EMBO/sd-character-level-ner huggingface dataset:
Figure 1Representative western-blots of the cortical (Ctx) or hippocampal (Hip) extracts from mice at the indicated time after seizure activity in pilocarpine (A,B) models. (C,F) Statistics of Gli1 or Shh expression levels shown in A,B or D,E respectively. n=8-14 mice in C and n=8-23 mice in F. Representative western-blots of the cortical (Ctx) or hippocampal (Hip) extracts from mice at the indicated time after seizure activity in kindling (D,E) models. For D and E, samples obtained from mice evoked with a single kindling stimulation to induce seizure activity as evidenced in EEG. (C,F) Statistics of Gli1 or Shh expression levels shown in A,B or D,E respectively. n=8-14 mice in C and n=8-23 mice in F. (G) Shh levels assayed by ELISA from mouse cortex and hippocampus at the indicated time after the initiation of status epilepticus (SE) induced by pilocarpine (n=7-10). (H,I) Shh levels assayed by ELISA in the medium of slices (H, n=9) or hippocampal neurons (I, n=6) incubated with picrotoxin (Pic) or Mg2+-free (0Mg) for the indicated times. (J) Representative western-blots and statistics of Gli1 expression levels from hippocampal neurons incubated with 0Mg for the indicated times (n=13-19). a-tubulin (a-Tub), a loading control. Data were mean + SEM. *p<0.05; **p<0.01; ***p<0.001 vs. Control (Ctrl) with student's t test.
Figure 7During ESC differentiation towards meiotic germ cells in vitro (X-axis), the X-chromosome inactivation status (Y-axis) differs between cell populations and is associated with distinct meiotic germ cell potential in PGCLCs. While somatic cells go through the most complete X-inactivation (red line), EpiLCs and subsequently PGCLCs reach moderate X-inactivation levels (PGCLC XGFP-), or escape X-inactivation entirely (PGCLC XGFP+). XGFP- PGCLCs, which have undergone moderate X-inactivation followed by gradual X-reactivation (green arrow, orange and red cells) are most efficient in differentiating into meiotic germ cells and can develop into oocytes and primary follicles. On the other hand, XGFP+ PGCLCs, which have never gone through X-inactivation and stayed constitutively active (light green cells), or XGFP- PGCLCs, which have reactivated too rapidly (dark green cells), do not show efficient entry into meiosis and mostly display an abnormal mitotic character.
Figure 9Heatmaps of ATAC-seq, anti-GFP (GFP-UBR7) ChIP-seq, and anti-H3K4me3 CUT&RUN in parental and UBR7KO cells. Plots are centered on TSS that­­­­­ show changes in ATAC-seq signal in UBR7KO versus parental cells. Sites are clustered by unsupervised k-means analysis. Cluster color key is defined by cluster labels. Representative genome track examples of clusters 1-4 as defined in (A). Antibody 1 H3K4me3 data are shown. C, D. Metaplots (left) of mean values per bin centered on all TSS, TSS not bound by GFP-UBR7, and GFP-UBR7 bound TSS. Dot plots (right) represent H3K4me3 (Antibody 1) from parental 293T cells (C) or loss of H3K4me3 in UBR7KO versus parental cells (D). Box limits represent 25th and 75th percentiles. Whiskers extend to 1.5x the interquartile range. Outliers are represented as black dots. A Kruskal-Wallis multiple comparisons test with Dunn post-hoc test was used to calculate Benjamini-Hochberg adjusted p-values (italics). Values p<0.05 are pictured. Experiment was performed with two independent anti-H3K4me3 antibodies
Figure 6A. Kaplan-Meier survival curves of WT and PRIMPOL KO mice after the administration of a single dose of MMC (7.5, 10 or 15 mg/kg). Dashed vertical lines represent median survival values for WT (blue) and KO (orange) mice. Statistical analysis was conducted with Log-rank (Mantel-Cox) test; p-value for the highest dose of MMC (15 mg/kg) is indicated. B. Haematoxylin-eosin (H&E) and Ki-67 immunohistochemistry (IHC) stainings of bone marrow sections derived from WT and PRIMPOL KO mice following the administration of 10 mg/kg MMC. Control mice (PBS) were sacrificed when MMC-injected mice reached the humane endpoint. Scale bar, 2.5 mm. In each image, a relevant area is shown at higher magnification (10x). C. Histograms show Ki-67-positive cellular area in the conditions shown in (B). Results are shown with three pairs of age- and gender-matched WT and KO littermates. When combined, the differences observed between MMC-treated WT and KO mice were statistically significant (two-way ANOVA and Bonferroni post-test, p-value 0.0044).
Figure 3(A) NuMA structure upon microtubule depolymerization. Nocodazole-treated HCT116 CMV-OsTIR1 CEP152-mClover-mAID cells were treated with 100 ng/ml nocodazole. Gray, red, green and blue represent α-tubulin, NuMA, CEP152 (mClover) and DNA, respectively. Scale bar, 5 μm. (B) Immunostaining of DHC1 and NuMA in centrinone-treated HCT116 TetOsTIR1 DHC1-3X-mAID-mClover cells. Gray, green, red and blue represent DHC1 (mClover), GT335, NuMA, and DNA, respectively. Scale bar, 5 μm. (C) Schematic illustration of target protein depletion using the AID system to analyze the requirement of dynein for acentrosomal spindle pole formation. (D) NuMA structure upon DHC1 depletion. Centrinone-treated HCT116 TetOsTIR1 DHC1-3X-mAID-mClover cells were treated with 1 μg/ml doxycycline (Dox) and 500 μM indole-3-acetic acid (IAA). Arrowhead indicates the spindle pole. Green, red, gray and blue represent DHC1 (mClover), NuMA, GT335 and DNA, respectively. Scale bar, 5 μm. (E) Frequency of acentrosomal pole patterns upon DHC1 depletion in (D). Values are mean percentages ± SD. from three independent experiments (N≧24 spindles in each experiment). ( (F) The distribution of NuMA upon DHC1 depletion. Arrowheads indicate the presence of NuMA on microtubules. Green, red, and blue represent α-tubulin, NuMA, and DNA, respectively. Scale bars, 5 μm. (G) Structure of acentrosomal spindle poles upon replacement of endogenous NuMA-mAID-mClover-FLAG with either mCherry-NuMA WT or 5A-3. Arrowheads indicate the assembled NuMA structure. Green, red, gray and blue represent endogenous NuMA (mClover), expressed NuMA (mCherry), GT335 and DNA, respectively. Z-projections of 21 sections, every 1 μm. Scale bar, 5 μm. (H) The number of NuMA structure in (G). Values are mean percentages ± SD. from four independent experiments (N≧15 spindles in each experiment). One-way ANOVA with Tukey's multiple comparisons test was used to obtain a P value.
Enhanced sensitivity of atg5, atg7, and nbr1 mutants to drought and salt stresses.Figure 7(A) Five weeks-old Arabidopsis Col-0 wild type (WT), atg5, atg7 and nbr1 mutant plants were placed into a growth chamber with approximately 50% humidity. The photograph of representative plants was taken 10 days after withholding watering. The experiment was repeated twice with similar results. (B) Seven days-old seedlings Col-0 WT, atg5, atg7 and nbr1 grown on solid MS medium were transferred to the same medium (control) or the same medium containing 0.16 M NaCl and photographed 5 days later. The survived seedlings were scored 5 days after the transfer and the average values and SE were calculated from three experiments. According to Duncan's multiple range test (P = 0.05), means do not differ significantly if they are indicated with the same letter.
Figure 3Cells were pretreated with endosomal-pH interfering drugs at the indicated concentrations and subsequently infected with SARS-CoV-2 as described in Fig. 2A and 2B in the continuous presence of NH4Cl (A), chloroquine (B), bafilomycin A1 (C), or concanamycin B (D). The proportion of infected cells was quantified by flow cytometry as described in Fig. 1D, and data were normalized to control samples that were not treated with inhibitors. n = 2-6 biological replicates. Binding of SARS-CoV-2 to Calu-3, Caco-2, A549*, and Vero cells (MOIs of 0.5, 0.6, 0.2, and 0.3, respectively) was synchronized on ice for 90 min. Subsequently, cells were rapidly shifted to 37°C to allow penetration. NH4Cl (50 mM for A549* and Vero cells, and 75 mM for Calu-3 and Caco-2 cells) was added at the indicated times to neutralize endosomal pH and block the acid-dependent step of SARS-CoV-2 infectious penetration. The proportion of infected cells was analyzed by flow cytometry, and data were normalized to that from control samples that had not been treated with NH4Cl. n = 2. Same as in (E) but using concanamycin B (50 nM) instead of NH4Cl. Uukuniemi virus (UUKV) was used at a MOI of 150 to control the efficiency of concanamycin B to neutralize endosomal pH in Caco-2 cells. n = 2. Same as in (E) but using concanamycin B (50 nM) instead of NH4Cl. Uukuniemi virus (UUKV) was used at a MOI of 150 to control the efficiency of concanamycin B to neutralize endosomal pH in Caco-2 cells. n = 2. Same as in (E) but using chloroquine instead of NH4Cl. n = 2.
Figure 2Representative dual-channel kymographs showing that Rheb enhances mitophagy in CCCP-treated axons. quantitative analysis showing that Rheb enhances mitophagy in CCCP-treated axons. The numbers of mitochondria (Mito), AVs, and Mito-AVs/mitophagosomes, the percentage of Mito within AVs, were quantified quantitative analysis the percentage of Mito within AVs, and the motilities of AVs and Mito-AVs along axons with and without elevated Rheb expression were quantified D,E Representative images (D) and quantitative analysis (E) showing that mitophagy in axons requires Rheb and Nix, but not Parkin. The number of mitophagosomes indicated by white arrows and the percentage of Mito within AVs in CCCP-treated axons expressing scrambled shRNA, Rheb shRNA, Nix shRNA, or Parkin shRNA were quantified, respectively (E). F,G Quantitative analysis (F) and representative blots (G) showing increased mitochondrial association with Rheb and LC3-II in mature cortical neurons upon ∆ψm depolarization. Cortical neurons at 14 DIV were incubated with DMSO or 10 μM CCCP for 24 hr and then subjected to fractionation into post-nuclear supernatant (P), mitochondria-enriched membrane fraction (M), and cytosolic supernatant (S). Equal amounts of protein (20 μg) were sequentially immunoblotted with antibodies against mitophagic/autophagic proteins Rheb, LC3, and Rab7, mitochondrial protein VDAC, and cytosolic protein GAPDH on the same membranes after stripping between each antibody application. The purity of mitochondrial fractions was confirmed by enrichment of VDAC and less abundance of GAPDH, compared to post-nuclear supernatant. Protein levels in mitochondrial fractions of CCCP-treated neurons were normalized to those in neurons incubated with DMSO. Data were quantified from four independent repeats. Quantitative analysis showing that Rheb RNAi augments axonal retention of oxidized mitochondria upon ∆ψm depolarization. The fluorescence of MitoROGFP was emitted at 510 nm and excited at 405 nm or 488 nm, respectively. Mean fluorescence intensity ratios evoked by the two excitation wavelengths at individual mitochondria in the CCCP-treated axons expressing scrambled shRNA or Rheb shRNA were quantified and normalized to those of control neurons transfected with scrambled shRNA (H). representative images showing that Rheb RNAi augments axonal retention of oxidized mitochondria upon ∆ψm depolarization. The fluorescence of MitoROGFP was emitted at 510 nm and excited at 405 nm or 488 nm, respectively. Ratiometric images were generated from fluorescence excited by 405 nm light relative to that excited by 488 nm light. The ratio has been false colored with the indicated heat map, with high intensity indicative of ROGFP fluorescence in a more oxidative level
VbP-treated T cells display hallmarks of pyroptosis (A) Macrophages and CD4 T cells from the same donor were subjected to the indicated treatments and morphologic changes as well as PI-uptake were monitored by live-cell imaging microscopy using a 25x objective.Figure 2A) Macrophages and CD4 T cells from the same donor were subjected to the indicated treatments and morphologic changes as well as PI-uptake were monitored by live-cell imaging microscopy using a 25x objective. Representative images from indicated time points are shown. Cyan color coding is used for the fluorescent PI-signal. One donor out of two is shown. (B) Representative images were acquired with a 63x objective at the end of the experiment shown in (A) at 16 hours. One donor out of two is shown. Scale bars: 25 µm. (C) CD4 T cells were treated with VbP or ABT737/S63845. Lysate and supernatant samples were collected at the indicated time points. Samples were analyzed by immunoblotting. αSS and αLS indicate the use of a small subunit or large subunit-specific caspase-1 antibody respectively. (Lys = lysate, SN = supernatant, FL = full length). One representative experiment out of three is shown.
A proposed model in which the 'LDB2-EGR' axis controls gene expression to regulate synaptic function ChIP-seq analyses identified the EGR family members as a functional partner of LDB2.Figure 9ChIP-seq analyses identified the EGR family members as a functional partner of LDB2. Our study suggested that LDB2 suppresses (blue) and upregulates (red) EGR-mediated gene expression in a context-dependent manner. See the main text for more detailed information.
SMO/HH activation promotes the phosphorylation of Smaug.Figure 3(A) HH/SMO promotes slow-migrating forms of Smaug. Western blot analysis of Cl8 cells that transiently express HA-SmaugWT alone, or together with either SMOWT-GFP or SMOPKA-SD-GFP, in the presence or in absence of HH. GMAP serves as a loading control. Here and in the other panels, the antibodies used are indicated on the left. Here and in (B,C), the black arrows indicate the unphosphorylated form of HA-SmaugWT and the brackets indicate the slower migrating phosphorylated forms of HA-SmaugWT. NT: not transfected. See a phosphatase assay in Fig EV4A. (B) Smaug phosphorylation requires its interaction with SMO. Western blot analysis of Cl8 cells expressing HA-SmaugWT, in presence of HH, in combination with SMO∆958-GFP, SMOPKA-SD-GFP or SMOPKA-SD, ∆978-GFP, as indicated. (C) FU controls Smaug phosphorylation. Extracts of Cl8 cells expressing SNAP-Smaug with SMOPKA-SD FU-SD-GFP (with HH) in combination with GFP-FUWT, GAP-GFP-FUWT, Myc-FUEE or GAP-null-FUDANA (as indicated) were labeled for the SNAP tag before electrophoresis. After quantification of the gels, the percentage (%) of phosphorylated Smaug (% of phosphorylated forms of SNAP-Smaug total amounts of SNAP-Smaug) was estimated. In C to E, the mean values and (SD) for independent biological triplicates (N=3) are shown in the graph at the bottom, and the statistical analysis was done by one tailed bivariate Wilcoxon rank test. See also fig EV4C, D. (D) Phosphorylation of Smaug by activated FU requires the co-expression of SMO. Mean values of the percentage of phosphorylated Smaug in extracts of Cl8 cells that express λN-SNAP-Smaug with GAP-GFP-FUWT and SMOWT alone or together (as indicated) N=3 (billogical triplicates). Note that the effect of GAP-GFP-FUWT and SMOWT together is lower than the one observed with GAP-GFP-FUWT and SMOPKA-SD FU-SD-GFP together (in panel C). This could have multiple nonexclusive causes, including the fact that SMOWT is present at much lower levels than SMOPKA-SD FU-SD [38] and/or the involvement of another kinase. See also Fig EV4B. Note that Smaug does not coIP FU unless SMO is present see Appendix Figure S2. (E) Forcing the interaction of Smaug with activated FU leads to a SMO-independent phosphorylation of Smaug. Mean values of the percentage of phosphorylated Smaug in Cl8 cells coexpressing λN-SNAP-Smaug with GFP-FU, FU-SBR, FUEE-SBR or FUDANA-SBR, with or without HH, as indicated. N=3 (biological triplicates). See also Fig EV4C and D. Note that the levels of Smaug phosphorylation observed in presence of FUDANA-SBR are not lower than those seen with GFP-FU. This suggests an incomplete inhibition of endogenous FU, which may be due to the trapping of FUDANA-SBR by Smaug. See a representative blot in Fig EV3C and a phosphatase assay in Fig EV4D.
Figure 7A) Fluorescencein situ hybridization with a probe against tf2 elements (green), and DAPI staining (blue) in WT and fft2Δ fft3Δ cells. B) The number of tf2 element signal clusters is significantly elevated (p=9.8x10-18, χ2-test) in fft2Δ fft3Δ cells, indicating a loss of retrotransposon clustering. Tf2 clustering was counted in wt cells (n=663), fft2∆ (n=556), fft3∆ (n=493), fft2∆ fft3∆ (n=636). Significance of difference between strains was assessed by Chi-square test.
Figure 1Expression of Bhlhe40 (grey) and Bhlhe41 (orange) in the indicated cell populations, data are from the Immgen Database (microarray data set). CMP - common myeloid progenitors. MΦ - macrophages. Flow cytometry detection of the Bhlhe40 (grey) and Bhlhe41 (orange) transcripts by the PrimeFlow RNA assay in alveolar (right) and peritoneal (left) macrophages. A probe specific for the Cd8a mRNA (black line) was used as a negative control. Results representative of at least four independent experiments. Cell surface phenotype and numbers of alveolar (C; digested lung) and peritoneal (D) macrophages in WT and DKO mice. Additional gating for CD11b+ cells was applied for peritoneal macrophages as indicated. Levels of CD11b expression on WT and DKO AMs are shown, and median fluorescent intensity for CD11b expression was quantified (C, bottom left; 3 mice per genotype). Absolute numbers of CD11chiCD11bloSiglecFhi alveolar macrophages from digested lungs (C) and CD11b+F4/80hi peritoneal macrophages (D) from WT and DKO mice are shown (bottom; 4 mice per genotype).
ATG3 and LC3B gene expression pattern in neutrophils after stimulation with PMA or rapamycin.Figure 2ATG3 and LC3B gene expression pattern in neutrophils after stimulation with PMA or rapamycin. ATG3 mRNA expression analysis (fold) after PMA (2.44±0.78) and rapamycin (RAP: 1.81±0.41) stimulation. LC3B gene alterations (PMA: 4.98±0.59) and rapamycin (RAP: 1.7±0.55). Effect of 3‐MA pre‐treatment, for both stimuli, was also assessed (PMA+3‐MA: ATG3: 1.36±0.64, LC3B: 1.5±0.49 and rapamycin+3‐MA: ATG3: 1.28±0.46, LC3B: 1.24±0.57). No other significant alteration in ATG genes mRNA levels was detected. Representative data of ATG7 mRNA expression analysis after treatment with PMA (1.21±0.42) and rapamycin (0.94±0.64). Data are representative of six independent experiments and presented as mean relative expression±SD. Wilcoxon matched‐pairs test, *p<0.05 and n.s.=not significant compared with control conditions (dot line) as indicated in the figure. Relative expression (folds) of each gene is derived from substitution of DCT values (normalized by GAPDH DCT value) in 2DDCT equation 36.
a, Northern blot analysis of lamp-2 expression.figf1a, Northern blot analysis of lamp-2 expression. Total RNA was hybridized using a lamp-2 cDNA2 probe and a murine glyceraldehyde-3-phosphate dehydrogenase probe. b, Western blot analysis of LAMP-2 expression. In control liver and kidney extracts the glycosylated LAMP-2 molecules were detected. In LAMP-2-/- tissues no LAMP-2 product was found. c, Increased mortality in LAMP-2 deficient outbred (C57B6/Jx129SV) mice. Forty-two control (closed circles) and LAMP-2-deficient mice (open circles) were monitored over 300 days. d, Loss of weight in early dying (n = 7 males) and surviving LAMP-2-deficient mice (n = 11 males) in comparison with control mice ( n = 12 males) monitored for 60 days after birth.
Figure 3(A) to (F) Expression of KUP9 as determined in the ProKUP9:GUS line. GUS staining of 5-day-old seedling (A), root maturation zone (B), transverse section of maturation zone (C) and (D), primary root tip (E), and root apex (F). Ep, epidermis; Co, cortex; En, endodermis; Pe, pericyle; Xy, xylem; Ph, phloem. Scale bars, 5 mm in (A), 1 mm in (B), 10 μm in (C), 5 μm in (D), 1 mm in (E), and 20 μm in (F). (G) and (H) Expression of KUP9 protein as determined in the kup9-1/ProKUP9:KUP9-GFP transgenic plants. Green fluorescence shows the subcellular localization of KUP9-GFP. PI (propidium iodide) and ER tracker were used to indicate the positions of the plasma membrane (PM) and endoplasmic reticulum (ER) and are shown as red and cyan fluorescence, respectively. Scale bars for root tip, 10 μm in (G) and 5 μm in (H). Scale bars for EZ and MAZ, 20 μm. (I) Co-localization analyses of KUP9-GFP with ER marker NIP1;1-mCherry in their crossing line. Scale bars, 20 μm. (J) Co-localization analyses of KUP9-GFP with the ER marker BIP2 using immunological staining. Scale bars, 5 μm.
Figure 6A Frequency of overlapping, adjacent, and separated inner kinetochores. Spermatocytes from Miwi+/-;Dicerf/+;Stra8-Cre, Miwi-/-;Dicerf/+;Stra8-Cre, Miwi+/-;Dicerf/-;Stra8-Cre, and Miwi-/-;Dicerf/-;Stra8-Cre mice were used to examine sister kinetochore configuration at meiosis I as shown in Fig 3A and 3B. n=3 mice for each genotype. The total numbers of counted cells in the four types of spermatocytes were 101, 117, 129 and 115, respectively. The total numbers of counted sister kinetochore pairs in the four types of spermatocytes were 4,040, 4,680, 5,160 and 4,600, respectively.C Frequency of abnormal arrangement in the meiotic metaphase I plate. The four types of spermatocytes in A and B were used to perform immunofluorescence as shown in Fig 1B and C. n=3 mice for each genotype. The total numbers of counted metaphase I plates in the four types of spermatocytes were 221, 181, 421 and 240, respectively.
Figure 5A) KLF5 immunoreactivity in normal human pancreas, human PDACs of different histological grade and mouse xenografts of CFPAC-1 and MiaPaca-2 cells. One representative patient out of nine is shown. Xenografts: n=4 for each cell line. B) Violin plot showing the fraction of tumor cell nuclei that were positive for KLF5 in the tissue microarray. Images were automatically acquired and quantified using the ImmunoRatio software. p values were calculated using a two-tailed Welch's t test. The number of tissue core sections for each grade is indicated (n total = 77). White central dots represent the median.C) Representative sections from the tissue microarray stained with the anti-KLF5 antibody are shown. The panels on the right were pseudo-colored for automated quantification. D) KLF5 expression in primary human samples of ADM and PanIN. One representative patient out of nine is shown.
Figure 3(A) Reno1 locus in six vertebrate species. Shaded region indicates the Reno1 locus. Blue gene models are from RefSeq or Ensembl, red gene models are based on PLAR transcript reconstructions (Hezroni et al., 2015). RNA-seq datasets from indicated tissues were taken from publicly-available datasets: BodyMap (Li et al, 2017a) (mouse), Reference Epigenome (human), FAANG (sheep), SRP011985 (opossum), SRP016501 (chicken), DRP000627 (coelacanth). RNA-seq data was scaled separately in each species to the highest coverage in the window, except for opossum and chicken, where a custom upper threshold was set as indicated. (B) The mouse Reno1 locus. Expression levels during neuronal differentiation of ES cells, in different cellular fractions of ES cells (data from (Engreitz et al, 2016)) and motor neurons (GSE90913), and in forebrain during different stages of embryonic development (data from the ENCODE project). Each set of tracks was normalized separately. (C) RENO1 locus in human. Expression levels in dorsolateral prefrontal cortex across six age groups, and in different cellular fractions in fetal and adult cells. Each set of tracks was normalized separately. (D) Reno1 smFISH signal (red) and DAPI staining (blue) in WT and Reno1m/m cells at day eight of neuronal differentiation, imaged using 100x objective. (E) Expression levels of Reno1 and Bahcc1 in 30 adult and embryonic tissues. Data from the ENCODE project, quantification was done using RSEM (Li & Dewey, 2011). (F) Targeted Chromosome Conformation Capture (4C) using the Reno1 promoter as bait. Top, smoothed trend lines and raw counts of the contact profile in ES cells (red) or differentiated neurons (black); bottom, domainogram showing mean contact per fragment end for a series of window sizes.