EMBO/sd-character-level-ner huggingface dataset:
Figure 5A. Stable islands in the cell cycle archipelago. The cell cycle and growth phenoprints were used to map the 397 mutant strains with at least one cell cycle or growth feature with a |s| ≥ 3, as well as the 240 independent WT replicates, in 2D using tSNE. As for the morpho archipelago, the data were clustered using the dbscan algorithm. The groups of strains clustering (dbscan parameters ε = 4.28, minPoints = 3) together in more than 90% of the maps defined an island. Dots in the scatter plot on the left represent strains and are colored with the same color code as for the island on the right graph. Light grey dots represent strains that were not consistently (less than 90% of the time) associated with one of the island. B. Heatmap showing, for each island, the average score of each cell cycle and growth feature used for the construction of the map. C. Islands shown in panel A were colored according to their average score for each listed feature. D. Shown are the enriched "biological process" GO terms associated with false discovery rate below 0.05. E. The islands in the cell cycle archipelago were colored according to the proportion of "y-genes" (genes of unknown function).
Figure 7(A) Dot plot showing the distance between parent centrioles pairs in A549 cells following the indicated treatments (ZM = ZM447439; REV = reversine). Nocodazole concentrations are 0.03 μM, 0.1 μM, 0.33 μM, 1 μM, 3.3 μM. Median (red) and 95% confidence interval thereof (black) are shown. N > 50 cells were analysed. (B) Immunoblot of A549 cells subjected to the indicated treatments for 24h as in (A). N = 3 independent experiments. (C) DNA content analysis of A549 cells subjected to the indicated treatments as in (A) either for 24h (left panels) or 48h (right panels). N = 2 independent experiments. (D) Schematic of the experimental conditions utilized to synchronize RPE1 cells and to specifically interfere with centrosome clustering after telophase. (E) Immunoblots of RPE1 cells synchronized (F) Representative fluorescence micrographs of RPE1 cells synchronized Centrosomal antigens were stained with the indicated antibodies. Blow-ups without Hoechst 33342 are magnified 2X. Scale bar: 5 μm.
DRP-1 induces autophagy in various cell lines.Figure 4.(A) Quantification of autophagy in 293 and MCF-7 cells. Graphs show the percentage of MDC-positive cells resulting from luciferase or DRP-1 Δ73 transfections (mean ± SD calculated from triplicates of 100 cells each; note that total cell number comprises transfected and nontransfected cells). These experiments were repeated three times with reproducible results. (B) Photographs of MDC-stained MCF-7 cells from the same experiment shown in A. Arrows point to MDC-positive cells. Transfections: luciferase (1a and 1b) and DRP-1 Δ73 (2a and 2b) at low (a) and high (b) magnifications.
a) Diagrams representing GFP-tagged WT HACE1 and mutant HACE1 (HACE1Δ HECT and.f4(a) Diagrams representing GFP-tagged WT HACE1 and mutant HACE1 (HACE1Δ HECT and HACE1Δ ANK) constructs transiently transfected into Hace1−/− MEF for protein aggregate clearance assay. (b-d) Representative confocal micrographs of (b) WT, (c) ΔHECT and (d) ΔANK MEF subjected to protein aggregate clearance assay by pulsing with puromycin for 4 h to induce protein aggregate formation followed by an 8-h chase to monitor for aggregate clearance. Vehicle-treated MEFs were used as control. Scale bars, 10 μm.
Figure 7(A, B) Representative immunohistochemical images of LC3 staining in skeletal muscle (A) and liver (B) after 80 min exercise in WT and PAQR3-deleted mice. Scale bar in (A): 20 μm. Scale bar in (B): 50 μm. (C, D) Immunoblotting analysis of skeletal muscle (C) and liver (D) from WT or PAQR3 knockout mice at rest or after 80 min exercise. (E) Abnormal limb-clasping of PAQR3 knockout mice when suspended by its tail. Quantification scoring of the limb clasping phenotype is shown on the right (n = 7 for each group; **p < 0.01). (F) Motor performance was measured using an accelerating rotarod apparatus, and the time of each mouse spent on the rotating rod until it fell off was recorded. Average time on the rod for each group is shown (n = 7 for each group; **p < 0.01). (G) Co-immunoprecipitation of Beclin1 or ATG14L with their binding partners in liver tissues from mice of indicated genotype. Immunoprecipitation (IP) and immunoblotting (IB) were performed using the antibodies as indicated. (H) The supernatants of liver lysates form WT or PAQR3 knockout mice were subjected to gel filtration analysis, followed by IB. (I) VPS34 complexes immunopurified with Beclin1 or ATG14L antibody in mouse liver were subjected to a quantitative PI(3)P ELISA assay (n = 5; **p < 0.01). (J) A schematic model of PAQR3 regulation on autophagy in response to glucosestarvation.
Hsf1 binds with high affinity but highly dynamic to heat shock elements (HSEs) and is dissociated from DNA by Hsc70 and DnaJB1.Figure 1A Domain structure of mammalian Hsf1. Intrinsically disordered regions are indicated with cylinders of smaller diameter. DBD, DNA binding domain; HR-AB, heptad repeat regions A and B; RD, regulatory domain; HR-C, heptad repeat region C; TAD, transactivation domain. B Equilibrium titration of Hsf1 binding to Alexa Fluor® 488-labeled HSE-DNA. Fluorescence polarization plotted versus the Hsf1 trimer concentration. KD = 4.7 ± 1.9 nM (n = 4). C Hsf1 rapidly switches between different HSE-containing double stranded DNA oligonucleotides. Trimeric Hsf1 was pre-incubated with Alexa Fluor® 488-labeled HSE-DNA in MgCl2-free buffer. At timepoint 0 (arrow) buffer, 4 mM MgCl2, 2 mM ATP+4 mM MgCl2, control DNA (DNACtrl at 10-fold molar excess over HSE-DNA), and HSE-containing DNA was added as indicated and fluorescence polarization followed over time. D Trimeric Hsf1 were bound to HSE-DNA and the indicated components added at timepoint 0. E Hsp70 dissociates Hsf1 from DNA with similar rates as Hsc70. Rates were determined The rate of Hsc70/DnaJB1-mediated dissociation of Hsf1 from HSE-DNA depends on temperature (for 25°C n = 6, for 37°C n = 5, T-test, p = 0.011) (F) The rate of Hsc70/DnaJB1-mediated dissociation of Hsf1 from HSE-DNA depends on concentration of Hsc70 and DnaJB1 (n = 3) (G) The rate of Hsc70/DnaJB1-mediated dissociation of Hsf1 from HSE-DNA depends on concentration of ATP (n = 3) (H) The rate of Hsc70/DnaJB1-mediated dissociation of Hsf1 from HSE-DNA depends on concentration of the nucleotide exchange factor Apg2 (HSPA4) (n = 3, ANOVA, **, p < 0.01; ****, p < 0.0001) (I). Shown are mean ± SD.
Figure 3A. Distribution of repeat elements around the transcription start site (TSS) regions of the SATS isoforms (SATS), common isoforms (common) and RefSeq genes (RefSeq). The Y-axis represents the proportion of isoforms (or genes) with each type of repeat element around their transcription start site regions. LTR, Long Terminal Repeat; SINE, Short Interspersed Nuclear Element; LINE, Long Interspersed Element; S_R, Simple Repeat; L_C, Low Complexity region; others, other types of repeats; NA, without repeat sequence. B. Expression comparison of the SATS isoforms (SATS), common isoforms (common) and Refseq genes (Refseq) in mESCs. The Y-axis represents the log2 transformed average FPKM values of each isoform or gene in three mESC lines (F145, F146 and F147). The FPKM values in each cell line was calculated by the reads mapped to the first exon and normalized by the exon length. Red dots represent the mean expression level of isoforms or gene in each group. The SATS and common isoforms from the same gene are connected by grey lines. C. Expression comparison of SATS genes in mESCs and somatic cells. The X-axis and Y-axis represent the log2 transformed average FPKM values of each gene of three mESC lines (F145, F146 and F147) and three somatic cell lines (ADSC, NSC, TTF), respectively. Big red dots, blue dots, yellow small dots represent SATS genes with > 2-fold expression difference and p-value < 0.05 (Two-tailed Student's t test), SATS genes with < 2-fold expression changes, and other genes with > 2-fold expression difference and p-value < 0.05 (Two-tailed Student's t test) between mESCs and somatic cells, respectively. R represents the Pearson correlation coefficient of gene expression between mESCs and somatic cells. D. Relationship between nucleotide and amino acid changes of the mESC-specific SATS isoforms as compared to corresponding common isoforms. Plus values represent nucleotide/amino acid expansions in the SATS isoforms, minus values represent nucleotide/amino acid deletions. The top and right boxplots are the quantile plots of nucleotide changes or amino acid changes, respectively. Black bars within the quantile box represent the median of changes. E. Examples of domain changes in proteins encoded by the SATS isoforms. Regions encoded by the common isoforms but not by the SATS isoforms are marked by red-dashed boxes. Numbers represent the orders of amino acids in the protein. F. Gene Ontology enrichment analysis of mESC-specific SATS genes. Circles are the enriched biological processes (p-value < 0.01, two-sided hypergeometric test with Benjamini-Hochberg correction) among SATS genes identified by ClueGO. The names of processes and their related GO terms are shown in the same colors. Circles are connected according to the hierarchical relationships of GO terms. The sizes of circles are negatively correlated with the enrichment p-values of GO terms.
Nucleosome stability at subtelomeres is linked to heterochromatic silencing.Figure 6A, B RT-qPCR analysis of transcript levels of TERRA (A) and tlh1+ (B) in WT strain and mutants as indicated (double mutants with ccq1Δ are indicated by blue dot). Data information: In (A, B), data is represented as mean ± SEM from 3 independent experiments and shown relative to WT leve
Figure 2(A) Representative H&E stained spleen, liver and kidney sections from 9-day old Nlrp3NeoR-A350V/WT and Nlrp3Mac-A350V/WT littermates. Scale bars 100 μm. (B-D) IL-1β and IL-18 protein levels measured in (B) spleen, (C) liver and (D) kidney homogenates from 6- to 9-day old indicated mice. Data information: Statistics in (B-D) were analyzed by student t-test (IL-18 graphs in B, C, D and IL-1β graph in D) or Mann-Whitney test (IL-1β graphs in B and C). Dots represent individual biological replicates. Error bars represent means ± SD. ns not significant; * p<0.05.
Increase in intracellular calcium [Ca2+]i concentration in HEK‐293T cells co‐expressing GC‐G and CNGA3 in response to cool temperaturesHEK‐293T cells expressing CNGA3 alone (upper panel) or together with GC‐G (full‐length in the middle panel and H+CYC domain in the lower panel) were loaded with the Ca2+ indicator Fura‐2 to monitor intracellular [Ca2+]i concentration.Figure 5HEK‐293T cells expressing CNGA3 alone (upper panel) or together with GC‐G (full‐length in the middle panel and H+CYC domain in the lower panel) were loaded with the Ca2+ indicator Fura‐2 to monitor intracellular [Ca2+]i concentration. Cells co‐expressing GC‐G and CNGA3 showed a rapid increase in [Ca2+]i when the temperature was lowered from 37 to 15°C (middle and lower panel); cells only expressing CNGA3 are not responsive to cool temperatures (top panel). Removal of extracellular Ca2+ (−Ca2+; middle panel) completely suppressed the response to cooling. ΔF represents changes in the ratio of the fluorescence intensity of Fura‐2 at 340/380 nm excitation. The ratiometric Ca2+ responses are representative of 30 cells recorded from three experiments.
Figure 4(A) LDs accumulate in touch neurons in atgl-1(xd314) visualized by SRS (Stimulated Raman Scattering). The zdIs5 reporter marks touch neurons. The white arrows indicate LDs in touch neurons in atgl-1(xd314). Scale bar: 10 μm. (B) atgl-1(xd314) and lid-1(xd288) show a gentle touch sensation defect, which is rescued by the neuron-specific expression of atgl-1 and lid-1, respectively. The fat-4 mutation enhances the touch sensation defect in atgl-1(xd314) and lid-1(xd288). Wild type and mec-4(u253) act as the negative control and positive control, respectively. The data were analyzed using Kruskal-Wallis test with Dunn's test. Asterisks denote significant differences as compared to wild type. ns: not statistically significant. **P < 0.01, ***P < 0.001, ****P < 0.0001. Data show mean ± SEM. # signify significant differences between the groups under the crossbars. ##P < 0.01. Data show mean ± SEM. Number of worms analyzed for each strain n =25. (C) The pathway of PUFA synthesis in C. elegans. LA: linoleic acid. ALA: alpha-linolenic acid. GLA: gamma-linoleic acid. STA: stearidonic acid. DGLA: dihomo-gamma-linoleic acid. ETA: eicosatetraenoic acid. AA: arachidonic acid. EPA: eicosapentaenoic acid. Fatty acids are also indicated by chemical abbreviations. For example, the abbreviation C18:2n-6 means 18 carbons with two double bonds and the first double bond is located at ω-6.
a) Suppression of USP33 facilitates TBK1–SEC5 complex formation.figf4(a) Suppression of USP33 facilitates TBK1-SEC5 complex formation. Flag-tagged SEC5 was introduced into HEK293T cells expressing empty vector (V) or HA-tagged RALB-G23V together with GFP shRNA or USP33 shRNA as indicated, and then immunoprecipitated with anti-Flag (M2) agarose followed by immunoblotting using antibody specific for TBK1. (c) RALB ubiquitylation at Lys 47 is crucial for formation of the RALB-SEC5 complex in response to poly(I:C) treatment. Flag-tagged SEC5 and the indicated HA-tagged RALB mutants were introduced into 293-hTLR3 cells. At 48 h after transfection cells were treated 100 μg ml−1 of poly(I:C) for 6 h. Flag-tagged SEC5 was immunoprecipitated with anti-Flag (M2), and analysed for co-precipitation by immunoblotting using anti-HA antibody. (c) RALB ubiquitylation at Lys 47 is crucial for formation of the RALB-SEC5 complex in response to poly(I:C) treatment. Flag-tagged SEC5 and the indicated HA-tagged RALB mutants were introduced into 293-hTLR3 cells. At 48 h after transfection cells were treated 100 μg ml−1 of poly(I:C) for 6 h. Flag-tagged SEC5 was immunoprecipitated with anti-Flag (M2), and analysed for co-precipitation by immunoblotting using anti-HA antibody. (d) RALB ubiquitylation at Lys 47 affects activation of TBK1 and IRF3. At 48 h after transfection with empty vector (V), Flag-RALB-G23V and Flag-RALB-G23V-K47R, 293-hTLR3 cells were treated with 10 μg ml−1 of poly(I:C) for 6 h. Immunoblot analysis was performed using the indicated antibodies. Uncropped images of blots are shown in Supplementary Fig. S7.
Figure 4Distribution of the plasma membrane reporter PH::GFP in control cells (A-A'') and in cells where dynamin activity had been blocked using a temperature-sensitive allele of dynamin (shibirets In (B), was inactivated at the onset of tube formation (C-D) Distribution of PH::GFP fluorescence intensity in control and shibirets cells. Data from 1-2 minute-interval time lapses was collected in windows of 20 minutes each (except for t=0). (C) Proportion of signal in the apical and in the basal membrane compartment over time in control cells (n=4) and also in cells where dynamin was inactivated (n=5), *p=0.015, Mann-Whitney test. (D) Total fluorescence intensity of PH::GFP over time in controls (n=4) and in cells where dynamin was inactivated (n=5), n.s.: p=0.063, Mann-Whitney test. Distribution of the plasma membrane reporter PH::GFP in cells where dynamin activity had been blocked using a temperature-sensitive allele of dynamin (shibirets in (E) it was inactivated ~15 minutes after the tube had begun to form. (F-G) Distribution of the luminal reporter Gasp::GFP in control cells (F) and in cells where dynamin was inactivated (G). Arrowheads point to protrusions sprouting from the lumen of the terminal cell. (H-I) Distribution of the general plasma membrane marker CD4::mIFP in combination with the Clathrin light chain fused to GFP (CLC::GFP, H) and with Dynamin::YFP (I).
Figure 4(A) Representative F4/80 immunohistochemical macrophage (brown) staining of spleen, liver and kidney sections from 9-day old Nlrp3NeoR-A350V/WT and Nlrp3Mac-A350V/WT littermates. Scale bars 100 μm. (B-D) Flow cytometric CD11b+F4/80+CD64+ macrophage quantifications in (B) spleen, (C) liver and (D) kidney from indicated 6-day old mice. Data information: Statistics were analyzed by student t-test (B, D, or Mann-Whitney test (C, Dots represent individual biological replicates. Error bars represent means ± SD. ns not significant; * p<0.05; ** p<0.01. (E-G) Flow cytometric CD11b+Ly6C+Ly6G- monocyte quantifications in (E) spleen, (F) liver and (G) kidney from 6-day old indicated mice. Data information: Statistics were analyzed by student t-test , E, F, G Dots represent individual biological replicates. Error bars represent means ± SD. ns not significant; * p<0.05; ** p<0.01. (H-J) Flow cytometric CD11b+Ly6C-Ly6G+ neutrophil quantifications in (H) spleen, (I) liver and (J) kidney from 6-day old indicated mice. Data information: Statistics were analyzed by student t-test J) or Mann-Whitney test , H and I). Dots represent individual biological replicates. Error bars represent means ± SD. ns not significant; * p<0.05; ** p<0.01.
Figure 7Once the E-S complex is formed, the first endopeptidase cut strongly destabilizes the helical transmembrane domain of APPC99, leading to the unwinding of the most C-terminal helical part of the Aβ substrate and providing the length to the "de novo" generated Aβ substrate to reach the active site (model originally proposed in (Szaruga et al, 2017)). The further unwinding of the Aβ substrate with each sequential cleavage, stretches the substrate and provides the length to fill the catalytic pockets but weakens the GSEC-Aβn interaction, until the eventual E-S dissociation triggers Aβ release. Here, we propose that the extracellular interface that includes NCT (241/242), APP (K28) and the first extracellular loop of PSEN1 anchors GSEC-APP/Aβn complexes during the sequential proteolytic mechanism, and the E-S interface is the target of selected imidazole-based GSMs.
Figure 6UCSC Genome Browser custom tracks of m6A-seq reads along the indicated mRNAs in WT, Mettl14 KO, KO+WT, and KO+RK mESCs. The y-axis represents the normalized number of reads. Blue reads are from non-immunoprecipitated input libraries, and red reads are from m6A-IP libraries. Above the custom tracks, the thick blue boxes represent the protein coding regions (CDSs), the thin blue boxes represent the untranslated regions (UTRs), and the blue lines represent introns. The bars at the bottom of the custom tracks indicate the amplicon locations for MeRIP (m6A-IP)-qPCR assays (B) and METTL14 RIP-qPCR assays (C) to detect m6A-positive (red) and negative (blue) regions. MeRIP (m6A-IP)-qPCR assays were performed for WT, Mettl14 KO, KO+WT, and KO+RK mESCs to validate the MeRIP-seq results. m6A-negative regions of the transcripts (blue) were included as negative controls. : In (B) , data from three independent replicates were analyzed by Student's t-test and shown as mean ± SD. *, p < 0.05; **, p < 0.01, ***, p < 0.001. METTL14 RIP-qPCR assays were performed for WT, Mettl14 KO, KO+WT, and KO+RK mESCs to compare the binding of WT and RK mutant METTL14 to mRNA targets. Primers (red color) that amplify m6A positive regions of the transcripts were used. In , (C), data from three independent replicates were analyzed by Student's t-test and shown as mean ± SD. *, p < 0.05; **, p < 0.01, ***, p < 0.001. The expression of ICL repair genes is reduced in mESCs expressing arginine methylation-deficient mutant (RK) METTL14. Total cell lysates from WT, Mettl14 KO, KO+WT, and KO+RK mESCs were subjected to Western blot analysis using the indicated antibodies. Polysome profiling was performed for KO+WT and KO+RK mESCs. Whole-cell extracts were fractionated through centrifugation in a sucrose density gradient. Optical scans (OD260) of the collected fractions are shown. Quantification of ribosome-bound mRNA for the indicated genes from individual fractions (as in (E)), relative to the amount of the total mRNA in all fractions. Gapdh was included as a negative control. : In (F), data from three independent replicates were analyzed by Student's t-test and shown as mean ± SD. *, p < 0.05; **, p < 0.01, ***, p < 0.001.
a) Ulk1 Ser 757 is required for mTORC1 to regulate the interaction of Ulk1 with AMPK in vivo.figf6(a) Ulk1 Ser 757 is required for mTORC1 to regulate the interaction of Ulk1 with AMPK in vivo. CBP/SBP tagged Ulk1 (wild type or S757C) was co-transfected with HA-AMPKα and Rheb into HEK293 cells as indicated. Ulk1 was purified by streptavidin beads and the co-precipitatedHA- AMPKα was examined by western blot (Rapa, 50 nM rapamycin treatment for 1 h before cell lysis). (b) Ulk1 Ser 757 is required for rapamycin to enhance the Ulk1-AMPK interaction in vitro. CBP/SBP Ulk1 proteins (wild type or S757C) were prepared from transfected HEK293 cells, which were pre-incubated with 50 nM rapamycin (Rapa, 1h) as indicated. The Ulk1 proteins were purified by streptavidin beads and the resulting Ulk1-bead was incubated with the bacterial purified AMPKα/β/γ complex. AMPKα protein levels in the in vitro pulldown assays were examined by western blot using AMPKα antibody. L.E.; long exposure. (c) Phosphorylation of AMPK sites Ser 317 and Ser 777 in Ulk1 are decreased in Tsc1−/− Tsc1. Tsc1+/+ (WT) and Tsc1−/− (KO) MEFs were starved of glucose (4 h), or treated with 50 nM rapamycin (Rapa, 1 h). Ser 317 and Ser 777 phosphorylation of endogenous Ulk1 was examined by western blotting with antibodies against Ulk1 phosphorylated at Ser 317 or Ser 777. (d) Rheb suppresses Ulk1 Ser 317 and Ser 777 phosphorylation in a manner dependent on mTORC1. HA-Ulk1, AMPKα kinase-dead mutant (DN), and Myc-Rheb were co-transfected into HEK293 cells as indicated. The cells were incubated with glucose-free medium (-Glu, glucose), in which either 20 μM compound C (C.C.) or 50 nM Rapamycin (Rapa) was added. Total cell lysates were probed with antibodies against Ulk1 phosphorylated at Ser 317, Ser 777, Ser 757, and HA, as indicated. (e) Rheb inhibits glucose starvation-induced Ulk1 activation. HA-Ulk1 and Myc-Rheb was transfected into HEK293 cells, which were incubated with glucose-free (-Glu), amino-acid-free (-A.A) medium, or 50 nM rapamycin (Rapa) for 4 h before lysis. HA-Ulk1 was immunoprecipitated and kinase assays were performed. Ulk1 activity was measured by 32P-autoradiogram and the protein level of HA-Ulk1 and GST-Atg13 used in the assay was determined by western blot and by Coomassie staining, respectively. Uncropped images of blots are shown in Supplementary Fig. S5.
a) Endogenous immunoprecipitation of NCOA4 with ferritin heavy (FTH1) and light (FTL) chains from DLD1 cell lysates.figf5(a) Endogenous immunoprecipitation of NCOA4 with ferritin heavy (FTH1) and light (FTL) chains from DLD1 cell lysates. b) Steady-state levels of FTH1 and FTL expressed in DLD1 cells growing in rich medium or starved for the times indicated (hours, h). (c) DLD1 cells were cultured in the presence or absence of bafilomycin A1overnight, fixed and processed for confocal immunofluorescence microscopy to determine the cellular distribution of endogenous FTH1 or FTL with LC3, LAMP2 or EEA1. Regions outlined with white dashed lines are magnified to the right of each panel. Scale bars in full and zoomed panels correspond to 10 and 2 μm respectively. (d) DLD1 parental or NCOA4−/− cells (clones 1 and 2; KO-1, KO-2) were starved for 6 h and the levels of NCOA4, FTH1, FTL and GAPDH determined. (e) DLD1 parental or NCOA4−/− cells were cultured in the presence or absence of bafilomycin A1overnight, fixed and processed for confocal immunofluorescence microscopy to determine the cellular distribution of endogenous ferritin (FTH1-FTL) and LAMP2. Regions outlined with white dashed lines are magnified to the right of each panel. Scale bars of full and zoomed panels correspond to 10 and 2 μm respectively. Uncropped images of blots are shown in Supplementary Fig. 7.