EMBO/sd-character-level-ner huggingface dataset:
Figure 7A-D Localization of PDE1-3Ty (A) shown by IFA using an anti-Ty antibody. Antibodies against GAP45, IMC1, cb-GFP, and myc-GAP70 were used as markers of the plasma membrane, IMC, residual body, and apical pole, respectively.Data information: Scale bars = 2 μm. A-D Localization of PDE2-3Ty (B) shown by IFA using an anti-Ty antibody. Antibodies against GAP45, IMC1, cb-GFP, and myc-GAP70 were used as markers of the plasma membrane, IMC, residual body, and apical pole, respectively.Data information: Scale bars = 2 μm. A-D Localization of GCα-3Ty (C) shown by IFA using an anti-Ty antibody. Antibodies against GAP45, IMC1, cb-GFP, and myc-GAP70 were used as markers of the plasma membrane, IMC, residual body, and apical pole, respectively.Data information: Scale bars = 2 μm. A-D Localization of PKG-3Ty shown by IFA using an anti-Ty antibody. Antibodies against GAP45, IMC1, cb-GFP, and myc-GAP70 were used as markers of the plasma membrane, IMC, residual body, and apical pole, respectively.Data information: Scale bars = 2 μm. E Western blot analysis indicates that GCα-3Ty is present in three distinct isoforms. A PKG-3Ty western blot also shows three immune reactive isoforms. Catalase or actin were used as loading controls and ΔKu80 as the parental strain. F PKG-3Ty parasites were solubilised in either PBS or 1% Triton X-100 (TX100) and split into soluble (S) and pellet (P) fractions. Total lysate is also shown. G, H The signal of Myc-ACβ-iKD followed by IFA (G) after 48 hours ± ATc.Data information: Scale bars = 2 μm. G, H The signal of Myc-ACβ-iKD followed by western blot (H) after 48 hours ± ATc. I Invasion of ACα-KO tachyzoites or Myc-Acβ-iKD after 48 hours ± ATc is not affected. However, addition of ATc for 48 hours reduces the invasion efficiency of ACα-KO/ Myc-Acβ-iKD (data is from three independent biological replicates).  J As for destabilisation of PKAc1-iKD or stabilisation of DDmyc-PKArG321E-Ty, ATc -KO/ Myc-Acβ-iKD tachyzoites treated with lytic cycle for 33 or 40 hours, exit host cells and invade and exit the monolayer of HFF cells leading to lysis while the non-treated parasites invade and initiate a new lytic cycle. Data information: Scale bars = 2 μm.
Figure 7A UCSC genome browser capture of lamin B1 ChIP-seq signal (log(LB1/Input)) and LADs discovered in wild-type (WT LAD) and R6/1 (R6/1 LADs) mice in combination with NPC lamin B1 DamID (top). Black arrows highlight differences in identified LADs between WT and R6/1 mice ChIP-seq data. Boxplots of LADs size (log10(LADs size + 1)) and of LADs score (log10(LADs score + 1)) obtained by EDD (bottom) from WT (N=3) and R6/1 (N=3) lamin B1 ChIP-seq data. Hipp, hippocampus. The bottom and top of the boxes are the first and third quartiles, and the line within represents the median. The whiskers denote the interval within 1.5 times the interquartile range (IQR) from the median. B Venn diagram of overlapping LADs between wild-type (WT, N=3) and R6/1 (N=3) mice hippocampus (top). Metaprofile of lamin B1 and Input datasets mean read density within common LADs for WT and R6/1 mice (bottom). C UCSC genome browser capture of lamin B1 ChIP-seq signal (log(LB1/Input)) LADs discovered in wild-type (WT LADs) and R6/1 (R6/1 LADs) mice; and common (green), wild-type (WT) specific (WT spec. LADs, red) and R6/1 specific (R6/1 spec. LADs, blue) LADs identified by EDD (top, WT (N=3), R6/1 (N=3)). Black arrows highlight WT specific LADs not identified in R6/1 mice. Boxplot of average size (log10(LADs size + 1)) for common, WT specific and R6/1 specific LADs (right). The bottom and top of the boxes are the first and third quartiles, and the line within represents the median. The whiskers denote the interval within 1.5 times the interquartile range (IQR) from the median. D Lamin B1 enrichment in common, wild-type (WT, N=3) specific (spec) and R6/1 (N=3) specific LADs (log10(LB1/Input reads +1) in hippocampus (Hipp; left). Bar graphs of significant (Benjamini's adjusted P-value < 0.05) Biological Processes terms from DAVID for genes within common and WT specific LADs (right). Gene-term enrichment was estimated by DAVID using a modified Fisher's exact test and Benjamini multiple correction test. Bars represents the -log10 (Benjamini's adjusted P-value). Exact P values are reported in Appendix Table S3.
Figure 3A-D. Same as Fig 2A-D, except that ACSF contained only 1 mM Ca2+, which is close to the estimated physiological concentration of extracellular Ca2+ (Forsberg et al., 2019). E-H. Same as Fig 2A-D, except that the slice were incubated with 0.2 mM EGTA-AM for 30 mins before recording, which introduces EGTA into the presynaptic terminals.
Proposed model of the force-dependent ubiquitin patterning of titin Hypothetical model of MuRF1-mediated ubiquitinated titin.Figure 5Hypothetical model of MuRF1-mediated ubiquitinated titin. Ubiquitin molecules are shown in gold and placed at the ubiquitination sites identified via mass spectrometry in this work. For simplicitly, only monoubiquitination is displayed. Stretch-induced unfolding of the NL sequence would cause the distancing of MuRF1 respects to its target sites in TK, thereby reducing or preventing their ubiquitination. In turn, this might alter the capability of TK to recruit Nbr1 and p62.
Conserved cysteines in LMF1 are important to LPL secretion A).Figure 5). The LMF1 protein sequence from multiple species was aligned using Clustal Omega (Sievers et al., 2011) ). Schematic of the membrane topology of LMF1 showing the location of all the cysteines. ). Secretion of LPL-V5 from HEK293∆LMF1 cells transiently transfected with LMF1 bearing the indicated cysteine to alanine mutations. Lipase levels in the lysate and pellet, and LMF1 levels in the pellet (α-HIS), are also probed. GAPDH serves as a loading control.
Figure 2Linear regression plot of in vitro limiting dilution assay (LDA) for GSC#9 treated with MALT1 inhibitor, mepazine (MPZ, 20 μM, 14 days). DMSO vehicle was used as a control. Data are representative of n=2. Stem cell frequency was calculated from LDA in GSCs #1, #4, and #12 treated with MPZ treatment (20 μM, 14 days). Data are presented as the mean + s.e.m. on 2 independent experiments. Tumorspheres per field of view (fov) were manually counted in GSCs #1, #4, #9, and #12 in response to MPZ (20 μM) and vehicle (DMSO), and in GSC#9 treated with Z-VRPR-FMK (75 µM) and vehicle (H2O) for 4 days. Data are presented as the mean + s.e.m. on 4 independent experiments for MPZ and 3 independent experiments for Z-VRPR-FMK. The expression of the stemness markers SOX2 and NESTIN was evaluated by western-blot and immunofluorescence (SOX2 in red NESTIN in green) in MPZ (+, 20 μM, 16 hours) and vehicle (-, DMSO, 16 hours) treated GSC#9. GAPDH served as a loading control. Scale bar: 10 μm. Cell viability was measured using Cell TiterGlo luminescent assay in GSCs #1, #4, #9, and #12, human brain endothelial cells (endo), human astrocytes (astro), and human neuron-like cells (neuron) treated for 48 hours with DMSO or MPZ (20 μM). Data were normalized to their respective DMSO-treated controls and are presented as the mean + s.e.m of 3 independent experiments in triplicate. FACS analysis of EdU staining was performed on GSC#1 treated overnight with MPZ (10 μM). Data are presented as the mean + s.e.m. on 3 independent experiments. FACS analysis of propidium iodide (PI) staining in GSC#9 treated for 48 hours with vehicle (DMSO) or MPZ (20 μM). Cell viability was measured using Cell TiterGlo luminescent assay in differentiated GSC#1 #4, and #9 (DGCs) treated for 48 hours with vehicle (DMSO) or MPZ (20 μM). Data were normalized to their respective DMSO-treated controls and are presented as the mean + s.e.m of 3 independent experiments. Morphology of GSCs #1, #4, #9 and DGCs #1, #4, #9 was shown using brightfield images. Heatmap of cell viability of GSC#9 using increasing doses (0, 5, 10, 20, 40 μM) of phenothiazines: mepazine (MPZ), fluphenazine (FLU), cyamemazine (CYAM), chlorpromazine (CHLO), pipotiazine (PIPO), alimemazine (ALI), promethazine (PRO), and doxylamine (DOXY). Data were normalized to their respective DMSO-treated controls. Nude mice were implanted with GSC#9 (106 cells) in each flank and randomized cages were treated with either vehicle (DMSO) or MPZ (8 mg/kg) daily i.p., for 14 consecutive days, once tumors were palpable. Tumor volume was measured from the start of treatment until 1 week after treatment was removed. Graph of tumor volume on day 21 post-treatment is presented. Data are presented as the mean + s.e.m. n=10/group. Cryosections from GSC-xenografted tumors were stained for the endothelial marker PECAM1 (red) and tumor marker NESTIN (green). Nuclei (DAPI) are shown in blue. Scale bar: 20 μm.
Figure 3(A) Schematic view of protein structure of RhpS. (B, C and D) Polyphenols bind to the RhpS-FL protein. The concentration of RhpS-FL protein was consistently measured as 0.6-μM. The binding curves of TA (B), PGG (C) and EGCG (D) are shown. The individual data points were shown (n=2 biological replicates). (E) Alanine-scanning mutagenesis of the RhpS sensor. RhpS containing a corresponding mutation was provided in the rhpS deletion strain on a pHM1 vector. LUX activity was estimated in the absence (black frame) and presence TA (50-μM), PGG (100-μM) or EGCG (2-mM). Error bars represent standard deviations (n=3 biological replicates). Data information: , * indicate significant differences between mock group and strains under the different test conditions as indicated. (Student's t test. *, P ≤ 0.05, **, P ≤ 0.01, and ***, P ≤ 0.001).
Figure 3(A) The bur1-∆C mutant, but not bur1-∆C vac17∆ double mutant, shows normal vacuole inheritance. Quantitative analysis of vacuole inheritance in wild-type, vac17∆, bur1-∆C, and bur1-∆C vac17∆ cells. Cells were pulse-chase labeled with FM4-64, and vacuole inheritance was assessed as the percent cells with inherited vacuoles. Error bars; SD calculated from three independent experiments with at least 100 cells counted in each strain/experiment. (B) The bur1-∆C vac17∆ double mutant cells have the ability to generate a vacuole de novo, which suggests that the cell-cycle delay is due to a defect in Bur1-dependent signaling. WT, vac17Δ, bur1-∆C, and bur1-∆C vac17∆ cells which express Vph1-GFP from its endogenous locus, were pulse labeled with the vacuole specific dye FM4-64. WT and bur1-∆C cells have both FM4-64 and Vph1-GFP signals in both mother and daughter cells. vac17Δ and bur1-∆C vac17∆ cells have both Vph1-GFP and FM4-64 on the vacuole in mother cells, however the daughter cells solely have a Vph1-GFP labeled vacuole, indicating that the vacuole was generated de novo, rather than inherited from the mother vacuole. Arrowheads; new vacuoles in daughter cells. Scale bar, 2 μm.
Figure 2A Methylation of LSD1 was assessed in MCF10A, MCF7 and MDA-MB-231 cells by immunoblotting using anti-LSD1 R838me2a. Protein expression levels of LSD1 in MDA-MB-231 cells transfected with CARM1 shRNAs or control vector, were assessed by western blotting (B) (Error bars represent the mean ± SD, n = 3 experimental replicates, ns = not significant, Student's t-test). relative mRNA expression levels of LSD1 in MDA-MB-231 cells transfected with CARM1 shRNAs or control vector, were assessed by qPCR analysis (C) (Error bars represent the mean ± SD, n = 3 experimental replicates, ns = not significant, Student's t-test). D MDA-MB-231 cells were treated with the indicated amounts of MS049 for 48 h, LSD1 R838me2a and LSD1 levels were assessed by western blotting using indicated antibodies. E MDA-MB-231 cells were treated with increasing amounts of MS049 for 48 h in presence of MG132 (10 μM) or DMSO, before LSD1 level was analyzed by immunoblotting. F MDA-MB-231 cells were transfected with control or CARM1 shRNAs and then treated with MG132 or DMSO for 10 h, and the cell lysates were analyzed by western blotting. G MDA-MB-231 cells were transfected with CARM1 shRNA#2 or control vector, and then treated with CHX (100 μg/ml) for the indicated time; LSD1 protein level was examined by western blotting. H MDA-MB-231 cells were transfected with control or CARM1 shRNAs and then treated with MG132 for 10 h. Cell lysates were immunoprecipitated by anti-LSD1 antibody and then analysed by immunoblotting. I MDA-MB-231 cells were treated with increasing amounts of MS049 for 48 h and MG132 for 10 h. The ubiquitination level of LSD1 was assessed by immunoblotting after IP with anti-LSD1 antibody. J HEK-293T that transfected with LSD1 WT or LSD1 R838A were treated with CHX for the indicated time. LSD1 were examined by western blotting analysis. K HEK-293T cells were transfected with the indicated plasmids and then treated with MG132 for 10 h. Cell lysates were immunoprecipitated using anti-Flag antibody and then subjected to immunoblotting. L HEK-293T cells expressing LSD1 WT or LSD1 R838A were treated with or without MS049 for 48 h and MG132 for 10 h. Co-IP assay was performed using anti-Flag and then subjected to western blotting.
Figure 5Figure 5. SLFN11 inhibits homologous recombination repair. (A-B) SLFN11 suppresses SCE. Representative metaphase spreads showing SCEs from wild type and SLFN11-deficient SF268 cells (red arrows) (A). SCEs were scored for 50 metaphase spreads for each cell line (B). Each point represents the total number of SCEs in a single metaphase spread, and black bars indicate the average value for all spreads. Statistical significance was determined using the Mann-Whitney test (***P < 0.0001). (C-E) Overexpressing of exogenous SLFN11 in U2OS cells results in decreased HR-directed DNA repair. A U2OS DR-GFP cell line stably expressing Flag-tagged SLFN11 was generated (C-D). The resulting cells were electroporated with an I-SceI expression plasmid. 48 hr after electroporation, cells were harvested and assayed for GFP expression by FACS analysis. Error bars are s.d.; n=3. (E).
Determination of Egg-NLS.Figure 4Schematic of the domain structure of Egg. The amino acid sequence alignment of the predicted Egg-NLS in fly, humans and mice is also shown. Asterisks, colons, and periods indicate identical, highly conserved, and weakly conserved residues, respectively. The letters in red indicate basic residues. Immunofluorescence analysis of Myc-Egg WT, Myc-Egg K987A, and Myc-Egg R988A mutants (red) in OSCs before and after Flag-Wde (green) co-expression. Anti-Myc and anti-Flag antibodies were used. DAPI (blue) shows the nuclei. Scale bar: 10 μm. Western blotting of Myc-Egg WT and its K987A and R988A mutants. Flag-Wde: Flag-Wde was co-expressed. SV40-NLS: SV40-NLS was added to Myc-Egg. Anti-Myc and anti-Flag antibodies were used. Immunofluorescence analysis of Myc-Egg K987A and Myc-Egg R988A mutants (red) in OSCs. Both mutants were fused to SV40-NLS. Anti-Myc antibody was used for detection. DAPI (blue) shows the nuclei. Scale bar: 10 μm. Protein-protein interaction assays. Myc-Egg WT, Myc-SV40-NLS-Egg WT, and Myc-SV40-NLS-Egg K1085R mutant were expressed with Flag-Wde in OSCs. Immunoprecipitation was conducted using anti-Flag antibody while anti-Myc (upper) and anti-Flag (lower) antibodies were used for western blotting.
Figure 7The ER-anchored IRE1 RNase is directly involved in the maturation of miR-2137, which targets and downregulates INPPL1. The reduction in INPPL1 protein results in the accumulation of PIP3, recruiting AKT to the plasma membrane where it can be phosphorylated by mTORC2 complex. Activated AKT signals to mTORC1 complex, leading to phosphorylation of p70S6K and 4E-BP1 and induction of anabolic pathways that promote cell growth and proliferation
Figure 2(A) RT-PCR analysis of the expression of miR-181a(-5p and -2-3p) in γδ T cells isolated from thymic biopsies, cultured for 11 days with IL-7 or IL-2 (n=12-15 independent biological samples). (B) RT-PCR analysis of the expression of miR-181a(-5p and -2-3p) in γδ T cells freshly isolated from thymic biopsies (Thymus) and PBLs (n=6-14 independent biological samples). (C-D) RT-PCR analysis of the expression of miR-181a(-5p and -2-3p) in γδ T cells isolated from thymus (C) or from PBLs (D) and cultured with the indicated cytokines, respectively for 11 days (C) or for 4-6 days (D). When indicated, γδ PBLs were also co-cultured with an anti-TGF-β blocking antibody. Data are normalized to the value obtained in IL-7 cultures (n=4-10 independent biological samples). (E-F) RT-PCR analysis of the expression of miR-181a(-5p and -2-3p) versus FACS analysis of the expression of TNF-α (left panel), IFN-γ (middle panel) and NKGD2 (right panel) in γδ T cells isolated from thymus (E) and PBLs (F). Samples were either freshly isolated or cultured for 11 days (γδ thymocytes) or for 6 days (γδ PBLs) with the indicated cytokines (n=5 independent biological samples).
Figure 4U2OS cells stably expressing H2B-EGFP were transfected with indicated siRNAs and after 48 h they were filmed in 5 min intervals. Time frame closest to the nuclear envelope breakdown (NEB) was set as 0 min. Representative examples are shown (n=3). Black bottom line indicates the time frames from NEB to the metaphase-to-anaphase transition. Scale bars indicate 10 μm. Progression through mitosis was categorized in cells from (A) (100/condition) as follows: nuclear envelope breakdown to the metaphase-to-anaphase transition (MAT), anaphase, telophase and interphase. Data from one of three experiments are shown, each bar indicates one cell. Cells that died during imaging are shown in grey. Time from the nuclear envelope breakdown to MAT was quantified in cells from (B). Shown is median ±SD (n=3). Statistical significance was determined by ANOVA (****p<0.0001). Abnormal nuclear organization was scored in cells from (B) that completed cell division. Representative images of aberrant nuclei are shown. Scale bar indicates 15 μm. Plotted is percentage of cells with aberrant nuclei. Shown is median ± SD (n=3). Statistical significance was determined by ANOVA (*** p<0.001).
Figure 6The gastric cancer risk-associated rs2978980 T>G genetic variant disrupts RORA binding to the intronic enhancer region of lncPSCA and down-regulates lncPSCA expression in cells. LncPSCA is a tumor suppressive lncRNA that acts by controlling DDX5 degradation through ubiquitination, resulting in released RNA Pol II from the DDX5-Pol II complex and activated transcription and expression of the P53 signaling genes that are important for gastric cancer progression.
Wnt dependent cytoplasmic to nuclear translocation of β-catenin de-represses translation.Figure 6(Top panel) Primary VSMCs were serum deprived overnight prior to stimulation with Wnt-3a (100 ng/mL) or its solvent (0.1% BSA in PBS) for 4 hr. The cells were treated with 0.5 µM puromycin for 15 min and harvested for Western blot analysis. Total β-catenin levels are indicated, and actin was used as a loading control. The average puromycin/actin ratio (n=3) was graphed. Error bars indicate standard deviation. An independent samples t-test was performed (2-tailed), with the p-value indicated above the error bar. (Bottom panel) Cells were treated similarly to above and fixed and stained for β-catenin (green) and nucleus (blue) The experiment in (A) was repeated using A10 cells (i) HEK 293T cells were serum deprived for 1 hr and then stimulated with Wnt-3a (100 ng/mL) or its solvent (0.1% BSA in PBS) for 4 hr. The cells were harvested and subjected to an endogenous coIP analysis using either β-catenin or FMRP antibody. The corresponding proteins in the precipitated immunocomplex were detected by Western blot analysis. Non-programed rabbit IgG was used as a control. Efficacy of Wnt-3a treatment was assessed by inhibition of GSK3 mediated phosphorylation of β-catenin (S33/37/T41) by Wnt-3a. (ii) HEK 293T cells were serum deprived for 1 hr and then stimulated with Wnt-3a (100 ng/mL) or its solvent (0.1% BSA in PBS) for 4 hr. Cell lysates were then subjected to m7GTP-agarose pull-down analysis. eIF4E and tubulin were used as positive and negative controls, respectively, and phospho-β-catenin (S33/37/T41) Western blots were used as controls for Wnt-3a stimulation. A working model for the relief of β-catenin mediated translational repression by Wnt stimulation
Mutant and non-mutant interfollicular keratinocytes have different proliferation and apoptotic rates during psoriasis-like disease progression A.Figure 2Schematic representation of the JunB and c-Jun double knockout mouse model (DKO*) with lineage tracing used to investigate the psoriasis-like disease development in mice. Briefly, DKO* psoriasis-like mouse model was generated by the cross of mice carrying the floxed JunB allele (JunBf/f) and floxed c-Jun allele (c-Junf/f) with the transgenic mice expressing the Cre recombinase-estrogen receptor fusion under the control of the basal keratinocyte specific K5 promoter (K5-Cre-ERT) to obtain JunBf/f c-Junf/f K5-Cre-ERT mice. To trace epidermal specific deletion of JunBf/f and c-Junf/f in DKO* mice, global double-fluorescent Cre reporter mouse Gt(ROSA) 26Sortm4(ACTB-tdTomato,-EGFP) Luo/J (referred as mT/mG) was crossed with DKO* mice (referred as DKO*-mT/mG). Experimental timeline to induce psoriasis-like disease in 8 week-old mice with four consecutive doses of tamoxifen injections (2 mg). Upon induction, non-mutant keratinocytes express Tomato, while mutant keratinocytes express GFP. GFP expression was analyzed at 0, 5, 7, 15 and 30 days post-induction. Composite immunofluorescent images showing GFP expression of whole-ear sections from DKO*-mT/mG mice at day 5, 7, 15 and 30. Increased GFP expression is shown at day 7 followed by progressive decrease of GFP+ keratinocytes (white dotted line represents basal layer, and red dotted line represents outermost skin layer). n=3 per time point. Quantification analysis of GFP expression (top) and epidermal thickness (bottom) of DKO* mice at different time points during psoriasis-like disease progression. n=6 per time point. Statistical significance *p<0.05, **p<0.01, ***p<0.001 (t-student two tailed-test relative to control group). Quantification analysis of cleaved Caspase-3 (cCas3 top) and Ki67 (bottom) in ear skin of DKO* mice at different time points during psoriasis-like disease progression. n=3 per time point. Intravital confocal time-lapse imaging of non-mutant Tomato keratinocytes and mutant GFP keratinocytes of DKO*-mT/mG at day 7 after first tamoxifen injection. Dotted circles emphasize areas in which mutant GFP+ keratinocytes are replaced by non-mutant Tomato+ keratinocytes. Percentage of GFP+ area eliminated during intravital confocal time-lapse imaging at day 5, 7 and 15 in DKO*-mT/mG mice (Time-lapse 1:8h). Positive value: Area of GFP increased. Negative value: Area of GFP reduced. Fluorescence imaging of DKO*-mT/mG ear skin at day 30 after first tamoxifen injection shows that remaining mutant GFP+ keratinocytes reside along the hair follicles. White dotted line separates epidermis and dermis. White arrows represent GFP+ hair follicles. Intravital confocal time-lapse imaging of DKO*-mT/mG at day 15 after first tamoxifen injection. Mutant GFP+ Keratinocytes (white arrows) are maintained around hair follicles.