EMBO/sd-character-level-ner huggingface dataset:
PC1 and PC2 express in Xenopus oocytes but yield no channel current.Figure 1A. Transmembrane topology of PC1 and PC2 proteins. The two proteins associate at the C-terminus through the coiled-coil domains and the extracellular side via the TOP domains. The GAIN domain and the GPS site in PC1 and the EF-hand motif in PC2 are indicated. The last six transmembrane domains of PC1 (shown in orange) share sequence similarity with PC2. B. Western blot of oocyte lysate (left) and biotinylation-purified surface (right) samples showing the expression of PC1 and PC2 in Xenopus oocytes and enhanced surface trafficking of the PC1/PC2 complex compared to either protein expressed separately. Anti-PC1 C-terminus antibody [29] recognized both full-length (asterisk) and GPS-cleaved CTF (open circle) of PC1. A higher-glycosylated 130 kDa PC2 (star) band was only seen when PC1 is coexpressed. C. Co-IP followed by western blot showing the association between PC1 and PC2 that were expressed in Xenopus oocytes. IP was done with an anti-FLAG antibody. Bands of full-length (asterisk) and GPS-cleaved CTF (open circle) of PC1 are indicated. Both 120 and 130 kDa bands of PC2 were seen in the IPed product. D. Representative current-voltage relationship (I-V) curves (left) and a scatter plot and bar graph (right) showing coexpression of WT PC1 and PC2 produced no current in TEVC recording. The current of the GOF PC2_F604P is included as a control. Currents at +60 mV are shown in the bar graph. Each point represents the recorded current from one oocyte. Oocyte numbers for scatter plot and bar graph are indicated in parentheses. Data are presented as mean ± SD (***P < 0.001, Student's t-test).
Figure 3A) Heatmap showing the normalized enrichment score (NES) measured by GSEA analysis of the listed pathways for FAPs, MuSCs and Macrophages (MAC) RNAseq in the comparison of OC vs YC (on the left) and for FAPs and MuSCs in the comparison of WO vs WY (on the right). GSEA results were filtered for significance by FDR q-value≤0.25. B) GSEA enrichment plots for gene expression profiles of YC and OC FAPs (on top) and MuSCs (on bottom) against the SASP gene list used in A). C) GSEA enrichment plots for gene expression profiles of YC and OC FAPs (on top) and MuSCs (on bottom) against the Cellular Senescence gene list used in A). D) Graph showing the relative expression of Cdkn1a Cdkn2a in WY, WO, WG, YC and OC FAPs and MuSCs, measured by qPCR. E) Graph showing the relative expression of Cdkn1a in WY, WO, WG, YC and OC FAPs and MuSCs, measured by qPCR F) Graph showing the quantification of β-Gal positive cells in WY, WO, WG, YC and OC FAPs and MuSCs compared to late passage IMR90 senescent cells as positive control (SEN) measured by flow cytometry analysis. G) Representative plots of β-Gal 488 fluorescence distribution in YC and OC FAPs and MuSCs and IMR90-SEN cells (in red) compared to their Fluorescence Minus One (FMO) negative control (in black). H) Graph showing the total number of FAPs and MuSCs available in YC and OC single mice.
The sugar-binding specificity of the lectin module.Figure 2A. A close-up view of the sucrose-binding pocket. Residues hydrogen-bonding with sucrose are shown as yellow sticks, and the sucrose molecule is shown as light pink stick. B. The glycan binding profile of Dln1. This profile represents FITC-labeled Dln1 at 200 μg/ml screened against 609 printed glycans on Mammalian Printed Array version 5.2 in replicates of six. A list of top 8 hits reacted with a concentration of 200 μg/ml is shown in Appendix Table S1. The two kinds of Dln1-binding glycan structures (No. 214 and 316) are illustrated in schematic. C&amp;amp;D. Close-up views of Manα1-2Man and Manα1-3Man binding sites. The mannobiose molecules are shown as cyan and gray sticks, respectively. The Fo-Fc electron density maps of disaccharides and the disaccharide-binding sites are shown as gray mesh and countered at 3.0 . The hydrogen-bonding residues are shown as yellow sticks. Polar interactions with the mannobiose are shown as dashed lines.
Figure 7(A) Requirements for en bloc transfer of linear ubiquitin dimer to maltoheptaose were investigated by leaving out the reaction components indicated. Reaction products were detected by staining with Coomassie. (B) Requirements for en bloc transfer of Met1-linked ubiquitin tetramer to maltoheptaose were investigated by leaving out the reaction components indicated. (C) Time-course assay performed at 30°C in the presence of Met1-linked ubiquitin oligomers in which the two most C-terminal residues were either glycine (normal ubiquitin) or alanine to prevent conjugation to substrates (GG/AA). (D) Maltoheptaose ubiquitylation was assayed for 60 s at 30°C in the presence of the indicated ubiquitin tetramers.
Proximity-induced pair-wise crosslinking.Figure 5A Mechanism of the nucleophilic substitution reaction between BrEtY and Cys. The product is a thioether, which is stable in reducing SDS-PAGE. B Ribbon representation of inactive (PDB 1G4M (Han et al., 2001)) and active (PDB 6U1N (Staus et al., 2020)) βarr1. Arrestin is in grey, M2R in pale green. C Western blot of whole cell lysates from HEK293T cells expressing βarr1 mutated as indicated in the top lines. All samples were run in parallel on the same gel. Pair-wise crosslinking between BrEtY-βarr1-3xHA and wt PTH1R. (D) Ribbon representation of active PTH1R as seen from the intracellular side (PDB 6NBF) (Zhao et al, 2019). Cys residues are shown in red. ECL3 (not resolved in the structure) is represent as a yellow dashed line. The approximate position of the Cys residue in this loop is marked with a fuzzy red circle. (E) Western blot of whole cells lysates stained with an α-HA antibody. Residues exchanged with BrEtY are indicated in the upper row, mutations at PTH1R are indicated on the left side. The red arrows indicate the two most prominent signals. Red squares indicate signals that vanish upon removal of specific Cys residues in PTH1R.
Serial transverse sections of the biopsied muscle from six DD patients,.figf4Serial transverse sections of the biopsiedmuscle from six DD patients, and samples from a XMEA patient and a normal control, were immunostained with antibodies against LAMP-2 and limp-I. LAMP-2 is absent in all DD muscles (patient 6 shown here), whereas it is present in the XMEA muscle. The antibody against limp-I highlighted vacuoles in both DD and XMEA muscles. LAMP-1 antibody faintly stained both DD and XMEA muscles, suggesting that the expression level of LAMP-1 might be lower in skeletal muscle than LAMP-2 or limp-I (data not shown). The muscle from the patient without a lamp-2 mutation also showed LAMP-2 deficiency on immunohistochemistry similar to genetically proven DD patients with primary LAMP-2 deficiency (data not shown). The immunohistochemical stains of LAMP-2, limp-I, and LAMP-1 in normal muscle shows numerous tiny particles and accentuates type 1 fibres. H&amp;amp;E, haematoxylin and eosin, original magnification ×150.
Figure 2A Closeup of the central symmetric dimer of the AMP-PNP particle from the membrane distal view. Interfaces within the symmetric dimer are formed intramolecularly between the FERM and kinase domains (F and K; F' and K'). Intermolecular interfaces are formed within the symmetric dimer between F and K', F' and K as well as K and K'. Intermolecular interactions that link symmetric dimers are formed between 2 FERM domains (F and F*; F' and F**). Coloring is as in Fig.1E. The FERM subdomains F1 and F3 (F2 is not visible from this view), kinase N- and C-lobes (KN, KC), AMP-PNP and the N- and C-termini (N, C) are labelled. B Superposition of W266 mediated FERM-FERM 3D-crystallographic dimers (Ceccarelli et al., 2006) (yellow; PDB: 2AEH) and the FERM-FERM contacts seen in the cryo-EM structure to link symmetric dimers (F-F*; F'-F**). The view is 90° rotated from panel (A). Subdomains and termini are labelled and disordered regions in the linker are shown as dashed line.
Figure 4A. Sufu protein levels in mouse embryonic fibroblasts (MEFs) Ptch1+/+, Ptch1+/- and Ptch1-/- transfected with a non-targeting siRNA (Control) or two siRNAs targeting mouse Fbxl17 (1) and (2). B. Quantification of Fbxl17 mRNA levels in MEFs Ptch1+/+, Ptch1+/- and Ptch1-/- transfected as in A. C. Analysis of Gli1 mRNA levels in MEFs Ptch1+/+ and Ptch1-/- upon Fbxl17 depletion using two different siRNAs. (mean ± SEM from 3 independent experiments, ***p<0.0005). D. Analysis of Bcl2 mRNA levels in MEFs Ptch1+/+ and Ptch1-/- upon Fbxl17 depletion using two different siRNAs. (mean ± SEM from 3 independent experiments, **p<0.005; ***p<0.0005). E. Detection of Flag-tagged Gli1 binding to HA-tagged Sufu in MEFs Ptch1-/- cells upon expression of Myc-tagged Fbxl17, or Fbxl17 depletion by two siRNAs. For each condition, 200 µg of whole cell lysate was immunoprecipitated. F. Immunostaining of Fbxl17 using anti-Myc antibody in Ptch1-/- MEFs stably transduced with a pBABE vector expressing Fbxl17 tagged with Myc either at the N-terminus [Myc(N)-Fbxl17] or at the C-terminus [Myc(C)-Fbxl17]. Scale bars: 20 m. G. Detection of Fbxl17 using anti-Myc antibody in Ptch1-/- MEFs stably expressing Myc(N)-Fbxl17 (Fbxl17 tagged at the N-terminus) and Myc(C)-Fbxl17 (Fbxl17 tagged at the C-terminus). H. Detection of HA-tagged Fbxl17 binding to endogenous Sufu immunoprecipitated from cytoplasmic and nuclear extracts from Ptch1-/- MEFs. Asterisk (*) indicates a non specific band. Identification of cytoplasmic and nuclear fractions was performed by Lamin A/C and GAPDH detection.
Figure 4Total expression of transposable element subfamilies in (A) Dppa2 KO compared to WT mESC clones. Total expression of transposable element subfamilies in (B) Dppa4 KO compared to WT mESC clones. (C) RNA-seq analysis of MERVL-int, L1Md_A and L1Md_T in mESCs sorted for expression of both Tomato and GFP reporters driven by MERVL and Zscan4 promoters, respectively, and the double-negative population. (D) Heatmap displaying the enrichment of DUX, DPPA2, DPPA4 and DPPA4ΔSAP binding at different transposable element families. Every subfamily enriched (pval < 0.05) in at least one DPPA2, DPPA4 or DPPA4ΔSAP replicate are shown. (E) MSA plot of DPPA2, DPPA4 and DPPA4ΔSAP ChIP-seq in mESCs showing enrichment over L1Md_A, L1Md_T and MERVL-int sequences. Gaps are in grey and sequences in white with overlap of color-coded ChIP-seq signals. Upper plot shows average coverage of the signal over the aligned TEs.
Figure 7(A-B) Nociceptive withdrawal thresholds in control (AviliDTR without the Cre) and Sst-Cre::AviliDTRmice after systemic diphtheria toxin injection. No significant differences in thermal (A) or mechanical (B) withdrawal reflexes after ablation of SstCre positive neurons. (C-F) Scratching behavior after a single intradermal injection of the indicated pruritogen observed for 1 hour. All compounds evoked a significant increase in the number of scratching bouts compared to vehicle alone in control (AviliDTR) or Sst-Cre::AviliDTRmice. Diphtheria toxin injection in control mice (AviliDTR) had no effect on scratching behavior evoked by histamine (C), chloroquine (D), IL-31 (E) or 5HT1F agonist Ly344864 (F) but significantly reduced responses to IL31, or Ly344864 in Sst-Cre::AviliDTRmice. n-numbers indicated in brackets, asterisk denotes p<0.05, two way RM ANOVA, Holm-Sidak multiple comparison. Error bars indicate SEM.
Figure 4Ser1660 is phosphorylated in response to DNA damage and required for the interaction of separase with γH2AX. Hek293 cells were arrested in G2 phase by sequential thymidine- and RO-3306 treatment, DRB- (+) or mock treated (-), and then analysed as indicated Myc-separase-WT or -S1660A expressing cells were subjected to (IP-)Western using, amongst others, a pan-specific antibody against phosphorylated serine (A, pan-pS Ser1660 is phosphorylated in response to DNA damage and required for the interaction of separase with γH2AX. Hek293 cells were arrested in G2 phase by sequential thymidine- and RO-3306 treatment, DRB- (+) or mock treated (-), and then analysed as indicated Myc-separase-WT or -S1660A expressing cells were subjected to (IP-)Western using, amongst other a separase antibody specific for phosphorylated Ser1660 (B, pS1660) Ser1660 is phosphorylated in response to DNA damage and required for the interaction of separase with γH2AX. Hek293 cells were arrested in G2 phase by sequential thymidine- and RO-3306 treatment, DRB- (+) or mock treated (-), and then analysed as indicated (C) DNA damage-induced Ser1660 phosphorylation of endogenous separase is largely blocked by ATM inhibition. G2-enriched Hek293 cells were treated with KU-55933 (0.3 μM) and/or DRB and analyzed by (IP-)Western 12 h thereafter using the indicated antibodies  (D) Preventing NES phosphorylation spoils nuclear localization of separase in response to DSBs. HeLaK cells expressing N-terminally NLS-eGFP-tagged separase variants were treated with DRB or carrier solvent (− DRB) for 4 h and then subjected to IFM using anti-γH2AX and anti-Nup153 to visualize sites of DNA damage and nuclear pore complexes, respectively. Transgenic separase was detected based on the eGFP autofluorescence. Note that due to their relatively high nuclear concentration co-localization of separase-WT and -S1660D with γH2AX-foci is not discernable. Scale bar = 5 μm  (E) In vitro phosphorylation of separase on Ser1660 by ATM kinase. Incubation of GST-p53 (amino acids 9-22), GST, separase-WT, or separase-S1660 with recombinant ATM-WT, ATM-KD (kinase dead), and/or KU-55933 in presence of [γ-33P]-ATP was followed by immunoblotting and autoradiography  (F) Arg-methylation of RG-repeats mediates recruitment of separase to DSB-containing chromatin. Myc-separase-WT or -KG expressing cells were treated with DRB as indicated and analysed by (IP-)Western and Coomassie staining  (G) In vitro Arg-methylation of separase's RG-repeats by PRMT1. Incubation of histone H4, separase-WT, or separase-KG with recombinant PRMT1 or reference buffer in presence of S-adenosyl-L-[methyl-3H]-methionine was followed by Coomassie staining and autoradiography
A functional network connecting 39 transcription factors with their targets.Figure 2A Number of target genes identified using differential expression for each assayed TF. *genes identified as essential for self-renewal of Hoxb8-FL cells either in (Basilico et al, 2020). B A force-directed graph displaying perturbed transcription factors (orange dots) and target genes (grey dots). Edges indicate if the target gene is differentially expressed, blue for genes downregulated and red for genes upregulated. Size of the nodes is proportional to their degrees.
IMMP1L-silencing alters MICU1 processing and basal [Ca2+]m levels Western blot analysis of endogenous MICU1 levels in HeLa cells upon silencing of IMMP1L and/or IMMP2L.Figure 5Western blot analysis of endogenous MICU1 levels in HeLa cells upon silencing of IMMP1L and/or IMMP2L. int: intermediate; m: mature form Western blot analysis of HeLa cells transfected with the indicated constructs and silenced with IMMP1L and/or IMMP2L. int: intermediate; m: mature form. Cells were treated with 5 μM MG132 for 4 h before lysis Western blot analysis of the supernatant (S) and insoluble pellet (P) fractions of control or IMMP1L-silenced HeLa cell mitochondria following carbonate extraction at pH 10. The established integral membrane protein TIM23 and the soluble protein cytochrome c (Cyt. c) were analyzed as markers Western blot analysis of the supernatant (S) and insoluble pellet (P) fractions of control or IMMP1L-silenced HeLa cell mitochondria transfected with HA-tagged MICU1 WT following carbonate extraction at pH 10. The established integral membrane protein TIM23 and the soluble protein cytochrome c (Cyt. c) were analyzed as markers. pre: precursor; int: intermediate; m: mature form. Cells were treated with 5 μM MG132 for 4 h before lysis Control or IMMP1L-silenced HeLa cells were transfected with HA-tagged MICU1 WT. Cells were incubated with 50 μg/ml cycloheximide (CHX) for the indicated times, collected and subjected to western blotting analysis as indicated. In the graph, the amount of HA (MICU1) is represented relative to the amount at time 0. (n=3 independent experiments) Representative kinetics (F) and analysis (G) of aequorin-based [Ca2+]m measurements in control or IMMP1L-silenced HeLa cells challenged with 10 μM cyclopiazonic acid (CPA) in the presence of 100 μM EGTA (n=3 independent experiments) Resting mitochondrial calcium levels, evaluated through ratiometric imaging of the mitochondrial targeted GCaMP6m, in control or IMMP1L-silenced HeLa cells (n=3 independent experiments; 37-55 cells).
Figure 6Experimental design for the ChIP samples. PAX6A TetOn and PAX6D TetOn cells were differentiated towards the retinal lineage. At day 6 the cells were treated with DOX to turn on PAX6A or PAX6D expression. After 3 days of treatment, the cells were collected at day 10 and used for ChIP experiment. The fragment enrichment evaluated by SES plot. GO analysis of the PAX6A potential targets and PAX6D potential targets identified by ChIP-Seq. PAX6A only, PAX6A and PAX6D shared, and PAX6D only targets are analyzed separately. Verification of PAX6D target genes using PAX6D TetOn cells. Two sets of genes (retinal and non-retinal neural genes) are directly regulated by PAX6D in opposite directions (n=3 biological replicates for each group).
Tfcp2l1 shields 2i-ESCs from spontaneous cell death but is dispensable for renewal and pluripotency.Figure 1A. Z-score shows transcription factors enriched in 2i-ESCs and FBS-ESCs. B. Immunoblotting shows protein levels of Tfcp2l1, Nanog, and H3K27me3 in 2i-ESCs and FBS-ESCs. C, D. Real-time PCR (C) and immunoblotting (D) showing Tfcp2l1 deletion by CRISPR-Cas9. n=5 clones; error bars indicate standard error of the mean (SEM); p values, t-test; five clones were pooled for immunoblotting. E. Alkaline phosphatase staining. The scale bar equals 100 µm. Left, representative stained colonies; right, a bar chart showing the percentage of differentiated and undifferentiated colonies of WT and Tfcp2l1_KO 2i-ESCs. F. Hematoxylin and eosin staining of teratoma formed by WT and Tfcp2l1_KO 2i-ESCs in NSG mice. Scale bars=100 µm. G, H. Propidium iodide staining to measure spontaneous cell death of WT and Tfcp2l1_KO 2i-ESCs (G) and ESC-FBS (H). A representative histogram is shown on the left and the bar chart on the right is the percentage of spontaneous dead cells. Error bars indicate SEM; n=3 biological repeats; p-values were calculated using t-test. I, J. Cumulative cell numbers of WT and Tfcp2l1_KO 2i-ESCs (I) and FBS-ESCs (J). Results are from three independent clones of WT and Tfcp2l1_KO ESCs.
The p62 bodies in the cytoplasm of HeLa cells are ubiquitin-containing protein aggregates.Figure 1.(A) HeLa cells were fixed and stained for p62. The different patterns of p62 distribution were scored in >200 cells as cytoplasmic with small bodies, cytoplasmic with large and intense bodies, equal nuclear and cytoplasmic staining, and nuclear enriched. The percentage of cells in the respective groups is indicated. (B) Expression level of GFP-p62 in HeLa cells stably expressing GFP-p62 (S-GFP-p62) compared with the level of endogenous p62 in the parent HeLa cells. (C) Cells stably expressing GFP-p62 display a cytoplasmic punctuate localization of GFP-tagged p62 similar to endogenous p62. Video microscopy of live cells demonstrated that the small, faint bodies (thin arrows) displayed directed migration, whereas the majority of the larger, intense bodies (thick arrows) were nonmigratory (Videos 1 and 2, available at http://www.jcb.org/cgi/content/full/jcb.200507002/DC1). A still image from Video 1 is shown here. (D) Bodies containing either endogenous p62 or transiently or stably transfected GFP-p62 all contain polyubiquitin. HeLa cells were fixed and stained with p62 and polyubiquitin (clone FK1) mAbs directly coupled with AlexaFluor555 (red) and AlexaFluor488 (green), respectively. Cells expressing GFP-p62 were only stained for polyubiquitin (red). Bars, 20 μm.
Cortactin is required for parkin-CCCP–induced mitochondrial clearance.Figure 5.MEFs were transfected with control or cortactin RNAi and parkin-GFP, and incubated with or without CCCP for 16 h as indicated. (A) Cells were subjected to immunostaining with antibodies for Tom20 and parkin. Bar, 10 µm. MEFs were transfected with control or cortactin RNAi and parkin-GFP, and incubated with or without CCCP for 16 h as indicated.(B) Cell lysates were subjected to immunoblotting with antibodies for Tom20, parkin, cortactin, and actin.
Figure 4(A) Average plots of Med8 cleavages around SAGA- and TFIID-dependent gene TSSs stratified into quintiles by the level of NET-seq signal in a 200 bp window downstream of the TSS. (B) Average plots of Med8 cleavage around the TSSs of the 87 most highly transcribed SAGA- and TFIID-dependent genes. (C) Boxplots of the transcription levels of the 87 most highly transcribed SAGA- and TFIID-dependent genes as determined by NET-seq. Significance was assessed by t-test. (D) Scatterplots of average NET-seq counts in a 200 bp window downstream of the TSS versus average Med8 cleavages in a 1 kb window upstream of the TSS for SAGA- and TFIID-dependent genes. Correspondence between the datasets was assessed by Spearman correlation.
Figure 2(A) Representative images of intestines from 8-week-old Srcap+/+ and Srcap-/- mice (n=6 per group). Length of 120 villi and 120 crypts (20 villi and 20 crypts per mouse) was measured and shown in right panel as means± S.D. ** P < 0.01 by two-tailed Student's t-test. Scale bar, 100 μm. Dotted lines indicate the boundary between crypts and villi. Arrows indicate the length of crypts or villi. (B) Intestinal tissues from 8-week-old Srcap+/+ and Srcap-/- mice (n=6 per group) were collected for immunofluorescence staining with indicated antibodies. Typical images were shown in left panel and statistical ISC numbers from 120 crypts (20 crypts per mouse) were shown in right panel as means± S.D. ** P < 0.01 by two-tailed Student's t-test. Scale bar, 50 μm. (C) 1×104 ISCs from indicated mice were collected for organoid formation. Scale bar, 200 μm. n=3 for each group. Organoid formation ratios was shown as means ± S.D. ** p<0.01 by two-tailed Student's t-test. (D) Srcap+/+ and Srcap-/- mice were treated with 10 Gy's radiation and sacrificed at different time points. Intestinal tissues were collected for H&E staining. Numbers of intact crypts were shown in lower panel as means± S.D. ** P < 0.01 by two-tailed Student's t-test. 50 fields (10 fields per mouse) were taken for each group (n=5). Scale bars, 100 μm. (E) Lgr5GFP-CreERT2;Rosa26lsl-YFP (LRYFP) were crossed with Srcapflox/flox mice, followed by administration of tamoxifen (TAM) for lineage tracing analysis. Mice were sacrificed 7 days after TAM induction and typical jejunum sections were stained. Numbers of traced crypt-villus units were shown in right panel as means± S.D. ** P < 0.01 by two-tailed Student's t-test. 50 fields (10 fields per mouse) were taken for each group (n=5). Scale bars, 50 μm. (F) Proliferation of Srcap+/+ and Srcap-/- ISCs were examined by Ki67 staining followed by flow cytometry analysis. Percentages of proliferated cells were counted as means ± S.D (lower panel). ** P < 0.01 by two-tailed Student's t-test. n=5 per group. (G) Multiple color staining was performed in Srcap+/+ and Srcap-/- intestine sections. AP-Red, DAB and AP-Blue were used for immunohistochemistray coloration. Numbers of each kind of epithelial cells from 50 crypts or 50 villi (10 crypts and 10 villi per mouse) were shown in right panel as means± S.D. ** P < 0.01 by two-tailed Student's t-test. Scale bars, 50 μm.