EMBO/sd-character-level-ner huggingface dataset:
a) Accumulation of MRs in cells treated with the PI(3)K inhibitor 3-MA.figf3(a) Accumulation of MRs in cells treated with the PI(3) K inhibitor 3-MA. Left panels show HeLa cells treated with 3-MA (4 mM) for 3 days. Cells were fixed and stained. Images are colour-inverted for better visualization. The quantification of this experiment is shown on the upper right (mean ± s.d. of three independent experiments; n = total number of cells counted). The lower right panel shows an enlarged area of 3-MA-treated cells in true colour. Scale bars are 20 μm. (b) siRNA-mediated depletion of Atg6 (Beclin-1) and Atg7 from HeLa cells causes MR accumulations. The right panel shows the quantification of MRs per cell (mean ± s.d. of three independent experiments); the left panel the efficiency of Atg6 and Atg7 depletion. (c) Inhibition of lysosomal acidification leads to MR accumulation. Panels on the left show HeLa cells left untreated or treated with chloroquine (20 μM) or ammonium chloride (10 mM) for 3 days. Scale bar are 20 μm. Cells were fixed and stained as indicated. A quantification of this experiment is shown on the left (mean ± s.d. of three independent experiments; n, total number of cells; P, probability of a two-tailed paired t-test).
(A,B) Western blotting analyses of the levels of autophagy-related proteins in BAT from mice at the normal temperature (30℃) or at 4℃ for 72 h (A).Figure 4(A,B) Western blotting analyses of the levels of autophagy-related proteins in BAT from mice at the normal temperature (30℃) or at 4℃ for 72 h (A). Expression of the indicated proteins was analyzed before and after 72 h cold exposure (4℃) (B). (C) Representative images from immunofluorescence staining of LC3B and CYCS in BAT from mice at the normal temperature (30 ℃) or 4℃ for 72 h. (D Representative transmission electron microscope (TEM) images of BAT cells isolated from mice at the normal temperature (30℃) or 4℃ for 72 h. Scale bar, 1 μm (D).The number of mitophagosomes per 100 mitochondria was counted and normalized (E). (F,G) BATs from mice exposed to normal conditions (30℃) or cold (4℃) were lysed and immunoprecipitated with antibodies against FUNDC1, LC3B or IgG, then immunoblotted with the indicated antibodies. The ratio between immunoprecipitated LC3B to FUNDC1 or FUNDC1 to LC3B were quantified and plotted (G).
Differential expression of individual TEs.Figure 5(a) Increased expression of TEs in stem cells of D7 seedlings according to qRT-PCR. Box plots outline the interquartile range (IQR) with the median and whiskers +/- 1.5 IQR. N = 2 (ATLANTYS3, ATCOPIA39, ATCOPIA49, ATCOPIA29, ATGP1-1), N = 3 (VANDAL6, ATGP2, ATGP1-2, ATLINE1, ATGP1-3), N = 4 (ATHILA6A, ATCOPIA83), N = 5 (VANDAL12, ATHILA3, ATCOPIA22), and N = 6 for CLAVATA. *p<0.01, **p<0.001 (t-test). (b) Heatmap of TE expression in several silencing mutants determined by qRT-PCR relative to a housekeeping gene. To be able to display data in log-scale also for samples with zero expression, a value of 1.0e-6 (factor 10 smaller than the smallest value) was added to all values. Data for nuclei and CLV3::H2BmCherry are the same as in (a). For seedling samples, N = 2 (ATCOPIA49, ATHILA3), N = 3 (ATCOPIA39, VANDAL6, ATGP2, ATLINE1, ATCOPIA29, ATGP1-1), and N = 4 (ATLANTYS3, VANDAL12, ATGP1-2, ATHILA6A).
LINC00941 and p63 control SPRR5, which is induced during differentiation and different from other human SPRRs A Differentially expressed genes in day 3 LINC00941 depleted epidermal tissue cluster within the epidermal differentiation complex (EDC) (n = 4-5 biological replicates/knockdown group).Figure 3A Differentially expressed genes in day 3 LINC00941 depleted epidermal tissue cluster within the epidermal differentiation complex (EDC) (n = 4-5 biological replicates/knockdown group). B LINC00941 knockdown on day 3 in calcium-induced keratinocyte differentiation cultures results in increased expression of SPRR5 (FC = fold change) (n = 3 biological replicates/knockdown group). Data is presented as mean ± SD. Statistical significance was tested by an unpaired t-test and corrected for multiple testing after Bonferroni (* = adj. p-value < 0.05, ** = adj. p-value < 0.01, *** = adj. p‑value < 0.001, and n.s. = not significant). C Detected fragments per million (FPM) for SPRR5 in control (siCtrl) as well as LINC00941 deficient (siLINC00941) epidermal tissue for all biological replicates on day 3 (D3) of differentiation as obtained by DeSeq2 (FC = fold change) (n = 4‑5 epidermal tissue cultures/knockdown group). SPRR5 induction during the course of keratinocyte (KC) differentiation is shown by northern blot (D) for four different primary keratinocyte isolates and compared to undifferentiated keratinocytes (n = 8 biological replicates). Data is presented as mean ± SD. SPRR5 induction during the course of keratinocyte (KC) differentiation is shown by qRT-PCR analysis (E) for four different primary keratinocyte isolates and compared to undifferentiated keratinocytes (n = 8 biological replicates). Data is presented as mean ± SD. F p63 knockdown on day 3 of keratinocyte differentiation leads to decreased SPRR5 transcript levels (FC = fold change) (n = 3-4 biological replicates /knockdown group). Data is presented as mean ± SD. Statistical significance was tested by an unpaired t-test and corrected for multiple testing after Bonferroni (* = adj. p-value < 0.05, ** = adj. p-value < 0.01, *** = adj. p‑value < 0.001, and n.s. = not significant).
FAK-dependent Akt-mTOR signaling inhibits the recruitment of LC3 to SCVs.Figure 4(A) WT and FAK−/− PEMs were incubated with S. typhimurium strain ΔinvG for 0-24 hours before immunoblotting with the indicated antibodies. (B) WT PEMs were incubated with Akt inhibitor AKTV/triciribine (10 µm) for 30 minutes before infection with S. typhimurium strain ΔinvG for 5 hours. Lysates were immunoblotted with the indicated antibodies. ( . (C) WT and FAK−/− PEMs were incubated with S. typhimurium strain ΔinvG for a total of 5 hours before analysis with antibodies recognizing LC3. DAPI was used to visualize nuclei and bacteria. Bars represent 10 µm. White boxes show enlarged regions in inset panels, bars represent 2 µm. White arrowheads indicate LC3-negative bacteria, yellow arrow heads indicate LC3-positive bacteria (panels c, f). Yellow arrowhead in panel f denotes an LC3+ cluster of bacteria. (D) The percentage of ΔinvG (first grouping) or ΔΣΠΙ1ΔΣΠΙ2 (second grouping) Salmonella co-localizing with LC3 at the indicated time points is displayed. At least 100 bacteria were counted per condition per time point. Values are means ± SEM, N = 3, *p<0.05. (E) WT and FAK−/− PEMs were incubated with S. typhimurium strain ΔinvG for 5 h with or without Bafilomycin A1 (300 ng) before immunoblotting with the indicated antibodies. Vertical white line indicate non-contiguous lanes generated from a single exposure. (F) FAK−/− PEMs were incubated with DQ-BSA-red for 1 hour before infection with S. typhimurium strain ΔinvG for 5 h. Cells were co-stained for LC3 and DAPI was used to visualize nuclei and bacteria. White boxes show enlarged regions in inset panel. G) WT PEMs were pretreated with the mTORC1 inhibitor rapamycin (4 µm) for 1 hour before incubation with ΔinvG Salmonella for a further 5 hours. Cells were lysed before immunoblotting with the indicated antibodies. (H) WT and FAK−/− PEMs were pretreated with rapamycin (4 µm), Akt inhibitor AKTV/triciribine (10 µm) or left untreated (-Tx) or before infection with S. typhimurium strain ΔinvG for a further 5 hours. WT macrophages were also pretreated with the FAK inhibitor PF228 (0.5 µm) for 1 hour prior to incubation with ΔinvG Salmonella for 5 hours. Cells were then assessed for the percentage of ΔinvG Salmonella co-localizing with LC3. At least 100 bacteria were counted per condition. Values are means ± SEM, N = 3, *p<0.05. N.s. not significant.
Figure 5A. Schematic overview for correcting trisomic cells. B. Karyograms of Di8, Di11 and Di15 ES cell lines. Di8 was the corrected ES cell line from Ts8, showing diploid karyotype and Y chromosome loss. Di11 and Di15 were from Ts11 and Ts15 ES cells respectively. C. AP staining of trisomic and the corrected diploid ES cells after LIF withdrawal (LIF 0 U/ml) for 5 days. Scale bar, 500 μm. D. Teratomagrowth curves of trisomic and the corrected diploid ES cells. The injected cell number was 5×105/site (8 sites/per group). Error bars, ±S.D. ** P < 0.01. E. Scatterplot of four-week teratoma weight. The teratomas were formed by the corrected diploid ES cells and their paternal trisomic ES cells. * P < 0.05.
Model for a role of Dyn2 in autophagic breakdown of lipid droplets in hepatocytes.Figure 8.Model for a role of Dyn2 in autophagic breakdown of lipid droplets in hepatocytes. After initiation of starvation, lipid droplets are enclosed by the double-membrane isolation membrane (phagophore), which engulfs the droplet, forming an autophagosome. Subsequent fusion with lysosomes results in the release of hydrolytic enzymes and lysosomal lipases into the autolysosomal compartment, which degrade the lipid droplets within. During the process of autophagic lysosomal reformation (top), nascent lysosomes are generated from membrane tubule extensions of this autolysosomal compartment. Dyn2 mediates the scission of these tubular structures. Depletion of Dyn2 by siRNA knockdown or inhibition of Dyn2 activity by pharmacological reagents (bottom) prevents tubular scission, eventually resulting in depleted lysosomal pools within the cell. Continued fusion of remaining lysosomes causes enlargement of the autolysosomal structures. A complete decrease in the recycled lysosomal population will prevent autophagic-mediated breakdown of hepatic lipid droplets.
Figure 6Immunoblot analysis of C99 V44Bpa crosslinking (CL) to PS1 WT or NTFs (circle) of normally endoproteolysed mutants in the presence of vehicle (DMSO) or RO7019009. Quantitation of crosslink efficiencies of C99 V44Bpa to the WT or mutant NTF in (A). Immunoblot analysis of C99 V44Bpa crosslinking to full-length (FL) PS1 (triangle) of endoproteolysis-deficient mutants and the corresponding M292D control in the presence of vehicle (DMSO) or RO7019009. Quantitation of crosslink efficiencies of C99 V44Bpa to the M292 or FAD mutant FL PS1 in (C).
F on RNAPII genomic stalling and transcription read-through.Figure 6A schematic illustrating the effect of transcription speed on RNAPII stalling at promoter proximal regions. Accelerated RNAPII is prone to move out of stalling regions (centered at the position of the first nucleosome) at 5' end of genes, resulting in reduced promoter proximal stalling peaks in RNAPII profile by plaNET-seq. A schematic illustration showing that accelerated RNAPII tends to evade from RNAPII stalling near gene poly-(A) sites (PAS). This leads to less efficient transcription termination and extended transcription read-through, reflected by the absence of RNAPII signal peaks downstream of PAS and elevated signal downstream of PAS stalling region by plaNET-seq.
Mitochondrial stress mediates germline apoptosis and lifespan through different cell non-autonomous mechanisms.Figure 8A Kaplan-Meier survival curves of wild-type (WT) and, rrf-1(pk1417), ppw-1(pk2505) strains fed bacteria transformed with either empty-vector (con) or with vector expressing dsRNA against isp-1 B Quantification of apoptotic corpses in wild-type (WT) and, rrf-1(pk1417), ppw-1(pk2505) strains fed as (A) and left untreated or treated with 600 J/m2 UVB. Data information: Data are presented as mean ± SEM, N=3, 9-10 worms per replicate and condition ** P < 0.01 vs con; ## P < 0.01 vs WT; ooo P < 0.00 and oooo P < 0.0001 vs untreated . Not significant if nothing is specified. 2-way ANOVA Tukey's multiple comparisons test. C C In the wild-type animals under normal conditions a signal from the germline limits lifespan D Systemic mitochondrial stress concurrently triggers a pro-longevity signal from the soma (independent from the germline) and a cell-autonomous germline signal partially inhibiting the limiting signal from the gonad, ultimately resulting in significant lifespan extension in the wild-type and further extending lifespan in germline less strains. Moreover, it induces a soma-to-germline signal that protects against IR-induced apoptosis in a brc-1/brd-1 dependent manner. In a germline competent strain, somatic isp-1 RNAi would only trigger the pro-longevity signal in the soma, which in the presence of the lifespan limiting effects from the germline turns into detrimental effects on lifespan. When isp-1 is instead only silenced in the germline, the anti-apoptotic effect is lost, while the absence of the pro-longevity signal from the soma and the partial suppression of the limiting signal in the germline would result in a lifespan extending effects, yet reduced compared to systemic MRC stress
Figure 2Analyses of the interaction between β-catenin and RAS. WCLs of DLD1 cells were immunoprecipitated with β-catenin or pan-RAS antibody and subjected to IB analyses IB analyses of indicated proteins in the samples immunoprecipitated with anti-β-catenin antibody with WCLs of HEK293 cells treated with Wnt3a-CM Left panel: MT-HCT116 cell extracts were fractionated as cytosolic and nuclear portions and subjected to IB analyses, respectively. Right panel: The cytosolic and nuclear fractions were immunoprecipitated with anti-β-catenin antibody and subjected to IB analyses β-Catenin-RAS interaction was visualized by in situ proximity ligation assay in HEK293 cells treated with L-CM or Wnt3a-CM. Scale bars, 20 µm. The proximity ligation signal was quantified in 10 randomly distributed microscopic images that contained > 100 cells In vitro pulldown analyses to identify β-catenin binding region of RAS. Upper panel: Schematic of the HRAS full-length (FL) or two deletion mutants. Lower panel: Purified GST-HRAS-FL, -ΔHVR, or -Δ131-165 proteins were pulled-down with purified His-β-catenin-FL protein and subjected to IB analyses IB analyses of IP with FLAG antibody in WCLs of HEK293 cells cotransfected with FLAG-β-catenin and Myc-HRAS-FL, -ΔHVR, or -Δ131-165 for 32 hr IB analyses of IP with FLAG antibody in WCLs of HEK293 cells cotransfected with FLAG-β-catenin and Myc-HRAS-WT, -2A, or -2E for 32 hr Upper panel: Schematic of the β-catenin-FL or two deletion mutants. Lower panel: Purified His-β-catenin-FL, ΔN86, or ΔC110 proteins were pull-down with purified GST-HRAS-FL protein and subjected to IB analyses IB analyses of IP with Myc antibody with WCLs of HEK293 cells cotransfected with Myc-HRAS and GFP-β-catenin-FL, -ΔN86, or -ΔC110 for 32 hr
Figure 2Sw.71 (left panel) and HPC cells (right panel) were treated with IFNβ to detect ISG20 mRNA expression. Data represent as mean ± SEM; n=3 biological replicates. NT, no treatment group; HPC, human primary culture. (A) Sw.71 and HPC cells were treated with different doses of IFNβ (3, 30, 300 ng/ml) for 8 h and RNA were collected for determining ISG20 mRNA expressions by qRT-PCR. Note the increase of ISG20 mRNA expression in a dose-dependent manner. *p < 0.05, **p < 0.01 by One-way ANOVA. (B) Sw.71 and HPC cells were treated with 300ng/ml IFNβ over time and RNA was collected for determining ISG20 mRNA expressions by qRT-PCR. Note the increase of ISG20 mRNA expression in a time-dependent manner. *p < 0.05 by One-way ANOVA. (C) Sw.71 (left panel) and HPC cells (right panel) were treated with IFNβ to detect ISG20 protein expression. (C) Sw.71 and HPC cells were treated with different doses of IFNβ (3, 30, 300 ng/ml) for 24 h and protein was collected for determining ISG20 protein expressions by western blot. Note the increase of ISG20 protein expression in a dose-dependent manner. β-actin served as a loading control. ( Sw.71 (left panel) and HPC cells (right panel) were treated with IFNβ to detect ISG20 protein expression. (D) Sw.71 and HPC cells were treated with 300ng/ml IFNβ over time and protein was collected for determining ISG20 protein expressions by western blot. Note that there was no ISG20 protein expression in the no treatment group in both Sw.71 and HPC, only after IFNβ treatment, ISG20 protein expression exhibited a time-dependent manner. β-actin served as a loading control.
Figure 6A Ccdc50+/+ and Ccdc50-/- mice were fed with 2.5% DSS in drinking water for 8 days and changed to regular water for 4-5 days. Body weights were measured daily (n = 10 mice each group). Data information: Data are representative of at least five biological replicates and shown as the mean with SEM *P <0.05, ***P < 0.001; two-tailed unpaired Student's t-test. B Representative images of colons of DSS-treated Ccdc50+/+ and Ccdc50-/- mice (Ccdc50+/+ mice, n = 8 mice; Ccdc50-/- mice, n = 7 mice) C The scatter plot depicted the colon length of Ccdc50+/+ and Ccdc50-/- mice treated with DSS (Ccdc50+/+ mice, n = 8 mice; Ccdc50-/- mice, n = 7 mice). Data information: Data are representative of at least five biological replicates and shown as the mean with SEM *P <0.05, ***P < 0.001; two-tailed unpaired Student's t-test. D Hematoxylin and eosin (H&E)-stained colon tissues removed from Ccdc50+/+ and Ccdc50-/- mice treated as in (A) on day 11 (Scale bars, 1000 μm). The rectangle was zoomed in 10 times and was shown at the bottom. E qRT-PCR analysis of Il6 and IL-1β mRNA levels in livers (Ccdc50+/+ mice, n = 8 mice; Ccdc50-/- mice, n = 8 mice) from DSS-treated Ccdc50+/+ and Ccdc50-/- mice. F qRT-PCR analysis of Il6 and IL-1β mRNA levels in colons (Ccdc50+/+ mice, n = 5 mice; Ccdc50-/- mice, n = 6 mice) from DSS-treated Ccdc50+/+ and Ccdc50-/- mice. Data information: mRNA data are normalized to GAPDH and relative to untreated wild-type mice. Data are representative of at least five biological replicates and shown as the mean with SEM , ***P < 0.001; two-tailed unpaired Student's t-test. G Immunohistochemical staining for IL-1β, IL-6 and TNFα in the colon tissues of DSS-induced Ccdc50+/+ and Ccdc50-/- mice; nuclei were stained with DAPI (Scale bars, 100 μm).
Figure 3A qRT-PCR analysis of levels of VEGF mRNA in HUVEC treated with Control-Fc or Spike RBD-Fc. For each group, n=3. Data are from 3 independent biological experiments. Data information: All data are shown as mean ± SD. For (A) P values are determined by Paired Student's t-test B ELISA analysis of VEGF concentration in the supernatant from Caco-2 cells treated with Control-Fc or Spike-Fc. For each group, n=4. Data are from 4 independent biological experiments. Data information: All data are shown as mean ± SD. For (B), P values are determined by Paired Student's t-test; C Immunofluorescence analysis of VEGF, cytokeratin (CK) and CD34 protein in the intestinal tissues of COVID-19 patients. CK and CD34 staining in green, VEGF staining in red and nuclear staining in blue. Scale bars, 50 µm. D Levels of ERK and pERK in Caco-2 cells treated with Control-Fc or Spike RBD-Fc by Western blot. Protein expression was normalized to β-actin. Data are from 3 independent biological experiments. Data information: All data are shown as mean ± SD. For (D), P values are determined by Paired Student's t-test E ELISA analysis of VEGF concentration in the supernatant from Caco-2 cells treated with Control-Fc, Spike RBD-Fc, or Spike RBD-Fc combined with SCH772984. For each group, n=4. Data are from 4 independent biological experiments. Data information: All data are shown as mean ± SD. ; for (E), P values are determined by Mann-Whitney test; F, G Immunohistochemical staining shows ERK (E) and pERK (F) expression in the duodenum of COVID-19 patients and healthy controls. Scale bars, 100 μm. For each group, n=7. n, biologically independent samples (human specimen). Data information: All data are shown as mean ± SD. For (F) and (G), P values are determined by Student's t-test H Immunohistochemical staining shows ERK and pERK expression in the intestinal tissues of mice treated with Control-Fc, Spike RBD-Fc or Spike RBD-Fc combined with SCH772984. Scale bar, 100 µm. For each group, n = 4. Data information: All data are shown as mean ± SD. for (H) P values are determined by one-way ANOVA. I The levels of Ras, c-Raf, pMEK, ERK, pERK and p-P90RSK in Caco-2 cells treated with Control-Fc, Spike RBD-Fc or Spike RBD-Fc combined with SCH772984 by Western blot. Protein expression was normalized to β-actin. Data are from 3 independent biological experiments. Data information: All data are shown as mean ± SD. for (I), P values are determined by one-way ANOVA.
Functional analysis of incomplete penetrance (see also Table EV8).Figure 5Stacked bar graph with fractions of penetrance mutants belonging to each penetrance bin for the four endocytic markers. act: actin; LE: late endosome; vac: vacuole. SAFE (Spatial Analysis of Functional Enrichment) of penetrance mutants grouped according to penetrance. Top: Bioprocess key for interpreting the global similarity network for yeast genetic interactions visualized using SAFE, which identifies regions of the network enriched for specific biological processes (Costanzo et al., 2016). Coloured dots denote the localization of the 4 marker genes within the global similarity network. Below: SAFE of penetrance mutants grouped according to their penetrance and marker. Orange: genes whose mutation caused high penetrance; grey: intermediate penetrance genes; blue: low penetrance genes. Numbers in brackets refer to the number of unique ORFs in each group.
Figure 6(A) Cryo-EM reconstruction of the eEF2 bound 80S ribosome adopting a rotated POST-1 state bearing chimeric ap/P- and pe/E-tRNA as well as a disordered eEF3. (B) Subunit rotation and head swivel observed in the S. cerevisiae eEF3-80S complex derived from the selected class from the 3D classification shown in (A). 18S rRNA structures illustrating the degree of rotation of the small subunit relative to the large subunit based alignments of the large subunit from a non-rotated structure reference (PDB ID: 6SNT) Matsuo et al., 2020(). The degree of head swiveling (right side) is illustrated based on alignments of the 18S rRNA body from a non-headed swivel reference structure (PDB ID: 6SNT) Matsuo et al., 2020(). (C, D) 80S ribosome in rotated (C) PRE-3 state containing an A/A- and P/E-tRNA and (D) PRE-4 state bearing the hybrid A/P- and P/E-tRNA and bound by the disordered eEF3 ligand. (E, F) Representation of the rotation of the subunit and the 40S-head swivel shown for volumes depicted in (C) and (D), respectively.
Figure 3A Scatter plot comparing the transcripts of 2-cell embryos from WT and Pabpn1l-/- females. Transcripts that increased or decreased by more than 3-fold in Pabpn1l-deleted oocytes or embryos are highlighted in red or blue, respectively. B Venn diagram and schematic showing the overlap in transcripts that are activated during zygote-to-2-cell transition in WT (FPKM[2-cell/zygote] > 3) and the transcripts that are decreased in Pabpn1l♀−/♂+ compared to WT at the 2-cell stage (FPKM[WT/Pabpn1l♀−/♂+] > 3 in 2-cell embryos). C 5-Ethynyl uridine (EU) fluorescence (green) showing RNA transcription in WT and Pabpn1l♀−/♂+ 2-cell embryos. Some WT embryos were treated with α-amanitin as early as the zygote stage and cultured to the 2-cell stage. The phosphorylated RNA polymerase II CTD repeat YSPTSPS (pS2) (red) is co-stained to label the RNA polymerase II activity. Nuclei are labeled by DAPI (blue). Scale bar = 20 μm. D Quantification of EU and pS2 signals in (A). The numbers of analyzed embryos are indicated (n). Error bars, S.E.M. ***P < 0.001 by two-tailed Student's t-test. E Relative intensity of tdTomato signals in (F). The numbers of analyzed embryos are indicated (n). Error bars, S.E.M. ***P < 0.001 by two-tailed Student's t-test. F Representative images of MuERV-L::tdTomato relative to GFP signal in the same embryo when WT embryos reached the corresponding stages. Zygotes were injected with the MuERV-L::tdTomato reporter plasmid and polyadenylated Gfp mRNA (as a positive control of microinjection), then allowed to develop in vitro. Cultured embryos were imaged at 24 h and 40 h after hCG injection. DIC, differential interference contrast. Scale bar = 100 μm.