EMBO/sd-character-level-ner huggingface dataset:
Figure 7A. β-galactosidase and H&E staining of lung sections from control (non-infected) and injured BASC viewer mice 7 days post infection (7 dpi) with influenza virus. Scale bar: 200 µm. B. Abundance of β-gal+ cells in control (non-infected) and injured (7 dpi) BASC viewer animals. Data depicted as mean±SD, n = 4, *p = 0.0286 (Mann-Whitney test), TB - terminal bronchiole. C. Club cell depletion/recovery after naphthalene treatment. Microscopic imaging of epifluorescence in lung sections from control and injured BASC viewer animals isolated 3, 7 and 21 days post naphthalene (dpn). Blue: DAPI. Scale bar: 200 µm. D. β-galactosidase staining of lung sections from control and injured BASC viewer animals 3, 7 and 21 days post naphthalene (dpn). Scale bar: 200 µm. E. Abundance of β-gal+ cells in control and injured BASC viewer animals. The percentage of terminal bronchioles containing 0, 1, 2, 3, 4, ≥ 5 β-gal+ cells is shown. Data depicted as mean±SD, n=3.
Figure 3 Figure 3. Computing stability in multistability regimes. A. Two plots that show the non-degeneracy of even and odd modes in an elliptical geometry for varying cell geometry and MinD recruitment rate. The degeneracy (light blue area) increases with the MinD recruitment rate. B. Schematic representation of the simulation process used to probe the stability of longitudinal and transverse patterns. The system is initialized with a homogeneous configuration and the gradient of the MinD attachment rate is aligned with the major or minor axis to direct pattern selection. After initialization the MinD attachment rate is equalized to allow the system to relax into the initialized state. If the initialized pattern persists in the absence of a stabilizing gradient, the gradient is reapplied to deflect the pattern from its preset alignment and study its stability vis-a-vis spatial inhomogeneities that break its symmetry. The stability towards all possible deflections with linear MinD attachment profiles is probed and the persistence of the initialized pattern is checked. C-D. Stability diagrams of the simulation procedure outlined in (B) for longitudinal (C) and transverse (D) patterns. White areas represent configurations where the respective mode was not initialized. The grey values show the fraction of all simulations (with different attachment templates) in which the respective pattern mode is sustained.
Figure 3A A schematic representation of PPLssKO-C99 translation intermediates used in an experiment shown in panel (B). Signal sequence and the TMD are shown in light and dark grey, respectively, whilst three stars indicate mutations of the PPL signal sequence that render it non-functional. B Variants of PPLssKO-C99 were translated in the presence of 2 μM HisTrx-SGTA using templates lacking a stop codon and terminating either before ("-TMD") or at indicated amino acid distance after the TMD. Pull-down with HisPur Cobalt resin was carried out as described in Materials and Methods, eluted material resolved by SDS-PAGE and results visualised by phosphorimaging. tRNA-bound species were verified by treating PPLssKO-C99 variant terminating immediately after the TMD (TMD + 0) with RNaseA (lane 9). translation reactions were carried out using RNAs coding for the indicated TA-proteins and lacking a stop codon. Sec61β 3R was used as a control protein without a functional TMD. Filled circles indicate tRNA-bound TA-protein species recovered with HisTrx-SGTA. D Rabbit reticulocyte lysate was left untreated ("untreat."), incubated with control immobilised antibodies ("contr. depl.") or immunodepleted of Hbs1L, a factor that together with Pelota and ABCE1 [4], mediates splitting of ribosomes stalled on truncated mRNAs (see diagram). These lysates were used to translate FLAG-tagged Sec61β from an mRNA lacking a stop codon in the presence of 2 μM HisTrx or HisTrx-SGTA and samples were processed as described for (B). E tRNA-bound species of FLAG-tagged Sec61β from experiment shown in panel (D) were quantified and expressed relative to the value obtained for the control-depleted RRL. Shown is the mean with standard error of mean for n=4 biological replicates. Statistical significance was calculated using unpaired t test with Welch's correction. ns - not significant.
Figure 3(A) Immunofluorescence images of RPE1 cells cultured in normal serum medium, and stained with antibodies against ENKD1 and Centrin and DAPI. Scale bar, 1 µm. (B) Immunofluorescence images and fluorescence intensity profiles of ENKD1 and CEP164 in serum-starved RPE1 cells. The arrow marks the centriole subjected to the line scan. Scale bar, 1 µm. (C) Immunofluorescence images and fluorescence intensity profiles of ENKD1 and Ninein in serum-starved RPE1 cells. The arrow marks the centriole subjected to the line scan. Scale bar, 2 µm. (D-F) 3D-SIM super-resolution images (D), quantification of the outer toroid diameters (E, n = 20 cells from three independent experiments), and a model illustrating the relative positions of γ-tubulin, Ninein, ENKD1, CEP164, CEP912, and Centrin (F) within a centriole. RPE1 cells were serum-starved and stained with antibodies against the indicated proteins. Scale bar, 1 µm. (G) Immunofluorescence images of RPE1 cells cultured in normal serum or serum-free medium, and stained with antibodies against ENKD1 and γ-tubulin and DAPI. Scale bar, 1 µm. (H) Immunofluorescence images of RPE1 cells cultured in serum-free medium, and stained with antibodies against ENKD1 and acetylated α-tubulin and DAPI. Scale bar, 1 µm. (I) Immunofluorescence images of RPE1 cells transfected with GFP-ENKD1 or GFP vector, cultured in a serum-starved condition, and stained with the antibody against acetylated α-tubulin and DAPI. Scale bar, 1 µm. (J) Schematic diagrams showing the centrosomal localization domain of ENKD1. (K) 3D-SIM images of ENKD1 localization at the centrosome in RPE1 cells cultured in serum-free medium (0, 12, 24, or 48 h of serum starvation), and stained with antibodies against ENKD1 and acetylated α-tubulin. Scale bar, 1 µm.
Figure 5(A) Genes up- (red) and downregulated (blue) in the tumor cells from lymph nodes (Vav1∆C LN) and spleen (Vav1∆C SP) of recipient mice transplanted with Vav1∆C-expressing CD4+ T cells. As comparative control, we used CD4+ T cells from spleen (C SP) and lymph nodes (C LN) of control recipient mice. Rows represent individual genes. Columns represent experimental replicas. Total number of genes is indicated on the left. Relative changes in abundance are shown in color gradients according to the color scale shown on the right. (B) GSEA showing enrichment of TFH-associated gene signatures in the transcriptome of Vav1∆C-transformed cells. The expression profile of the top 25 leading-edge genes in the transcriptome of CD4+ T cells from healthy control (C) and Vav1∆C-tumor-bearing mice (Vav1∆C) is shown. The normalized enrichment scores (NES) and false discovery rate values (FDR, using q values) are indicated inside the GSEA graph. Relative changes in abundance are shown in color gradients according to the color scale shown at the bottom. q-val, q value. (C) Main functional categories encoded by the Vav1∆C-dependent transcriptome. For all of them, P  ≤  0.001 (Fisher's exact test). (D-I) GSEAs showing enrichment of indicated gene signatures (top) in the transcriptome of Vav1∆C-transformed cells. In each case, we show the expression profile of the top 25 leading-edge genes in healthy control (C) and Vav1∆C-tumor-bearing mice (Vav1∆C). NES and FDR values (FDR, using q values) are indicated inside each GSEA graph. Relative changes in abundance are shown in color gradients according to the same color scale shown in panel I. (J) Dot plot of the Vav1∆C-dependent gene signature fit score in the indicated experimental groups (bottom) that were retrieved from a previous work (Zang et al., 2017). Dots represent values from an individual sample. Bars represent the mean enrichment value ± SEM for the overall sample set. ***, P ≤ 0.001 (according to Tukey's honest significance difference test). (K) Venn diagrams representing the number of up- (left) and down-regulated (right) genes from the Vav1∆C -dependent transcriptome shared with the indicated datasets. (L) Level of enrichment of indicated gene signatures that are either Vav1∆C-specific (red) or shared with the different datasets (blue) used in panel J.
Effect of nocodazole on autophagy. Vegetatively growing cells, YW5-1B and TN124, were treated with 10 μg/ml nocodazole in YEPD medium for three hours. A, YW5-1B cells were transferred to 0.17% yeast nitrogen base without amino acid and ammonium sulfate containing 1 mM PMSF and 10 μg/ml nocodazole, and further incubated for 4.5 h. The accumulation of autophagic bodies was examined under a light microscope. Arrows indicate autophagic bodies. B, TN124 cells were starved in SD(−N) containing 10 μg/ml nocodazole for 4.5 h. ALP activity was measured before (open bars) and after (filled bars) starvation.
Fig.Figure 4(A) Sagittal planes of whole-mount immunostaining and quantification for TUBG on WT mouse embryos from E3.5 to E6.5. The insets are magnifications of selected centrosomes from the dotted areas denoting the inner cell mass (E3.5) or epiblast (E5.5 and E6.5). Bars represent mean ± s.d (two-tailed student's T-test or one-way ANOVA with Tukey's multiple comparisons tests). Scale bars: E3.5 and E5.5 = 15 μm; E6.5 = 30 μm and 3 μm (insets). E3.5: 88 ± 26% (n=73 cells from 5 embryos); E5.5: 92 ± 10% (n=270 from 3 embryos); E6.5: 79 ± 12% (n=12,082 from 3 embryos). (B, C) Representative gSTED super-resolution images of immunostaining for TUBG and either CEP164 (B) or ODF2 (C). Whole-mount staining was used for the CEP164 data as well as for the E3.5 blastocysts for ODF2, whereas staining on sections was used for ODF2 at E5.5 and E6.5. Quantification of the percentage of centrosomes (TUBG) with CEP164 or ODF2 are shown on the right. **** p < 0.0001, *** p < 0.001, * p < 0.05 (two-tailed student's T-test or one-way ANOVA with Tukey's multiple comparisons tests; the ODF2 data between E5.5 and E6.5 was not significant using the latter test). Bars represent mean ± s.d. Scale bar = 0.5 μm. CEP164 at E3.5: 2 ± 2%, (n=92 centrosomes from 4 embryos); E5.5: 50 ± 2% (n=249 from 3 embryos); E6.5: 62 ± 0% (n=856 from 3 embryos). ODF2 at E3.5: 20 ± 9% (n=108 from 5 embryos); E5.5: 51 ± 4% (n=294 from 5 embryos); E6.5: 60 ± 2% (n=1,183 from 4 embryos). (D) Immunostaining and quantification of the area of TUBG and PCNT signals from E5.5 and E6.5 centrosomes in epiblast cells. The separate channels for the marked centriole are shown in the insets. *** p < 0.001, ** p < 0.01 (two-tailed student's T-test). Bars represent mean ± s.d. Scale bar = 2 μm. Quantification of PCNT at E5.5: 0.22 ± 0.01 μm (n=103 centrosomes from 3 embryos); E6.5: 0.31 ± 0.02 μm (n=342 from 3 embryos). Quantification of TUBG at E5.5: 0.15 ± 0.02 μm (n=108 from 4 embryos); E6.5: 0.25 ± 0.01 μm (n=311 from 3 embryos).
Figure 6A. Cumulative distribution plots showing the difference in distribution of transcript optimality between upregulated and downregulated transcripts in K562 cells subject to ILF2 CRISPR interference targeting ILF2. Transcript quantities are indicated in the figure legend. Data information: Wilcoxon signed rank tests were performed on the upregulated and downregulated groups against the control group. P-values are denoted (right). B, C. HEK293 Tet-off experiments showing the degradation of REL-OPT (B) and REL-WT (C) transcripts with ILF2 and ILF3 siRNA and Control (CTR) siRNA treatment, post-doxycycline addition. Data information: data is representative of 3 independent experiments in which the data represents the mean ± SD for 3 biological replicates. A two-way ANOVA with Holm-Sidak multiple comparisons was performed. P-values are denoted as follows: p < 0.05 (*), p<0.01 (**). D. Representative immunoblot of FLAG-tagged REL-OPT and REL-WT expressed in HEK293T cells under ILF2 and ILF3 siRNA treatment. The immunoblot is representative of 3 independent experiments. ACTB is shown as loading controls. E. Representative immunoblot of FLAG-tagged REL-OPT and REL-WT in HEK293T cells co-expressed with two different isoforms of ILF2. The immunoblot is representative of 3 independent experiments. ACTB is shown as loading controls.
Phosoholipase C gamma1 (PLCγ1)-deficient mouse embryos show a deficit in mesencephalic axon guidance (A) Mouse embryos were immunostained with anti-neurofilament antibody (blue), showing mesencephalic projection.Figure 1(A) Mouse embryos were immunostained with anti-neurofilament antibody (blue), showing mesencephalic projection. Dispersed axon projections (arrows point to the area of the superior colliculus) and decreased axon fibers (arrowheads point to the area of the pretectal commissure) were observed in Plcg1f/f;Nes-Cre mice at E11.5 (n = 20). The areas of the superior colliculus is shown magnified in each inset (scale bar = 200 μm) (B) Representative images between the posterior and anterior borderlines of the superior colliculus (a and b). The red boxes were drawn as rostrocaudal region of interest (ROI) based on the upper part of the pretectal commissure in the embryonic mesencephalon (scale bar = 100 μm) (C and D) The axons passing through the ROI was quantified by measuring the fluorescence intensity in the ROI, and the distribution of axons was statistically analyzed (n = 5; t-test; **P < 0.005) (E) Whole-mount immunostaining images show neurofilament (red), netrin-1 (purple), and DCC (green), respectively. An E11.5 Plcg1f/f;Nes-Cre embryo showed shortened dorsal ramus and opthalmic nerves (white and yellow boxes). Scale bar: 200 μm (F - G) Enlarged views of the opthalmic nerve (yellow arrow heads) and dorsal ramus (white arrow heads) (scale bar: 50 μm) (H) Nerve length measurements; Op: ophthalmic nerve; DR: dorsal ramus (n = 3; t-test; **P < 0.005). Data are presented as the mean ± s.e.m (I) Double immunostaining images. The pattern of expression and colocalization of DCC (green)- and TH (red)-positive axon bundles extending toward the alar thalamus. Scale bar: 100 μm
AMPK-CLIP-170 signal at the intercalated disc controlled the cell shape in cardiomyocytes.Figure 4(A) GFP time-lapse images of neonatal rat cardiomyocytes expressing EGFP-CLIP-170 WT 0, 2 and 3 hours after treatment with 2 μM MYK-461. (B) Immunostained images of neonatal rat cardiomyocytes 4 hours after treatment with 0.01% DMSO (Control, upper row) or 2 μM MYK-461(MYK-461, bottom row). These cells were stained with an α-tubulin (green) and a plakoglobin (red) antibody. (C) ﻿Box and whisker plots of the cell size of a cardiomyocyte 2 hours after treatment with or without 2 μM MYK-461, showing the 25th percentile (bottom line of each box), median (middle line of each box), 75th percentile (top line of each box), maximum and minimum (each whisker). Adenovirus expressing EGFP-CLIP-170 (adCLIP) mutant was used (WT, S311A, S311D). Numbers in the graph indicate n number of the cells from 2 independent experiments. Control (-/-): n=784, MYK-461: n=939, CLIP WT: n=656, CLIP WT + MYK-461: n=619, CLIP S311A: n=405, CLIP S311A + MYK-461: n=635, CLIP S311D: n=613, CLIP S311D + MYK-461: n=735. Data means ±S.D. Differences among multiple groups were compared by one-way ANOVA, followed by a post hoc comparison using the Tukey method. **, P<0.01 versus Control, ††, P<0.01 versus CLIP WT, n.s., not significant.
The middle region of Atg16L1 is required for interaction with FIP200.Figure 3(A) Schematic representations of the wild‐type Atg16L1 and its mutants. Human Atg16L1α (NP_060444) has an amino‐terminal Atg5‐interacting domain (grey box), a coiled-coil domain (shaded box) and seven WD repeats (black boxes). Star indicates the position of the Crohn's disease‐associated T281A mutation. (B, C) HEK293T cells were co‐transfected with the indicated constructs. Cell lysates were analysed by IP. IP, immunoprecipitation; ND, not determined.
Figure 3A Glucose tolerance test of mice that received AAV control (GFP) or PLTP at 10 weeks of HFD. After eight hours of fasting, 2 g/kg body weight of glucose was administered. n=9 for GFP and n=7 for PLTP. *P<0.05, **P<0.01 relative to GFP by two-way ANOVA repeated measures followed by Bonferroni's test. B Insulin tolerance test of mice in (A) at 10 weeks of HFD. Mice were fasted for four hours and administered insulin (0.75 U/kg body weight). n=9 for GFP control and n=7 for PLTP. *P<0.05, **P<0.01 relative to GFP by two-way ANOVA repeated measures followed by Bonferroni's test. C Fasting insulin levels of mice in (A) at 10 weeks of HFD. Blood samples were harvested after eight hours of fasting. n=5 for GFP and n=4 for PLTP. *P<0.05 relative to GFP by Student's t-test. D Glucose tolerance test of mice that received AAV control (GFP) or PLTP at 6 weeks of HFD. Note that no difference was found in body weight between the groups by two-way ANOVA repeated measures followed by Bonferroni's test.. After eight hours of fasting, 2 g/kg body weight of glucose was administered. n=7 for GFP control and n=10 for PLTP. AUC of glucose is shown in the right graph. *P<0.05 by Student's t-test. E 18F-FDG PET/CT scan images of mice that received adenovirus GFP (control) or PLTP at 14 weeks of HFD. Mice received CL316,243 at dose of 1 mg/kg of body weight 30 min before the PET/CT images. Representative images of n=5 for both groups. Yellow arrows indicate anatomical references as bladder, heart and interscapular BAT. F Quantification of 18F-FDG uptake in indicated tissues from mice in (E). After the 18F-FDG PET/CT imaging, indicated tissues were harvested and the radioactivity was measured by scintillation counter. n=5 for both groups. *P<0.05 relative to GFP by Student's t-test. N.S., not significant.
Arginine-rich DPRs have preferential interaction with endogenous EXOSC10 A.Figure 7A. Schematic diagrams of codon optimized (lacking extensive G4C2 repeat) plasmids expressing N-terminally EGFP fused 89 repeats of either poly-GR, poly-PR or poly-GA under the control of CMV promoter. B. Immunoblot validation of successful fractionation using antibodies against HDAC1 (nuclear marker) and GAPDH (cytoplasmic marker). C. Co-immunoprecipitation of GFP-DPR interacting proteins in cytoplasmic fraction using GFP-trap beads. Immunoprecipitants were served for immunoblot with anti-EXOSC10 antibody. The same membrane was reprobed with anti-GFP antibody. D. Quantification of the relative intensities of "EXOSC10 in co-IP fraction" / "EXOSC10 in input". E. Co-immunoprecipitation of GFP-DPR interacting proteins in nuclear fraction using GFP-trap beads followed by immunoblot using anti-EXOSC10 antibody. The same membrane was reprobed with anti-GFP antibody. F. Quantification of the relative intensities of "EXOSC10 in co-IP fraction" / "EXOSC10 in input".
Figure 4Hippocampal autaptic neurons of munc18-1null mutant mice were rescued with lentiviruses expressing M18WT, M18S241A or M18S241D. Electrophysiology recordings were performed between DIV14-17.(A) Amplitude of excitatory synaptic response (EPSC) (M18WT: 1.37±0.21 nA, n=35; M18S241A: 2.53±0.31 nA, n=45; M18S241D: 0.24±0.05 nA, n=17, ***p<0.001, **p<0.01, *p<0.05). Hippocampal autaptic neurons of munc18-1null mutant mice were rescued with lentiviruses expressing M18WT, M18S241A or M18S241D. Electrophysiology recordings were performed between DIV14-17.(B) Percentage of cells that do not respond to electrical stimulation (M18WT: 15.5%; M18S241A: 0%; M18S241D: 56.9%). These cells are not included in (A). Insert: representative recording of single EPSCs. Scale bar represents 10 ms and 0.5 nA. Hippocampal autaptic neurons of munc18-1null mutant mice were rescued with lentiviruses expressing M18WT, M18S241A or M18S241D. Electrophysiology recordings were performed between DIV14-17.(C) Typical examples of spontaneous vesicle release. Scale bar represents 0.1 s and 25 pA.(D) Spontaneous release frequency (M18WT: 7.05 ± 1.28 Hz, n=32; M18S241A: 14.26 ± 2.04 Hz, n=37; M18S241D: 0.64 ± 0.29 Hz, n=11; ***p<0.001, **p<0.01).(E) Amplitude of spontaneous release events (M18WT: 15.40 ± 1.06 pA, n=17; M18S241A: 15.03 ± 0.61 pA, n=20; M18S241D: 10.98 ± 0.72 pA, n=10; **p<0.01). Hippocampal autaptic neurons of munc18-1null mutant mice were rescued with lentiviruses expressing M18WT, M18S241A or M18S241D. Electrophysiology recordings were performed between DIV14-17.(F) Size of the readily releasable pool (RRP) assessed with single hyperosmotic sucrose application (500 mM, 3.5 s, M18WT: 0.74 ± 0.15 nC, n=7; M18S241A: 1.6 ± 0.33 nC, n=10; M18S241D: could not be determined; * p < 0.05). Hippocampal autaptic neurons of munc18-1null mutant mice were rescued with lentiviruses expressing M18WT, M18S241A or M18S241D. Electrophysiology recordings were performed between DIV14-17.(G) Neurons were stimulated with two consecutive pulses with varying inter-stimulus interval to assess release probability. Paired pulse ratio calculated as ratio of the second to the first synaptic response is plotted as function of inter-stimulus interval (M18WT, n=25; M18S241A, n=19; M18S241D, n=17); * p < 0.05, ** p < 0.01). All data are expressed as mean ± SEM. See also Figure EV2 and EV3.
Figure 8GFP based reporter assay showing the efficiency of NHEJ repair pathway under indicated siRNA-depleted conditions in HEK293 cells. Graph represents the quantifications from three independent experiments (*P<0.05, **P<0.01, ***P<0.001; one-way ANOVA, n=3, mean +SEM). GFP based reporter assay showing the efficiency of HR repair pathway under indicated siRNA-depleted conditions in HEK293 cells. Graph represents the quantifications from three independent experiments (ns P>0.05, **P<0.01; one-way ANOVA, n=3, mean +SEM). CFA showing the IR sensitivity of 53BP1-proficient and deficient MCF7 cells under indicated siRNA-depleted conditions. Data was collected from two individual experiments with triplicates. (ns P>0.05; two-way ANOVA, n=2, mean ±SEM). CFA showing the IR sensitivity of BRCA2-proficient and deficient DLD1 cells under indicated siRNA-depleted conditions. Data was collected from two individual experiments with triplicates. (**P<0.01, ***P<0.001; two-way ANOVA, n=2, mean ±SEM). Representative IF-images showing Rad51 foci in S-phase (Edu positive) U2OS nucleus after 5h of IR (2Gy) exposure. (Scale bar; 10µm).   Graph represents the quantification of (E) showing average number of Rad51 foci per S-phase (Edu positive) nucleus. (ns P>0.05, ****P<0.0001; unpaired t-test, n=2, mean +SEM, at least 50 nuclei per condition and experiment).   CFA showing the rescue of cell survival in IR treated, ATX3-depleted (siRNA), HeLa cells, after co-depletion (siRNA) of RNF8. (ns P>0.05, *P<0.05, ****P<0.0001; two-way ANOVA, n=3, mean ±SEM). Model representing p97-ATX3 complex maintaining RNF8 homeostasis under soluble environment and promoting RNF8 chromatin extraction at sites of DSBs to promote NHEJ.