EMBO/sd-character-level-ner huggingface dataset:
Figure 5A. SR-SIM images depicting invading parasites with and without the presence of the F-actin ring at the tight junction and invading adf cKD Cb-EmeraldFP parasites. SAG1 staining (in red) was performed prior to permeabilisation to specifically label extracellular part of the parasite. Note the accumulation of actin (blue arrow) around the nucleus and the posterior pole of the parasite, irrespective of F-actin ring formation at the TJ (orange arrowhead). 3D rendering is shown for each presented case (inlet). Data information: Scale bar represents 5 µm for SR-SIM images White arrow points to direction of invasion. B. SR image of an invading parasite before nuclear entry. F-actin forms a continuous meshwork around the nucleus that appears connected to the posterior pole. C. F-actin localisation in relation to the TJ during invasion. F-actin (white arrow) is in close vicinity to the Ron2 ring (light blue arrow) during invasion. Data information: cale bar represents 5 µm for SR-SIM images hite arrow points to direction of invasion. D. Quantification of perinuclear actin in indicated parasites. Parasites were counted by assessing Cb EmeraldFP signal as an actin marker around or in the vicinity of the nucleus. Still images to the right represent what was considered as F-actin - nucleus association. The orange arrowhead points to F-actin surrounding the nucleus in the posterior end of the parasite, the red and yellow arrowhead shows F-actin in the vicinity of the nucleus, hinting at a close interaction between the two. Three biological replicates were analysed with a minimum of 25 parasites per replicate. Data information: Error bars represent standard deviation. Scale bar represents 5 µm for SR-SIM images White arrow points to direction of invasion. E-G. SR-SIM images showing three stages of invading wt parasites. SiR-tubulin staining for microtubules (in red) was performed prior to fixation to specifically label microtubules in the parasite. During invasion, the microtubules (MT) are deformed at the TJ area (white arrowhead). Accumulation of actin (green arrow) at the posterior forms a F-actin meshwork. The nucleus is deformed when it enters the TJ area (orange arrowhead). H-J. In the case of the myoA KO strain, the MTs also deform when passing through the TJ (white arrowhead). However, F-actin is not concentrated at the posterior pole or the TJ, but evenly distributed within the cytosol of the parasite, with some peripheral location. K-M. In case of adfKD, MTs are deformed at the TJ area (white arrowhead). F-actin is accumulated at both ends of the parasite (green arrowheads), with no accumulation at the TJ. Data information: Scale bar represents 5 µm for SR-SIM images White arrow points to direction of invasion.
Figure 3(A) Real-Time RT-PCR analysis for IL-1β mRNA levels in PC3 cells with control or CTH knockdown. Recombinant IL-1β (10ng/ml) was added for 24h in serum-free medium. Data information: All data shown represent the means ± SD (n=3 biological replicates). ANOVA followed by Tukey's post-hoc test was used for the statistical analysis. (*P<0.05; **P<0.01; ***P<0.001). (B) ELISA assay for IL-1β protein level in the culture medium of PC3 cells with control or CTH knockdown. Data information All data shown represent the means ± SD (n=3 biological replicates). Student's t-test (B) was used for the statistical analysis. (*P<0.05; **P<0.01; ***P<0.001). (C) PC3 cells transfected with control or CTH knockdown were incubated for invasion (24h) assay with recombinant IL-1β (10ng/ml) added to the medium of the lower chamber. Data information: All data shown represent the means ± SD (n=3 biological replicates). ANOVA followed by Tukey's post-hoc test was used for the statistical analysis. (*P<0.05; **P<0.01; ***P<0.001). (D) PC3 cells transfected with control or CTH siRNA were exposed to IL-1β (20ng/ml) for 1hr. Subcellular localization of p65 was detected by immunocytochemistry. Nuclei were counterstained with DAPI. The representative images are shown. Scale bars: 25μm. (E) The percent of nuclear translocation for p65 was scored by counting the number of nuclear positive stained cells of p65 to the total number of cells in random microscopic fields. Data information: All data shown represent the means ± SD (n=3 biological replicates). ANOVA followed by Tukey's post-hoc test was used for the statistical analysis. (*P<0.05; **P<0.01; ***P<0.001). Real-Time RT-PCR analysis for VEGF (F) mRNA level in PC3 cells with control or CTH knockdown. Recombinant IL-1β (10ng/ml) was added for 24h in serum-free medium. Data information: All data shown represent the means ± SD (n=3 biological replicates). ANOVA followed by Tukey's post-hoc test was used for the statistical analysis. (*P<0.05; **P<0.01; ***P<0.001). Real-Time RT-PCR analysis for MMP-13 (G) mRNA level in PC3 cells with control or CTH knockdown. Recombinant IL-1β (10ng/ml) was added for 24h in serum-free medium. Data information: All data shown represent the means ± SD (n=3 biological replicates). ANOVA followed by Tukey's post-hoc test was used for the statistical analysis. (*P<0.05; **P<0.01; ***P<0.001).
PLCβ1 and PTPRN2 act upstream of Cofilin-mediated actin dynamics.Figure 6(A) LM2 cells transfected with siRNA targeting PTPRN2, PLCβ1, or control siRNA were stained with phalloidin (red) and DAPI (blue) and analyzed using fluorescence microscopy. Right, mean fluorescence intensity quantification of whole cell phalloidin signal. N = 40 cells/group. Scale bar, 10 μm. (B) Mean fluorescence intensity quantification of whole cell phalloidin signal in MDA-MB-231 cells overexpressing PTPRN2, PLCβ1, or control vector. N = 40 cells/group. (C,D) MDA-MB-231 cells were transfected with siRNAs targeting CFL1 or a control siRNA in the setting of control or PTPRN2 overexpression and subjected to the migration (C) or invasion (D) assays. N = 5 inserts/group. (E) MDA-MB-231 cells were transfected with siRNAs targeting CFL1 or a control siRNA in the setting of control or PLCβ1 overexpression were subjected to the migration assay. N = 5 inserts/group. (F) Bioluminescence imaging quantification of 40,000 MDA-MB-231 cells overexpressing PTPRN2 or control vector and transfected with control siRNA or siRNA targeting CFL1. Right, H&amp;amp;E staining of representative lung sections. N = 5 mice/group. (G,H) MDA-MB-231 cells overexpressing PTPRN2 (G) or PLCβ1 (H) were transfected with siRNA targeting the 3'UTR of CFL1 to deplete endogenous CFL1. Cells were further transfected with plasmids encoding either GFP-CFL1-WT or GFP-CFL1-Lck and subjected to the migration assay. N = 5 inserts/group.
In vivo vascularization of CI, FI, PI and PI+MVF.Figure 6A Schematic illustration of the experimental setting: dorsal skinfold chambers are implanted on day -2 followed by transplantation of FI, CI, PI and PI+MVF on day 0. Intravital fluorescence microscopy is performed on days 0, 3, 6, 10 and 14 after islet transplantation. On day 14, the tissue is harvested for immunhistochemical stainings. B Observation window of a dorsal skinfold chamber with 8 neutral red-stained transplanted FI on day 0. Scale bar: 5 mm. C Take rate (% of transplanted islets) on day 14 after islet transplantation into the dorsal skinfold chamber (n = 8 each). Mean ± SD. One-way ANOVA and Tukey's multiple comparisons post hoc test were used for statistical analysis. +P < 0.05 vs. PI. D Representative intravital fluorescent microscopic images of FI, CI, PI and PI+MVF within the dorsal skinfold chamber on day 0 and 14. FITC-labeled dextran is used for the visualization of blood-perfused microvessels. Grafts are marked by broken lines. Scale bar: 150 μm. E, F Quantitative analysis of the functional capillary density (cm/cm-2) (E) and revascularized area (mm2) (F) of transplanted FI, CI, PI and PI+MVF on day 0, 3, 6, 10 and 14 (n = 8 each). Mean ± SD. One-way ANOVA and Tukey's multiple comparisons post hoc test were used for statistical analysis. *P < 0.05 vs. FI; #P < 0.05 vs. CI; +P < 0.05 vs. PI. G Representative intravital fluorescent microscopic images of FI, CI, PI and PI+MVF within the dorsal skinfold chamber on day 14. Rhodamine 6G is used to visualize blood perfused vital endocrine cells (bright signals). Scale bar: 150 µm. H Quantitative analysis of the rhodamine 6G-positive area (% of islet size) within transplanted FI, CI, PI and PI+MVF on day 0, 3, 6, 10 and 14. Mean ± SD. One-way ANOVA and Tukey's multiple comparisons post hoc test were used for statistical analysis. *P < 0.05 vs. FI; #P < 0.05 vs. CI; +P < 0.05 vs. PI.
Figure 3(A) Ribosome profiles from PABPDHFR cells grown in the presence or absence of TMP for 12 hours. Lysates were subsequently resolved by ultracentrifugation on 5-50% sucrose gradients and fraction were collected during which time UV absorbance at 254nm (Abs 254 nm) was monitored for tracing. 40S and 60S subunits, monosomes (80S) and polysomes positions are indicated above tracings. PABP-expressing and -depleted cells are denoted by a black and blue trace lines, respectively. (B) SUnSET assay of PABPDHFR cells grown in the presence or absence of TMP for 12 hours to maintain or deplete PABP. Cells were subsequently pulsed with either puromycin or puromycin and cycloheximide (control), lysed and equal protein amounts were resolved by SDS-PAGE. Western blot analysis was performed using a monoclonal antibody against puromycin or actin (loading control). (C) Ribosome profiles from PABPDHFR cells grown in the absence of TMP for 12 hours to deplete PABP and resolved on sucrose gradients containing low KCl [(150 nM) traced in dark blue] or high KCl [(500 mM) traced in turquoise]. (D) Scatter plot comparing log2 fold-changes [(-) PABP vs (+) PABP] in polysome-associated mRNA (y-axis) to corresponding changes in total cytoplasmic mRNA. Transcripts identified as regulated via altered translation efficiency (orange and red) or abundance (light and dark green) according to anota2seq are visualized together with non-regulated transcripts (grey). (E and F) Top ten Wikipathway (WP) terms (Slenter et al., 2018) significantly enriched among proteins coded for by mRNAs that were decreased (E) or increased (F) in abundance in PABP-depleted cells relative to cells expressing PABP. The number above each column represents the number of genes associated with its corresponding term.
Figure 2(A) Quantification of chromosomal cargo gradients (top) and average Rango-3 E in oocytes injected with increasing concentrations of Ran mutant mRNA concentrations. Data from oocytes imaged side-by-side within one experiment. Means ± SDs, one-side ANOVA, Tukey's test. Numbers of analyzed oocytes are indicated in the brackets in the top panel and are identical for both panels. (B) Representative images of importin β cargo gradients detected by Rango-3 FLIM/FRET imaging. Scale bars, 20µm. (C) Linescans of the estimated Rango-3 FRET efficiency (E) in 10 µm-wide areas (D) Quantification of the Rango-3 E gradients (top panels) and average cellular Rango-3 E (bottom) oocytes that were treated side-by-side by the injection of mRNA for Rango-3, alongside with buffer (controls) or 2000ng/µl mRNA for RanT24N or RanT24N, T42A. Means ± SDs, one-side ANOVA, Tukey's test, oocyte numbers indicated in brackets.
Fig.Figure 7(A) CFU in homogenates from the distal small intestines of infant rabbits infected with the indicated strains 18, 28 and 38 h following inoculation. Black horizontal line indicates mean CFU (n=7-10 animals/group). One-way ANOVA was used for statistical analysis. *P<0.05 for the indicated comparison. (B) H&E stained sections from the distal small intestine from the rabbits infected with the indicated strains 18 hours after infection. Black arrows point to bacterial colonies closely attached to the epithelium. (C) Fluid accumulation in rabbits infected with the indicated strains 38 hours post inoculation. Error bars represent mean ± standard deviation (n=7 animals/group). One-way ANOVA was used for statistical analysis. *P<0.05 (when compared to the rabbits infected with WT).
Figure 2(A-B) Volcano plots visualizing the differentially expressed genes in ARLmon/Y (A) and AR-/Y (B) testes compared to testes from WT males. The dotted horizontal lines represent a q-value of 0.05, while the dotted vertical lines represent a FC of 1.5. Genes that are downregulated compared to WT are shown using blue dots, while red dots represent the upregulated genes. Hsd17b3 and Star are indicated as yellow and green stars, respectively. (C) Box plots showing testicular expression from the bulk RNA-seq data (biological replicates, n = 5) of SC- and LC-specific genes extracted from the single-cell RNA-seq data Expression levels are normalized to average expression in WT for each gene. (D) RT-qPCR analyses of Rhox5 and Insl3 of testes from 13-week old WT males, ARLmon/Y and AR-/Y. Expression levels are normalized to WT.
Figure 1Example of a simulated hemifusion to fusion pore transition in the presence of wild-type vacuolar SNARE complexes. This example represents a scenario were fusion occurs between two highly curved membrane dimples (1/8 nm-1). Shown are the (A) stalk, (B) inner-leaflet contact, (C) hemifusion diaphragm, and (D) fusion pore. Nyv1 is shown in blue, Vam3 in red, Vti1/Vam7 in green, the linkers in dark grey, and the TMDs in yellow. The enlarged highlighted ends of the TMDs represent the C-termini. Lipid head groups are depicted in orange, the inner-leaflets of the membrane(s) in gray, and outer-leaflets in white.
a) DLD1 cells were incubated in the presence or absence of DFX (30 μM) and PIK-III (5 μM) and the levels of FTH1 and FTL were analysed using western blotting.figf7(a) DLD1 cells were incubated in the presence or absence of DFX (30 μM) and PIK-III (5 μM) and the levels of FTH1 and FTL were analysed using western blotting. . (b) DLD parental or NCOA4−/− cells were incubated in the presence or absence of DFX and the levels of FTH and FTL were determined. (c) Ncoa4−/− mouse embryonic stem cells were analysed for Ncoa4 and Fth1 by western blotting. Note the complete absence of the lower Fth1 band consistent with earlier findings. (d) Representative haemotoxylin and eosin staining of spleens from control or Ncoa4−/− mice is shown and brown pigmented regions apparent in Ncoa4−/− sections are indicated (white arrows). (e) The presence of iron in spleens from control or Ncoa4−/− mice was determined using Perls' Prussian blue staining. Representative micrographs (left panels) or magnifications (right panels) are shown. Scale bars correspond to 100 μm and 20 μm in full and magnified panels respectively. (f) Quantitative image analysis of the data shown in e was performed as described in the Methods. Each data point represents relative iron levels in a single mouse spleen section and horizontal lines are means of each group (n = 6 mice per group; ∗P 0.0006 for unpaired t-test). Uncropped images of blots are shown in Supplementary Fig. 7.
Figure 7(a-c) Slices through defocus cryo-tomograms of the endpiece in pig (a), horse (b), and mouse (c) sperm. Insets show digital zooms (black boxes) and subtomogram averages (white boxes) of microtubule tips. Note the presence of a plug (arrowhead) extending into the lumen. (d-f) Representative examples of the mechanisms by which doublets can transition into singlets. Doublets can split into two complete singlets (panels i), or the B-tubule can terminate abruptly with the A-tubule extending as a singlet (panels ii). White arrowheads in the left panels indicate approximate locations of transverse slices shown in the right panels. Asterisks mark approximate locations of the change in A-tubule luminal density. (g-i) In situ structures of singlet microtubules from pig (g), horse (h), and mouse (i) sperm endpieces. Note how the MIP binds to two tubulin monomers (white arrowheads).
Figure 2A: SSEA-1+ TNG E-tdT ESCs were cultured for three days in GMEMβ/FCS/LIF and sorted into GFP+/tdThigh (Nanoghigh/Esrrbhigh) or GFP-/tdTlow fractions (Nanoglow/Esrrblow). The purity of the sorted populations (>99.5%) is shown (day 0). After replating in GMEMβ/FCS/LIF, SSEA-1+ cells were analysed daily for Esrrb-tdTomato and Nanog:GFP expression. B,C: Immunofluorescent detection of Nanog and Esrrb protein in wild-type E14Tg2a ESCs cultured in N2B27/LIF/2i and released in GMEMβ/FCS/LIF for 1, 2 or 3 days. Quantification of the mean Nanog and Esrrb fluorescence levels measured in nuclei identified by automatic segmentation of single optical plane images obtained by confocal microscopy. Oct4 negative cells, also identified by immunostaining, were excluded from the analysis. (B) The red vertical and horizontal lines indicate the negative thresholds defined by staining ESCs differentiatied for 3 days in the absence of LIF and presence of retinoic acid. The red trendline represents the moving average of the data distribution at day 3. Representative of two independent experiments each including at least 1000 nuclei. (C) Histogram plots of the data presented in B. : Sorted SSEA-1+ and Esrrb-GFPhigh/Nanog-mCherryhigh NER ESCs were replated in GMEMβ/FCS/LIF and SSEA-1+ cells were analysed daily for Esrrb-GFP and Nanog-mCherry expression. Wild-type E14Tg2a set the negative thresholds. E: SSEA-1+ TNG E-tdT ESCs from the sorted populations used above (panel A, day 0) were replated at clonal density in GMEMβ/LIF/FCS or N2B27/LIF/2i and stained for alkaline phosphatase activity after 6 days. Error bars: standard deviation of the number of colonies observed in two independent experiments.
AML-EV increase the relative frequency of LT-HSC, upregulate P53 expression and confer quiescence.Figure 2Schematic diagram of methods: the in vivo AML-xenografts (tail-vein injection of 105 Molm-14 cells or vehicle per mouse) and the intra-femoral (IF) injection of AML-EV into one femur with a contralateral control vehicle-injected femur of the same mouse. EV were isolated by serial high-speed centrifugation (at 2x, 10x, 100 x 103 g) from AML cell-line culture media or AML patient plasma. Bone marrow was flushed from long bones at the indicated time-points and immunophenotypic analysis was performed by flow cytometry. Flow cytometric analysis showing the frequency of LK and KSL cells in lineage-negative cells (left panel) and LT-HSC in KSL (right panel) in: Molm-14-xenograft (red, n=10) vs. control (black, n=10). Data were obtained from at least two independent experiments. Flow cytometric analysis showing the frequency of LK and KSL cells in lineage-negative cells (left panel) and LT-HSC in KSL (right panel) in: IF injection of EV from Molm-14 cells (red, n=8), AML plasma EV (Orange, n=6) and human CD34 EV (Blue, n=4) relative to the vehicle-injected contralateral femurs. Data were obtained from at least two independent experiments. Cell-cycle histograms of KSL (upper panel) and LT-HSC (lower panel) from Molm-14-xenografts or control using Hoechst-33342 staining. Flow cytometric analysis of the Ki67-ve percentage of LT-HSC representing the G0 phase of cell-cycle in: (left panel) Molm-14-xenografts (red, n=7) vs. control (black, n=9); (right panel) IF injection of Molm-14-EV, U-937 EV, HL-60 EV (red, n=5,4,4) vs. human CD34 EV (blue, n=3) vs. controls (black). Flow cytometric analysis of intracellular P53 levels shown in histograms (F) of LT-HSC in: (left panel) Molm-14-xenograft, n=6 red vs. non-engrafted control, n=4 black, or (right panel) IF injection of EV from Molm-14, U-937, HL-60 (red, n=5,5,3) or human CD34 cells (blue, n=6) normalized to vehicle-injected contralateral femurs. Flow cytometric analysis of intracellular P53 levels shown in histograms of LT-HSC in: (left panel) Molm-14-xenograft, n=6 red vs. non-engrafted control, n=4 black, or (right panel) IF injection of EV from Molm-14, U-937, HL-60 (red, n=5,5,3) or human CD34 cells (blue, n=6) normalized to vehicle-injected contralateral femurs.
Figure 3(A) Acetylated cohesinHalo488 was loaded onto DNA in the presence of Scc2-Scc4 and washed in high-salt buffer. DNA-bound cohesin was further incubated with Wapl-Pds5 plus Sororin, and then treated with buffer, Plk1, or Plk1 plus BI4834. CohesinHalo488 particles were observed in the presence of ATP and 100 mM KCl. MSD versus time is shown. D indicates the diffusion coefficient (n = 45, mean ± s.e.m.). (B) Acetylated cohesinHalo488 was treated as in (A), or treated with CDK1 or Aurora B rather than Plk1. Sororin intensity on DNA was measured after immunofluorescence microscopy with anti-Sororin antibody (n > 100, *p < 0.0001, two-tailed Mann-Whitney test). (C) Acetylated cohesinHalo488 was treated as in (A) but treated with Aurora B rather than Plk1. CohesinHalo488 particles were observed in the presence of ATP and 100 mM KCl. MSD versus time is shown. D indicates the diffusion coefficient (n = 45, mean ± s.e.m.).
Figure 8LDL-cholesterol is taken up via LDL receptor mediated endocytosis and the liberated free cholesterol is incorporated into late endosomal limiting membrane via NPC2 and NPC1. ORP2 delivers cholesterol from late endosomes to FAK/integrin recycling endosomes where cholesterol facilitates FAK association with the PI(4,5) P2 containing membrane. Activated FAK increases the activity of endosomal PIPKIγ which generates PI(4,5) P2, accelerating the recycling of active integrins. ORP2 unloads PI(4,5) P2 from FAK/integrin endosomes and delivers it to NPC1 containing late endosomes, regulating their tubulo-vesicular dynamics. This coupling of ORP2 and FAK activity drives efficient cholesterol delivery to the plasma membrane, stimulating FA dynamics and cell migration.
Figure 4A: Western-blot analysis of Pitrm1 protein in brain, muscle and liver of two 4-mo male Pitrm1+/- mice and two Pitrm1+/+ littermates. Densitometric analysis is reported in the histograms below the blots. Pitrm1+/+ is in blue, Pitrm1+/- in red. Error bars indicate ±SD B: representative hindlimb-clasping phenomenon is shown in a 4 mo Pitrm1+/- male mouse, consisting in strong adduction of the hindlimbs when the animal is suspended by the tail; a littermate Pitrm1+/+ control displays the normal reflex, consisting in wide abduction of the limbs. All examined Pitrm1+/- animals displayed this abnormal reflex from 2 months of age. C: rotarod test. Blue and red lines refer to Pitrm1+/+ (n=9) and Pitrm1+/- (n=7) 4 mo animals, respectively. Error bars indicate ±SD. Statistical analysis was by unpaired, two-tail Student's t test. *p<0.05; ** p<0.01. D: negative geotaxis test. The experiment was carried out on the same group of animals as in panel B. Error bars indicate ±SD. Statistical analysis was by unpaired, two-tail Student's t test. * p<0.05. E: cylinder test. The experiment was carried out on the same group of animals as in panel b. Error bars indicate ±SD. Statistical analysis was by unpaired, two-tail Student's t test. *** p<0.001. F: pole test. The experiment was carried out on the same group of animals as in panel B. Error bars indicate ±SD. Statistical analysis was by unpaired, two-tail Student's t test. ***p<0.001.
Figure 6A) hSSU72 downregulation results in telomere DNA damage foci (TIF). Cells with indicated shRNAs were fixed and IF-FISH analysis was performed using a 53BP1 antibody and a PNA-telomere probe. (Right) Quantification of cells with more than 5 telomeric 53BP1 foci: n=3; **p ≤0.01 based on a two-tailed Student's t-test to control sample. Error bars represent standard error of the mean (SEM). B) hSSU72 is required for efficient loading of hSTN1 at human telomeres. ChIP was preformed using anti-FLAG antibodies and Southern blotting was performed using a human telomeric probe. Quantification of 3 independent ChIP experiments *p ≤0.05 **p ≤0.01 based on a two-tailed Student's t-test to control sample. Error bars represent standard error of the mean (SEM).