomic DNA (right). Mean±SEM of five mice per group/experiment. Data of 7 dpi is pooled from two separate experiments. The vBcl-2 Δα1 and ΔBH2 mutants did not yield significantly different results when compared to the WT in infectious virus titers [for Δα1, P = 0.82 (day 5); P = 0.08 (day 7); for ΔBH2, P = 0.49 (day 5); P = 0.54 (day 7); unpaired t-tests] and in viral genome loads in the lungs [for Δα1, P = 0.25 (day 5); P = 0.28 (day 7); for ΔBH2, P = 0.21 (day 5); P = 0.37 (day 7); unpaired t-tests]. n.s., not significant.
Multimodal cortical interneuron migration is modulated by neurotransmitter signaling A.Figure 6A. Heatmap depicting normalized values for 48 migration parameters for all cells tracked. Parameters are grouped in 4 categories - speed, shape, pausing and direction - and 9 subgroups, which are color-coded and described. Cell tracks are clustered into 9 different clusters based on their expression values for the different parameters. The clusters are color-coded and numbered. Clusters are further grouped into 3 major categories - directed, exploratory and confined. The z-score scale describes the normalized expression values. In total 3979 moving cell tracks with the following number of tracks per group: Control - 696, Bicuculline-Methiodide - 1189, Saclofen - 527, TPMPA - 138, AP-5 - 429, CNQX - 500, Strychnine - 98, Serotonin - 180, and SR57227 - 222, were analyzed. B. Representative visualization of cell tracks in the various clusters identified in Figure 6A. For visualization purposes, only cells from the control group are used. C. Mean square displacement (MSD) in μm2 over the delay in minutes (min) for all cell tracks in a cluster. The curves are color-coded to denote the corresponding cluster. For visualization purposes, the MSD is shown for a time delay corresponding to 24 hours of imaging. D. Velocity auto-correlation (VAC) over the delay in minutes (min) for all cell tracks in a cluster. The curves are color-coded to denote the corresponding cluster. For visualization purposes, the VAC is shown for a time delay corresponding to 24 hours of imaging. The dashed box depicts the VAC for each cluster for 4 hours of imaging to visualize the initial decay. E. Stacked bar plot depicting the relative distribution of cell tracks among the three cluster groups - directed, exploratory or confined - for control and treatment groups. Bars are color-coded to denote the corresponding cluster group. F. Bar plot depicting the fold-change in composition of cluster groups - directed, exploratory or confined - of treatment groups relative to the control distribution. Bars are color-coded to denote the corresponding cluster group. The dotted line represents the control baseline for visualization of the fold changes between the groups.
Inhibition of canonical Wnt-signaling perturbs hindbrain, but not spinal cord cell fates.Figure 1One-cell stage embryos were injected animally with mRNA encoding dkk1 (35/45 pg) protein. Total RNA was isolated from ten control embryos at neurula stage 17 (lane 2) and ten embryos from each dkk1 injected group (lanes 3-4). Various neural AP markers were examined by sqRT-PCR: cement gland and forebrain (xag1, xanf1, otx2), hindbrain (krox20, hoxb3), hindbrain/spinal cord border (hoxb4), and spinal cord (hoxb9, cdx1-2-4, hoxd10). Ef1α serves as a control for quantitating RNA levels. -RT-PCR was performed on total RNA isolated from control embryos (lane 1). One-cell stage embryos were injected animally with the zygotic-expressing Nxfz8/pCS2 encoding plasmid (100/200 pg). Total RNA was isolated from ten control embryos at neurula stage 17 (lane 2) and ten embryos from each Nxfz8 injected group (lanes 3-4). Various neural AP markers were examined by sqRT-PCR: cement gland and forebrain (xag1, xanf1, otx2), hindbrain (krox20, hoxb3), hindbrain/spinal cord border (hoxb4), and spinal cord (hoxb9, cdx1-2-4, hoxd10). Ef1α serves as a control for quantitating RNA levels. -RT-PCR was performed on total RNA isolated from control embryos (lane 1). Embryos from the same experiment as in (A) underwent in situ hybridization. Expression of the hindbrain marker krox20 (upper two panels) was reduced by Dkk1 (45pg) in 83% of the embryos (n=18). Expression of spinal cord markers (hoxb9, cdx4, hox10) was normal, being unchanged in 89% of the embryos (n=18 for each marker). Control embryos, (CE).
UBQLN1 co-localizes with Mtb that are cleared through autophagy.Fig 4(A) BMDMs treated with IFN-γ and infected for 24h with GFP-Mtb were immunostained for UBQLN1 and either ubiquitin (using the FK2 antibody) or p62. GFP-LC3 BMDMs infected with DsRed-Mtb were immunostained for UBQLN1. (B) Quantification of those Mtb that co-localize with at least one of the cellular markers shown in A. More than 375 total bacteria were counted in two independent experiments, and the number co-localizing with at least one marker is indicated as "total" under each bar. (C) Wt macrophages (Atg16L1flox/flox; Cre-) and autophagy-deficient macrophages (Atg16L1flox/flox Lyz-cre; Cre+) were activated with IFN-γ or not, followed by infection with the live/dead Mtb reporter strain. % viable was quantified after addition of AnTc. **P<0.005, ***P<0.0001, Fisher's exact test (in which the proportions of viable to non-viable Mtb was compared). (D) Atg16L1flox/flox Cre+ and Cre- BMDMs treated with IFN-γ and infected for 24h with GFP-Mtb were immunostained for UBQLN1 and p62. The number of Mtb co-localizating with the cellular markers is shown. (C-D) At least 100 bacteria were analyzed in three independent experiments. (E) Images from IFN-γ treated BMDMs of indicated genotype infected with GFP-Mtb and immunostained with UBQLN1 and p62. Arrows indicates p62+ UBQLN1+ Mtb.
Figure 6(A) Vinculin was knocked down as described (Figure 5D) in MCF10A cells and cells were stained for LATS1 and TRIP6. Merged images show LATS1 (green), TRIP6 (red) and DNA (blue). Scale bar=20µm. (B) Vinculin was knocked down as described (Figure 5D) and TRIP6 immune complexes and lysates were probed by western blotting for LATS1, TRIP6, and vinculin and the relative levels quantified (Mean ± SD; n=3; **P≤0.01, T-test). (C) MCF10A cells were subjected to different treatments as described (Figure 4A) and TRIP6 immune complexes and lysates were probed by western blotting for vinculin and TRIP6. (D) Quantification of TRIP6-vinculin binding from part (C) is shown. (Mean ± SD; n=3; *P≤0.05, **P≤0.01, ***P≤0.001, T-test). (E, F) MCF10A cells were treated separately (or not) with Latrunculin B (E) and by serum starvation (F) and LATS1 immune complexes and lysates were probed by western blotting for vinculin, LATS1, and TRIP6. Quantification of LATS1-TRIP6-vinculin binding is shown. (Mean ± SD; n=3; *P≤0.05, **P≤0.01, ***P≤0.001, T-test). (G) Wild type and T12 mutant of vinculin were co-expressed with LATS2 and FLAG-TRIP6 and anti-FLAG antibody was used to isolate immune complexes. Immune complexes and lysates were probed by western blotting for vinculin, LATS2 and FLAG-TRIP6. Quantification of LATS2-TRIP6-vinculin (WT or T12) binding is shown. (Mean ± SD; n=3; *P≤0.05, **P≤0.01, ***P≤0.001, T-test). (H) Vinculin was knocked down as described (Figure 5D) in MCF10A cells grown at high density on PDMS membranes. The membranes were stretched at 17% elongation for 2 hours and fixed while under tension and stained for TRIP6. Merged images show TRIP6 (red) and DNA (blue). Scale bar=20µm.
Oscillations in isotopic labeling of TCA cycle metabolites throughout the cell cycle from [U-13C]-glutamine show induced oxidative and reductive glutamine metabolism in S phase.Figure 4Oscillations in aspartate (a) concentrations throughout the cell cycle when feeding [U-13C]-glutamine (representing oxidative TCA cycle activity). Oscillations in malate (b) concentrations throughout the cell cycle when feeding [U-13C]-glutamine (representing oxidative TCA cycle activity). (c) Uniform malate m+4 labeling throughout the cell cycle (combined with the increase in malate concentration in S phase representing increased oxidative TCA cycle flux in S phase). (d) Oscillations in pyrimidines m+3 labeling throughout the cell cycle when feeding [U-13C]-glutamine (representing de novo pyrimidine biosynthesis). (e) Oscillations in lactate m+3 labeling throughout the cell cycle when feeding [U-13C]-glutamine (representing malic enzyme activity). (f) Oscillations in malate m+3 throughout the cell cycle when feeding [U-13C]-glucose (representing pyruvate carboxylase activity). (g) Oscillations in citrate m+5 when feeding [U-13C]-glutamine throughout the cell cycle (representing reductive IDH flux). (h) Oscillations in acetyl-CoA m+2 when feeding [U-13C]-glucose, representing oxidative glucose metabolism. Oscillations in acetyl-CoA m+2 when feeding [U-13C]-glutamine, representing reductive glutamine metabolism (i). For measurements of metabolite concentrations (a-b), red marks represent mean and s.d. of n=5. For measurements of fractional isotopic labeling (c-i), red marks represent mean and s.d. of n=3.
Figure 3A, Detection of GFP, Mito-DsRed, mitochondria marker (ATP5B, blue) and nuclei (Hoechst, white) in wildtype C57BL/6J recipient retinas 21 days after transplantation with Mito-DsRed-expressing Nrl::GFP donor photoreceptors. B-D'', Higher magnification of regions in A (indicated by yellow dashed boxes) showing colocalization of ATP5B and Mito-DsRed-labelled mitochondria in the outer segments (B), soma (C), and the axon (D) of acceptor photoreceptors.
ROS inhibit NFAT1 translocation via oxidation of calcineurin A WM3734 cells were pre-incubated for 5 min with extracellular H2O2 (0 - 500 µM).Figure 4WM3734 cells were pre-incubated for 5 min with extracellular H2O2 (0 - 500 µM). Tg-stimulated NFAT1-GFP nuclear translocation was measured as F/F0. The IC50 of H2O2 inhibition of NFAT1 nuclear import was determined to be 13.2 µM (red mark). Tg-induced NFAT1-GFP nuclear import in transient TMX1 silenced WM3734 cells and after pre-incubation with 100 µM N-acetylcysteine (NAC) for 48 h. Endpoint quantification of the data from (C) and upon treatment with NAC, PEG-catalase (50 U/mL), or DTT (1 mM). data are presented as mean ± SEM (n values: control=73, control+NAC= 19, TMX1 kd=57, TMX1 kd+NAC=63, TMX1 kd+catalase=58, TMX1 kd+DTT=22). Cytosolic calcineurin activity (CaNAR2) in WM3734 cells with transient knockdown of TMX1 or TMX3 measured upon Tg stimulation. Quantification of the basal calcineurin activity and the maximal calcineurin activity (plateau-basal). data are presented as mean ± SEM (n values: control=49, TMX1 kd=48, TMX3 kd=63). Normalized Tg-induced cytosolic calcineurin activity in Mel Juso cells after 5 min pre-incubation with extracellular H2O2 (0 - 200 µM). Quantification of the maximum cytosolic calcineurin activity (plateau-basal). Quantification of the basal calcineurin activity and the maximal calcineurin activity (plateau-basal) 8 h after treatment with the antioxidant NAC (100 µM) in WM3734 with transient knockdown of TMX1. data are presented as mean ± SEM (n values: control=15, TMX1 kd=24, TMX1 kd+NAC=19).
Clinical phenotype characterization of the Apopt1 knockout mice A Body weight measured in female and male animals at 3, 6 and 12 months of age.Figure 2 A Body weight measured in female and male animals at 3, 6 and 12 months of age. Data are presented as mean ± SEM (n = 5 per group) Distance run by the tested female and male mice on the treadmill at 3 and 12 months of age. Data are presented as mean ± SEM (n = 6 per group). The asterisks represent the significance levels calculated by two-way ANOVA with Sidak's multiple comparisons test: Females - ****P < 0.0001 (3 months), ***P = 0.0004 (12 months, Males - ****P < 0.0001 (3 months), **P = 0.0019 (12 months) Time in seconds spent by the female and male mice on the Rotarod cylinders before falling at different ages. Data are presented as mean ± SEM (n = 5 per group). The asterisks represent the significance levels calculated by two-way ANOVA with Sidak's multiple comparisons test: Females - **P = 0.0059 (3 months), **P = 0.0083 (6 months), *P = 0.0391 (12 months), Males - **P = 0.0069 (12 months) Number of entries in each arm of the Y maze performed at different ages. Data are presented as mean ± SEM (n = 10 per group). The asterisks represent the significance levels calculated by two-way ANOVA with Sidak's multiple comparisons test: ****P < 0.0001  E Total spontaneous horizontal and vertical movements of 12-month old mice measured in an activity cage. Data are presented as mean ± SEM (n = 6 per group). The asterisks represent the significance levels calculated by two-way ANOVA with Sidak's multiple comparisons test: ****P < 0.0001 (horizontal)
Impaired synaptic vesicle size, number of PSD and exocytosis in cortical neurons.Figure 7(a) example TEM images of synapses in wild-type and Trpml1-/-micebraincortex. (b) the distribution of synaptic vesicle size determined in 10 and 14 images obtained from 3 wild-type and 3 Trpml1-/-mice, respectively. (c) example TEM images at low magnification of wild-type and Trpml1-/-micebraincortical slices with the dark PSD profiles marked by white arrows. (d) average number of PSD/µm2 analyzed in at least 10 images obtained from 3 mice of each line. (e) Glutamate levels and secretion by cultured cerebral cortical neurons. Total glutamate measured in non-stimulated wild-type (black) and Trpml1-/- (green) neurons and neurons stimulated by depolarization with 30 mM KCl (blue and red). (f) Rescue of glutamate levels and secretion by expression of TRPML1 in cultured cerebral cortical neurons. Total glutamate measured in non-stimulated Trpml1-/- neurons (green) and Trpml1-/- neurons transfected with TRPML1 (black) and untransfected (red) and TRPML1-transfected (clue) Trpml1-/- neurons stimulated by depolarization with 30 mM KCl. (g) averaged Co-IP of SNAP25 and VAMP2 in three brain regions of wild-type and Trpml1-/- mice. The images of all blots are given in Extended view Figure E4.
Retrotransposition occurs genome-wide in the fly midgut A The bioinformatic pipeline used to identify somatic TE insertions in short-read sequencing datasets.Figure 2A The bioinformatic pipeline used to identify somatic TE insertions in short-read sequencing datasets. Two types of supporting reads are identified genome-wide: mate support reads, where one of the paired-end reads is mapped to the reference genome, while the other mate (not shown) is associated with a TE, and clipped reads, which span the insertion site and map partially to the reference genome and partially to a TE. Isolated reads were then clustered and assembled to map individual insertion sites. Only insertions with a valid target site duplication (TSD) were retained, sample specific calls were filtered and manual validation of each candidate insertion was performed on IGV. B The frequency of gut specific somatic insertion sites in the Pros>2XGFP and Delta>nlsGFP genetic backgrounds. C The distribution of TE classes active in the two genetic backgrounds studied. TEs were categorized in four main classes: LTR - long terminal repeat retrotransposons, LINE-like - non-LTR retrotransposons, TIR - terminal inverted repeat DNA transposons, and FB - fodlback element D TSD length distribution for somatic insertions of most frequent TE families. Insertions from both genotypes were pooled. E The target site motif found around (+/- 10bp) rover-LTR insertions sites recovered from the clonal gut samples. (E-value was calculated with MEME software (Bailey et al, 2009), where E-value < 0.05 is considered statistically significant)
Biochemical analysis and cryo-EM structures of yeast ABCE1-containing initiation complexes.Figure 1A Volcano plot representing the statistical analysis of the fold enrichment of proteins after affinity purification in HEPES epes buffer of ABCE1-TAP followed by label-free quantification (LFQ) using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Proteins above the curved lines show a statistically significant enrichment according to the t test value. B, C Sucrose density gradient UV-profile after in vitro splitting assays (B) and relative abundance of 80S and subunits as calculated from triplicates and displayed as mean ± SD. (C); SF = splitting factors including Dom34, Hbs1, ABCE1 and eIF6; (+) = 4-fold molar excess of eIF3j; (++) = 20-fold molar excess of eIF3j. D, E Cryo-EM maps low-pass filtered at 6 Å and models of the yeast subclasses representing an ABCE1- and eIF3j-containing 43S PIC (D) and an ABCE1- and eIF5-containing partial 48S IC (E).
PGC-1α regulates FUNDC1 expression (A-C) Western blotting analysis of proteins related to mitochondrial biogenesis in BATs from mice at 30℃ (Normal) or following 72 h at 4℃ (Cold) (A).Figure 2Western blotting analysis of proteins related to mitochondrial biogenesis in BATs from mice at 30℃ (Normal) or following 72 h at 4℃ (Cold) (A). Relative protein expression levels were normalized to Actin (B). mRNA expression levels of the indicated genes were assayed by real-time PCR and normalized to Actb (C). Flag-PGC-1α was introduced into PGC-1α knockdown brown preadipocytes for 24 h and samples were collected for western blotting analyses of the indicated proteins (D). Expression levels of the indicated proteins were quantified (E) (mean ± SEM; n=3). Scramble, scramble shRNA. Samples were collected for real-time PCR analysis. The expression of Fundc1 was normalized (F). (G-H) Scramble and PGC-1α knockdown preadipocytes were induced to differentiate into mature adipocytes and samples were collected for western blotting analyses (G) and quantification of the indicated proteins (H)
Figure 1(A) THP-1 cells were subjected to knockdowns as indicated and treated with 100 ng/mL LPS overnight, followed by 0.25 mM LLOMe for 3 h, and the levels of IL-1β were determined in supernatants. (B) THP-1 cells were subjected to GABARAP knockdown and processed as in A. (C) IL-1β levels in supernatants of THP-1 cells subjected to TRIM knockdowns and treated with LPS and then with LLOMe. (D) Confocal microscopy of THP-1 cells treated sequentially with LPS and LLOMe, and stained for LC3B and TRIM16. Line tracings correspond to arrows. Arrowheads indicate colocalization. Scale bars, 5 μm. (E) IL-1β levels in supernatants of THP-1 cells subjected to TRIM knockdowns and treated with LPS and then with LLOMe. (F) Levels of IL-1β were determine in supernatants from THP-1 cells subjected to knockdown as indicated, treated with 100 ng/mL LPS for overnight and then with 0.25mM of Silica, Alum, or monosodium urate (MSU). (G) IL-1β in supernatants of primary human macrophages (MDM) subjected to knockdowns and sequentially treated with LPS and LLOMe. Data, means ± SEM; n≥5 (except for B where a representative data set from 3 repeats). *P < 0.05 (t test or ANOVA).
Figure 1m6A-related proteins identified by HSATIIII-ChIRP-MS (cf. Table EV3 in Ninomiya et al., 2020). HSATIII lncRNA-dependent localization of m6A-related factors. Control and HSATIII knockdown HeLa cells were exposed to thermal stress (42°C for 2 h and recovery for 1 h at 37°C) and stained by HSATIII-FISH and immunofluorescence using an anti-WTAP antibody (B) or anti-YTHDC1 antibody (C). The nuclei were stained with DAPI. Scale bar: 10 µm. Enrichment of m6A RNAs in nSBs. Thermal stress-exposed HeLa cells were stained by HSATIII-FISH and immunofluorescence using an anti-m6A antibody. The nuclei were stained with DAPI. Scale bar: 10 µm. 3-DZA inhibits localization of m6A RNAs HeLa cells were exposed to thermal stress in the presence of 3-DZA (100 µM) and stained as described above. Scale bar: 10 µm. 3-DZA inhibits localization of YTHDC1 (F) and WTAP (G) in nSBs. HeLa cells were exposed to thermal stress in the presence of 3-DZA (100 µM) and stained as described above. Scale bar: 10 µm. Box plot of relative intensities of m6A (H) , YTHDC1 (I) to HSATIII within nSBs in individual nuclei. nSB areas were defined by binarized images of HSATIII-FISH. The mean is indicated with X. The first and third quartiles are the ends of the box, the median is indicated with a vertical line in the box, and the minimum and maximum except for the outliers are the ends of the whiskers. The outliers are indicated with open circles. P-value (Mann-Whitney U-test) is shown above (n=30). Box plot of relative intensities WTAP (J) to HSATIII within nSBs in individual nuclei. nSB areas were defined by binarized images of HSATIII-FISH. The mean is indicated with X. The first and third quartiles are the ends of the box, the median is indicated with a vertical line in the box, and the minimum and maximum except for the outliers are the ends of the whiskers. The outliers are indicated with open circles. P-value (Mann-Whitney U-test) is shown above (n=30). YTHDC1 accumulation within nSBs was arrested by METTL3 knockdown. HeLa cells were stained as described in Figure 1C. Scale bar: 10 µm. Box plot of relative intensities of YTHDC1 to HSATIII within nSBs in individual nuclei (n=30). The mean is indicated with X. The first and third quartiles are the ends of the box, the median is indicated with a vertical line in the box, and the minimum and maximum except for the outliers are the ends of the whiskers. The outliers are indicated with open circles. P-values (Kruskal-Wallis test, followed by Dunn's multiple comparison test) are shown above.