EMBO/sd-character-level-ner huggingface dataset:
Figure 1(A) The hair coats of Sirt7−/− and Sirt7+/+ mice were clipped on P60 in the mid second telogen phase. Images were captured at P60, P120 and P150. (B) The postnatal day when the hair coat recovered by 25%, 50%, 75% and 100% in shaved Sirt7−/− and Sirt7+/+ mice. n = 6 mice/genotype. Box-and-whisker plots: mid-line, median; box, 25th and 75th percentiles; whiskers, minimum and maximum. (C) H&E staining of the skin in unshaved Sirt7−/− and Sirt7+/+ mice on P90 and P130. (D) Representative immunofluorescence images showing Ki67 and p-cadherin (Pcad, labeling HG cells) expression in Sirt7−/− and Sirt7+/+ HFs on P74 and P90. The HF boundary and dermal papilla (DP) is denoted by the dotted and dashed lines, respectively. (E) Percent Ki67 positive cells in each hair germ in D; n = 3 mice and 10 HGs were counted in each mouse. (F) Telogen duration in Sirt7−/− and Sirt7+/+ mice. n = 6 mice per genotype. Box-and-whisker plots: mid-line, median; box, 25th and 75th percentiles; whiskers, minimum and maximum. (G) The hair coats of Sirt7-TG and WT mice were clipped on P45 and images were captured on P56 and P90. (H) The postnatal day when the hair coat recovered by 25%, 50%, 75% and 100% in shaved Sirt7-TG and WT mice. n = 6 mice per genotype. Box-and-whisker plots: mid-line, median; box, 25th and 75th percentiles; whiskers, minimum and maximum. (I) Representative immunofluorescence images showing Ki67 and Pcad expression in Sirt7-TG and WT HFs at P60 and P65. The HF boundary and DP is noted by the dotted and dashed lines, respectively. (J) The percentage of Ki67 positive cells in each HG; n = 3 mice and 10 HGs were counted in each mouse. (K) H&E staining of skin tissues in unshaved Sirt7-TG and WT mice on P65. (L) Telogen duration in Sirt7-TG and WT mice. n = 6 mice per genotype. Box-and-whisker plots: mid-line, median; box, 25th and 75th percentiles; whiskers, minimum and maximum. (M) Abstracted HF cycling in Sirt7−/−, Sirt7+/+, Sirt7-TG and WT mice mentioned above. The red star indicates anagen entry in Sirt7−/− and Sirt7-TG mice; the black arrows show the date of doxycycline feeding.
Figure 8(A) Immunofluorescence of HA-tagged DVL3 localization in MDA-MB-468 cells. The cells were probed with an HA antibody (red). The nucleus was stained with DAPI (blue) and the actin filaments (green) were stained with Phalloidin. Merge of actin (green) and nuclear staining (blue) is shown as ACTIN/DAPI, merge of DVL3 (red) and nuclear staining (blue) is shown as HA/DAPI, merge of DVL3 (red) and actin (green) is shown as HA/ACTIN, and, merge of DVL3 (red), nuclear staining (blue) and actin (green) is shown as MERGE for empty vector (EV), HA-DVL3-WT and HA-DVL3-NESm transient transfected cells. Scale bar: 10µm. (B) RT-qPCR-based analysis of expression changes of DVL3 in stable MDA-MB-468 (EV, HA-DVL3-NESm) cells. (C) RT-qPCR-based analysis of expression changes of KMT2D in stable MDA-MB-468 (EV, HA-DVL3-NESm) cells. (D) RT-qPCR-based analysis of expression changes of ARAP1 , RFX5, GBAT2, WDR74, SNORD3D, HAUS5, TAP1, HLA-A, PDG1, and, MR1 in stable MDA-MB-468 (EV, HA-DVL3-NESm) cells. (E (E) Images and quantification of spheroid areas in stable MDA-MB-468 cell line (EV, HA-DVL3-WT and HA-DVL3-NESm). The data are presented as the mean ± SD, n = 8 showing statistical difference after 32h for HA-DVL3-WT respect to EV and48h for HA-DVL3-NESm respect to EV *p < 0.05. Independent t tests were performed per time point for statistical comparison. Scale bar: 300µm. (F) Proposed model of the role of DVL3, colocalizing with transcription factors such as ZBTB7B and chromatin modifying enzymes as KMT2D in transcriptional regulatory region and regulate transcription.
Figure 4(A) Absolute numbers of NK cells in one of the 4 draining lymph nodes in mice treated or not with the anti-CD62L antibody to inhibit NK cel ingress in the lymph node from blood. Where indicated mice were challenged with LPS. (B) Absolute numbers of IFN-γ+ NK cells in one of the 4 draining lymph nodes before and after LPS administration in mice in which NK cell entry in the lymph node was blocked or not by anti-CD62L treatment. (C) (left panel) Absolute numbers of endogenous and adoptively transferred (CFSE+) NK cells in the draining and contralateral lymph node 5 hours after LPS administration; (right panel) percent of endogenous and adoptively transferred NK cells on total lymphocytes in the draining and contralateral lymph node 5 hours after LPS administration. (D) Flow cytometry analysis of LPS draining and contralateral lymph node from mice that received CFSE labeled cells at the moment of LPS administration. Lymph node cells were stained with anti-CD3, anti-DX5 and anti-IFN-γ antibodies and the analysis performed on gated CD3-DX5high cells. One representative of 4 independent experiments is shown. (E) Tumor volume in mice treated or not with LPS, where indicated mice received anti-CD62L antibody 12h before LPS treatment. (F) Percent of IFN-γ+ NK cells within the tumor in mice treated or not with LPS and with the anti-CD62L antibody.
Fig.Figure 2(A) Summary of ChIP-Seq results obtained using HPC7 cells and an antibody to total STAT5. For each STAT5 occupied region, the occupancy of STAT5 in unstimulated (T=0 mins) and in TPO stimulated (T=30 mins) cells is indicated within a 5-kb window centered on the total STAT5 bound region. (B) Examples of loci containing ChIP-Seq peaks from Cluster 1 (ie uSTAT5 peaks) or Cluster 2 (ie pSTAT5 peaks) showing loss of the former and appearance of the latter following TPO stimulation. (C and D) ChIP-Seq results were validated by ChIP-qPCR using antibodies against total STAT5 or pSTAT5. Data represented as means ± SD of 3 independent experiments. (E) De novo motif discovery using MEME with STAT5 binding sites as input. The most significantly enriched motifs in unstimulated and TPO-treated HPC7 cells are shown. (F) Venn diagram showing the overlap between STAT5 and CTCF binding. (G) ChIP-Seq genome-browser views showing co-localization of uSTAT5 binding with CTCF. (H) Immunofluorescence analysis of STAT5 wild type and knockout MEFs with 4′,6-diamidino-2-phenylindole (DAPI) staining (blue) and anti-STAT5 antibody (red). Scale bars represent 25μm. (I) Western blot analysis of cell lysates isolated from STAT5 wild type and knockout MEFs. **, p<0.01; ***, p<0.001.
Structural model of full-length Bak derived from NMR(a) NMR backbone resonance assignments labeled in a 2D-[15N,1H]-HSQC spectrum of BakΔTM.Figure 2(a) NMR backbone resonance assignments labeled in a 2D-[15N,1H]-HSQC spectrum of BakΔTM. (b) NMR backbone resonance assignment of the Bak-TMH in MSP1D1ΔH5 nanodiscs. (c) 3D-15N-edited-[1H,1H]-NOESY spectrum containing all sequential amide connectivities as well as the contacts to lipid tails and water exchange. The location of the transmembrane helix is indicated above. (d) Overlay of the 20 lowest-energy NMR structures of the Bak-TMH in MSP1D1ΔH5 nanodiscs. A transmembrane helix is formed between Pro187 and Phe209 as indicated in the picture. (e) Structural model of full-length Bak obtained by an MD simulation and by utilizing our structural information. The arrow indicates the flexibility of the 4-amino-acid linker between the soluble domain and the TMH.
Figure 4Growth phenotype comparison of BRI1-mCit/bri1/ubp12i/ubp13 and BRI125KR-mCit/bri1/ubp12i/ubp13. The indicated transgenic lines were grown on DEX medium for 18 days. Scale bars: 10 mm. Western blot analysis monitoring the BRI1 protein accumulation in BRI125KR-mCit/bri1/ubp12i/ubp13 plants with the α-BRI1 antibody. Total proteins were isolated from the roots of the lines indicated (A). PEPC was used as a loading control. Hypocotyl length relative to the mock control. Seedlings of the indicated lines were grown on DEX medium containing 100 nM BL or mock in the light for 6 days (Appendix Fig S7
Figure 3A, B. Menin knock down (KD) in INS-1 cells using a Men1-specific CRISPR sgRNA upregulated Pbk protein levels (A) and mRNA levels (B). qPCR data was from three independent experimets (n=3). **P = 0.0093, ***P = 0.0004 (two-tailed unpaired student's t-test). C, D. Men1 knock out (KO) in PIME cells through Cre-mediated gene editing upregulated Pbk protein level (C) and mRNA level (D). qPCR data was from three independent experimets (n=3).**P = 0.0059 (two-tailed unpaired student's t-test). E, F. JunD KD in INS-1 cells using shRNA upregulated Pbk protein levels (E) and mRNA levels (F). qPCR data was from three independent experimets (n=3).***P = 0.0001 (shRNA-#1), ***P = 0.0007 (shRNA-#2) (two-tailed unpaired student's t-test). G, H. JunD KD in PIME cells using shRNA increased Pbk protein levels (G) and mRNA levels (H). qPCR data was from three independent experimets (n=3).*P = 0.0236 (shRNA-#1), *P = 0.0175 (shRNA-#2) (two-tailed unpaired student's t-test). I, J. JunD KD in menin null PIME cells moderately suppressed Pbk protein levels (I) and mRNA levels (J). qPCR data was from three independent experimets (n=3).*P = 0.0030 (shRNA-#1), *P = 0.0065 (shRNA-#2) (two-tailed unpaired student's t-test). K. The predicted JunD binding sites within Pbk promoters of mouse, human, and rat genomes via software PROMO3.0. L. HEK293T cells were co-transfected with a JunD-expressing plasmid and luciferase reporter constructs driven by different wild-type mouse JunD binding sites (JBSs) for 36 h, followed by luciferase assay. Three independent experimets (n=3). **P = 0.0011 (site 2), **P = 0.0032 (site 3), ***P = 0.0001 (site 4) (two-tailed unpaired student's t-test). ns, not statistically significant difference. M. Luciferase reporters driven by WT or mutant JBS4 were transfected into HEK293T cells with a JunD expression plasmid or with or without menin expressing plasmid. Luciferase assays were performed 36h after transfection. Three independent experimets (n=3). N. JunD overexpression in PIME cells suppressed Pbk protein levels (M, Lane 2 vs Lane 1), but did not affect Pbk expression in menin-null PIME cells (M, Lane 4 vs Lane 3). O. JunD KD suppressed PIME cell growth, but not menin-null PIME cells. Two independent experimets (n=2). **P = 0.0025 (Two-way ANOVA). ns, not statistically significant difference.
WASH is localized in autophagosomes.Figure 3(A) WASH colocalizes with GFP‐LC3 upon starvation. HeLa cells stably expressing GFP‐LC3 were stained with anti‐WASH antibody after treatment with EBSS or culture medium (CM) for 1 h. (B) Autophagy‐related WASH does not colocalize with EEA1. HeLa cells stably expressing GFP‐LC3 were cultured with EBSS for 1 h and stained with antibodies against EEA1 and WASH. (C) WASH localizes to unclosed autophagosomes. HeLa cells stably expressing GFP‐Atg5 were transfected with Atg4B(C74A) mutant, followed by stimulation with EBSS for 1 h. Cells were stained with anti‐WASH antibody (red). (D) WASH does not localize in autolysosomes. HeLa cells were treated with CM or 1 h in the presence or absence of 20 nM BafA1 at 37°C for 1 h. WASH and LAMP1 were visualized by staining with anti‐WASH and anti‐LAMP1 antibodies. (E) Colocalization of WASH and autophagy substrate p62 during autophagy. HeLa cells treated with or without EBSS were stained with anti‐p62 and anti‐WASH antibodies. For (A-E), scale bar, 10 μm. (F) Immuno‐electron microscopy analysis of EBSS cells treated with EBSS for 1 h. Black arrowhead indicates WASH particle, red arrowhead indicates autophagosome, and black arrow denotes autolysosome. In all, 73±2% of autophagosomes contains WASH particles. Scale bar, 100 nm. (G) WASH is not localized in autolysosomes. Autolysosomes and early endosomes were separated from HeLa cells and immunoblotted with indicated antibodies. The above experiments were repeated for three independent times with similar results.Source data for this figure is available on the online supplementary information page.
miRNAs are critical for glycolytic metabolic signatures of ESCsCellular glucose uptake, lactate production, and oxygen consumption rates were measured in WT and Dgcr8−/− ESCs.Figure 1A Cellular glucose uptake, lactate production, and oxygen consumption rates were measured in WT and Dgcr8−/−ESCs. B Relative mRNA levels of metabolic enzymes were determined by qRT-PCR in WT and Dgcr8−/−ESCs. C Western blotting analysis of metabolic enzymes in WT and Dgcr8−/−ESCs. Actin serves as a loading control.
Figure 6(A) Immunoprecipitation and immunoblotting showing the interaction between endogenous ENKD1 and CP110 in mouse retinal tissues. Immunoprecipitation was performed with the anti-ENKD1 (upper panel) or anti-CP110 (lower panel) antibody or the corresponding control IgG. (B-D) Immunoprecipitation and immunoblotting showing the interaction of GFP-ENKD1 with Flag-CP110 (B and upper panel of D) or HA-CP110 (C and bottom panel of D) in HEK293T cells. Immunoprecipitation was performed with antibodies against GFP (B and C), Flag (upper panel of D), or HA (bottom panel of D). (E) Identification of the domains of ENKD1 mediating its interaction with CP110. (F) Identification of the domains of CP110 mediating its interaction with ENKD1. (G) Schematic diagrams showing the domains of ENKD1 and CP110 that mediate their interaction. (H) GST pull-down showing the interaction of purified GST-CP110-N with purified His-ENKD1-M. (I, J) Immunoprecipitation and immunoblotting (I) and quantification (J, n = 3 independent experiments) showing the interaction of HA-CP110 with Flag-CEP97 in HEK293T cells transfected with the indicated plasmids. The intensity of each Flag-CEP97 band was normalized to that of the β-actin band.
Figure 7(a) Schematic outlining Lgr5 Apc Huwe1 stem cell transformation / tumour model. (b) Kaplan-Meier survival plot of cohorts of induced Lgr5 Apc and Lgr5 Apc Huwe1 mice. Deletion of Huwe1 led to a significant reduction in survival of these animals (Log Rank, p < 0.001, n ≥ 15). (c) Lysozyme IHC demonstrating increased lysozyme positive cell numbers in tumours from Lgr5 Apc Huwe1 mice. Scale bars = 200 µm (low magnification), 100 µm (high magnification inset). (d) OLFM4 IHC demonstrating expanded stem cell population in Lgr5 Apc Huwe1 tumours. Scale bars = 200 µm (low magnification), 100 µm (high magnification inset).
Figure 6Immunoblot images (A) and quantification (B) show GPRC5A 46/41 kDa ratios in OC cells treated with 0-10 µM cisplatin and with 25 µM LJH685 alone or as a combination for 72 h. The ratio in mock cells was set to 1. N = 4. Confocal micrographs of EphA2 (red) and GPRC5A (green) in TYK-nu and TYK-nu.R treated with 5 µM cisplatin and 10 μM LJH685 as indicated for 72 h. Arrows point co-localization of the receptors. Scale bars: 20 μm. EphA2 (total and phosphorylated), RSK1/2 and GPRC5A in TYK-nu were assessed by immunoblotting after RSK1/2 knockdown for 2 d and following treatment with 0-5 μM cisplatin for 72 h. Asterisks indicate unspecific bands. EphA2 (total and phosphorylated), RSK1/2 and GPRC5A and these proteins coupled with PARP and PCNA in TYK-nu.R were assessed by immunoblotting after RSK1/2 knockdown for 2 d and following treatment with 0-5 μM cisplatin for 72 h. Asterisks indicate unspecific bands. Chart illustrates the viability of the RSK1/2 depleted TYK-nu.R after cisplatin treatment. N = 3.
Figure 6Schematic representation of the metabolic changes in RIRCD. We propose a mechanism that takes place in three phases. Phase 1 consists in FGF21 increase concomitant with the activation of the integrated stress response which has a protective effect leading to increased expression of proteins involved in amino-acid transport and offers a metabolic rescue by increasing lipid oxidation and TCA. The second phase consists of mTOR activation which leads to increased mitochondrial biogenesis and protein synthesis and in phase 3, the recovery, where we have a decrease in all stress markers.
Figure 4A-B EEF2K contributes to the decreased translation observed with NFR without impacting translation in presence of TM. A. Quantification of newly synthesized proteins at 0, 2, 4 and 6h after 20 μM NFR (left panels) or 10 μg/ml TM (right panels) treatment in MEF eEF2K +/+ and eEF2K-/-. Treated cells were labeled for 15 minutes with 35S-methionine and visualized by SDS PAGE and subsequent autoradiography. Autoradiographies from four different experiments (see also source data) were quantified and results show percentage of translation compared to untreated cells. The mean and s.e.m of four independent metabolic labeling experiments are shown. *P value ≤ 0.05, ** P value ≤ 0.01, *** P value ≤ 0.001 obtained using 2way ANOVA analysis of variance (in red) followed by Bonferroni posttest (in black). B. eEF2K+/+, eEF2K-/-, eIF2αWT and eIF2αS51A MEFs were treated for 6h with indicated doses of NFR, 200 nM rapamycin, or 10 μg/ml TM, or for 30 min with 10 μg/ml CHX. 35S-methionine incorporation was measured by liquid scintillation counting. Data are shown as the percentage of translation compared to untreated cells. *P value ≤ 0.05, ** P value ≤ 0.01, *** P value ≤ 0.001 obtained using 2way ANOVA analysis of variance followed by Bonferroni posttest. C Representative polysome profiles of eEF2K WT and KO MEF treated 6h with NFR or TM. Area under curve for Sub-polysomes (S) and Polysomes (P) used to calculate the P/S ratio were indicated (see also raw data provided). Bar graph represents ratio normalized to untreated cells. OD254 nm is optical density at 254 nm. Data showed mean ±s.e.m. of P/S ratio calculated from 3 independent experiments. P values were calculated using 1way ANOVA analysis of variance followed by Bonferroni posttest; *P value ≤ 0.05, ** P value ≤ 0.01. D The ribosome half-transit time in MEFs eEF2K+/+ and eEF2K-/- was determined as described in Materials and Methods. Incorporation of 35S-Methionine into total protein within the PMS and PRS was obtained by linear regression analysis. Presented graphs are representative of two (CHX 1 μg/ml for 30 min and Starvation 30 min in PBS in eEF2K+/+ cells) to four (NFR 20μM for 6h in eEF2K+/+ and eEF2K-/- cells) independent experiments. Indicated values represent the x displacement measurement (in time) between the PMS line at 300sec and the PRS line (see also raw data provided). Histogram represents mean and s.e.m. of the ribosome half transit time from four independent experiments. P values were calculated using two tails unpaired Student's T-tests; *P value ≤ 0.05.
Transient expression of SipB in COS-2 cells.Figure 3.Transient expression of SipB in COS-2 cells. (A) A plasmid expressing YFP-tagged SipB was transfected into COS-2 cells, and its localization was examined under a confocal microscope.  (B) SipB colocalized with mitochondrial markers in transfected cells. COS-2 cells were transfected with a plasmid encoding either SipB-CFP (top) or SipB-YFP (bottom). Transfected cells were stained with MitoTracker® Red (Molecular Probes, Inc.) to visualize mitochondria and were examined by confocal microscopy. The SipB-induced structures were readily stained with mitochondrial markers (top). Mitochondria were also seen contained within the SipB-induced structures (bottom).  (C) SipB-induced structures were labeled with the ER marker Sec-61. COS-2 cells were cotransfected with plasmids encoding SipB-YFP and Sec61-CFP, and transfected cells were examined by confocal microscopy.
Activation of LRRC8 channels by pro‐apoptotic stimuli.Figure 5Activation of LRRC8 channels by pro‐apoptotic stimuliA-CCisplatin‐induced iodide influx into WT (black ■), but not HAP1−/− (green ▼) HEK cells indicates VRAC halide current activation during apoptosis. Cells expressing an iodide‐sensitive YFP variant were exposed to 200 μM cisplatin for periods of 0.5 h (A), 4.5 h (B), or 8.5 h (C) before adding extracellular I− (50 mM final). The difference in slopes of YFPfluorescence quenching between control and cisplatin‐treated cells semiquantitatively reflects VRAC current activation. Note that increased YFP quenching with cisplatin preincubation is not due to large non‐specific leaks resulting from cell morbidity. Such leaks should lead to a fast component of YFP quenching in WT, but not HAP1−/− cells after the pipetting artifact that immediately follows addition of iodide (indicated by arrows).DSwelling‐induced iodide influx into WT (black ■) and HAP1−/− (green ▼) HEK cells for comparison. Iodide (50 mM final) was added in isotonic or hypotonic (230 mOsm final) solution at the time indicated by arrow.E, FTime course of VRAC activation by 200 μM cisplatin (E) or 4 μM staurosporine (F) determined as in (A-C). Averaged maximal slopes of YFP quenching from eight wells (E) or 16 wells (F) each were evaluated to estimate iodide influx rates. WT (black ■) and HAP1−/− (green ▼). Data are presented as mean ± SEM.
Figure 1(A) Primary hippocampal neurons (DIV6+3) were transfected with shRNA targeting TDP-43 (shTDP) or control (shCtrl) together with GFP-RAB11 to visualize trafficking of recycling endosomes (RE). At least four dendrite segments per neuron were live imaged at 5 Hz for 1 minute to analyze recycling endosome transport. Representative dendrite segments and kymographs of GFP-RAB11 vesicle movement. Quantitative analysis of vesicle motility (B) and total number (C) from kymographs. (D) iPSC-derived human neurons were transduced with shRNA targeting human TDP-43 (shTDP) or control (shCtrl) seven days after thawing for three days. TDP-43 levels were analyzed by quantitative RT-PCR. Expression was normalized to the housekeeping gene YWHAZ and PGK1. (E) iPSC-derived human neurons (DIV7+3) were transduced with shTDP or shCtrl and GFP-RAB11 and at least three dendrite segments per neuron analyzed as in (a). Quantitative analysis of vesicle motility (F) and number (G) from kymographs. Mean +/- s.e.m., n = 3, unpaired two-tailed t-test: * denotes p < 0.05, ** denotes p < 0.01, *** denotes p < 0.001.
Simultaneous quantification of transcription and translation of endogenous genes and a synthetic genetic construct.Figure 3Comparison of protein synthesis rate of endogenous E. coli genes measured using Ribo-seq from this study (in molecules/s units) and from that by Li et al. (Li et al, 2014) (in molecules/generation units). Each point corresponds to a single gene and color denotes the ratio of transcription initiation rate to translation initiation rate (giving RNAP/ribosome) capturing whether transcription (light yellow) or translation (dark blue) is more dominant. Transcription (bottom) and translation (top) profiles for yggN, rpoH and greA, computed from the RNA-seq and Ribo-seq data without induction. Positions of the genetic parts and gene are shown below the profiles. (C) Promoter strengths in RNAP/s units and RBS initiation rates in ribosome/s units. Transcription (bottom) and translation (top) profiles for lacZ. Profiles are shown for cells in the absence and presence of IPTG (1 mM). Position of genetic parts and gene is shown below the profiles. RBS is omitted from the genetic design due to its size. (E) Measured promoter strength in RNAP/s units, RBS initiation rate in ribosomes/s units, and the transcriptional terminator and translation termination efficiency for lacZ. Data shown for cells in the absence and presence of IPTG (1 mM).