EMBO/sd-character-level-ner huggingface dataset:
Figure 7A) Binding of NDP52 to ubiquitin tetramers of different linkage types. Beads coated with the indicated GST-tagged proteins were incubated with pooled M1-, K63-, and K48-linked tetra-ubiquitin chains. Anti-ubiquitin Western blot of input and proteins bound to beads. B-D) Kinetics of S. Typhimurium replication in Tbk1-/- MEFs complemented with the indicated Flag-tagged TBK1ΔC fusion proteins. Infected cells were lysed at the indicated time points post inoculation (p.i.) and bacteria were enumerated by their ability to form colonies on agar plates. Mean and s.d. of triplicate MEF cultures and duplicate colony counts. Data are representative of at least two repeats. Statistical differences to Tbk1-/- MEFs expressing TBK1ΔC are shown. *p<0.5, **p<0.01, ***p<0.001 one-way ANOVA with Dunnett's multiple comparisons test. Western blot for Flag-tagged TBK1 variants in post nuclear cell lysates. B-D) Kinetics of S. Typhimurium replication in Tbk1-/- MEFs complemented with the indicated Flag-tagged TBK1ΔC fusion proteins. Infected cells were lysed at the indicated time points post inoculation (p.i.) and bacteria were enumerated by their ability to form colonies on agar plates. Mean and s.d. of triplicate MEF cultures and duplicate colony counts. Data are representative of at least two repeats. Statistical differences to Tbk1-/- MEFs expressing TBK1ΔC are shown. *p<0.5, **p<0.01, ***p<0.001 one-way ANOVA with Dunnett's multiple comparisons test. Western blot for Flag-tagged TBK1 variants in post nuclearcell lysates. B-D) Kinetics of S. Typhimurium replication in Tbk1-/- MEFs complemented with the indicated Flag-tagged TBK1ΔC fusion proteins. Infected cells were lysed at the indicated time points post inoculation (p.i.) and bacteria were enumerated by their ability to form colonies on agar plates. Mean and s.d. of triplicate MEF cultures and duplicate colony counts. Data are representative of at least two repeats. Statistical differences to Tbk1-/- MEFs expressing TBK1ΔC are shown. *p<0.5, **p<0.01, ***p<0.001 one-way ANOVA with Dunnett's multiple comparisons test. Western blot for Flag-tagged TBK1 variants in post nuclear cell lysates.
Figure 6 Figure 6. TGF-β1 secreted from Tregs induces Il-17rb expression of breast cancer cells via Smad2/3/4 signaling. (A) CD4+CD25+ Tregs were isolated from inguinal LN of BALB/c mice injected with 4T1 cells for three weeks, and were transwell cocultured with 4T1 cells in the presence of control IgG or neutralizing antibodies against IL-10 or TGF-β1. After 5-day coculture, 4T1 cells at lower well were analyzed for Il-17rb expression by RT-qPCR analysis. Gapdh was used as internal control. (B and C) Western blotting analyzed Il-17rb in 4T1 cells treated with recombinant TGF-β1 (B) proteins for 5 days. (B and C) Western blotting analyzed Il-17rb in 4T1 cells treated with recombinant IL-10 (C) proteins for 5 days. (D) Western blotting analyzed Il-17rb in shLacZ or shTgfbr1 4T1 cells treated with recombinant TGF-β1 for 5 days. (E) 4T1-injected BALB/c mice were sacrificed at three weeks post initial injection. Total cells isolated from inguinal lymph node tissues were transwell co-cultured with 4T1 cells. After 5-day coculture, soft-agar colony forming activity was determined in co-cultured shLacZ or shTgfbr1 4T1 cells at lower well. (5x102 cells/well, n = 6 wells per group). (F-H) Western blotting analyzed Il-17rb in shLacZ, shSmad2 (F)4T1 cells treated with recombinant TGF-β1 for 5 days. (F-H) Western blotting analyzed Il-17rb in shLacZ,shSmad3 (G)4T1 cells treated with recombinant TGF-β1 for 5 days. (F-H) Western blotting analyzed Il-17rb in shLacZ, shSmad4 (H) 4T1 cells treated with recombinant TGF-β1 for 5 days. (I and J) WT 4T1 (I) or Il-17rbDel 4T1 (J) cells were treated with or without recombinant TGF-β1 for 5 days. After treatment, nuclear translocation of NF-κB p65 was assayed by Western blotting in cells treated with recombinant Il-17b for 2 hours. Nuclear Matrix Protein p84 was used as a loading control of nuclear extracts.f triplicate measurement. In (B, C, D, F, G, H, I, and J), the intensity of each band was quantified using the Image J software. Gapdh, α-tubulin, or p84 was used as a loading control. Relative expression (RE) of protein levels in each sample to control 4T1 cells or Il-17rbDel 4T1 clone is indicated.
Figure 4A. On the left side, a schematic overview of Oct6 and its mutants used for iPSC generation. POUS and POUHD are shown as orange bars connected by the linker. Sites mutated to the respective Oct4 residues and Oct4 linker are denoted in green, and the mutation in the Sox-Oct interface is in black. The efficiency of these constructs for iPSC generation from MEFs is depicted as the absolute number of GFP-positive colonies on the right. Error bars represents standard deviations of two biological replicates run in parallel. B. Oct4-GFP-positive colonies of two O6SKM iPSC lines, expanded from single colonies of pluripotent cells that were generated by the contribution of the synthetic Oct6 molecule (Oct67K,22T,LinkO4,151S). Scale bars: 250 μm. C. Genotyping of two O6SKM iPSC lines. Two stable lines of iPSCs generated with the Oct6, Sox2, Klf4, and c-Myc retroviral set are positive for these four transgenes but negative for the Oct4 transgene. OSKM iPSCs and OG2-MEFs were used as PCR controls. D. Expression analysis of viral transgenes in two O6SKM iPSC lines done by qRT-PCR using specific primers. OG2-MEFs were harvested 5 days after infection and used for comparison as a positive control. E. Two O6SKM iPSC lines immunostained for pluripotency markers Sox2 and Nanog. Oct4 expression was confirmed by the Oct4-GFP transgene. DNA stained by DAPI. Scale bars: 100 μm. F. Bisulfite sequencing of genomic Oct4, Nanog, and Col1a1 promoter regions of O6SKM iPSCs, OSKM iPSCs, and OG2-MEFs. White and black circles represent unmethylated and methylated CpG sites, respectively. G. Pairwise scatter plots comparing the global gene expression profile of O6SKM iPSCs1 with OG2 ESCs (left) and O4SK iPSCs (right). Black lines represent a two-fold change in gene expression levels between the paired cell lines. On the right side of the plots, the color bar indicates scattering density. Red and green dots represent up- and downregulated genes, respectively. Positions of selected pluripotency-related genes are highlighted as orange points. H. Hierarchical clustering of O6SKM iPSC lines with OG2 ESCs and O4SK iPSCs, illustrating the close relationship between their global gene expression profiles.
Figure 3(A) Cell model composed of superimposed 3D reconstructions of high-resolution TEM tomograms covering different regions of the cell. Each square is one 3.2x3.2x0.17 µm 3D reconstruction. The tomograms were assigned to two categories for quantitative evaluation: those including the tip of the tube (orange) and the rest (purple). (B) Distribution of three commonly seen classes of vesicles, expressed as their density per area of cytoplasm screened, in the two regions of the cell. Box plots represent median, interquartile range (IQR), and IQR*1.5 below and above the IQR. n=62 tomograms from two different cells. (C) Representative examples for each vesicle class are shown next to the quantification plots. (D) Distribution of endocytic events at the apical or basal membrane compartment, expressed as density per membrane surface screened. Left shows tomograms corresponding to the cell body and branch, and right shows tomograms corresponding to the tip of the cell. Box plots represent median, interquartile range (IQR), and IQR*1.5 below and above the IQR. n=48 tomograms from two different cells.
Figure 3(A) Mitochondria were isolated from cells that were transfected with a plasmid that encoded COA7HIS or an empty vector. Mitochondria were solubilized and analyzed by reducing SDS-PAGE and Western blot. Mitos, mitochondria. (B) Radiolabeled [35S]TIMM8A and [35S]COX19 precursors were imported into mitochondria that were isolated from cells that were transfected with a plasmid that encoded COA7HIS or an empty vector. The samples were analyzed by reducing SDS-PAGE and autoradiography. The results of three biological replicates were analyzed, quantified, and normalized to control mitochondria at 30 min. The data are expressed as a mean ± SEM (n=3). IAA, iodoacetamide. (C) Mitochondria were isolated from cells that were transfected with a plasmid that encoded COA7HIS or an empty vector under reducing (DTT) and non-reducing (IAA) conditions and analyzed for levels of MIA40 by Western blot. (D) Proteins from HEK293 cells transfected with empty plasmid or RESA1HIS were modified with PEG-PCMal. Control cells were pretreated with the thiol oxidizing agent diamide (DAM) or the reductant DTT. The samples were analyzed by SDS-PAGE and Western blot. (E) Flp-In T-REx 293 cells induced to express MIA40FLAG were transfected with a plasmid that encoded COA7HIS or an empty vector. The affinity purification of MIA40FLAG was performed, and eluate fractions were analyzed by SDS-PAGE and Western blot.
Figure 6(A) The relative mRNA expression levels of EMT-related genes in RAD21-overexpressing MDA-MB-231 cells were quantified and normalized by 18s transcript. Empty vector-expressing MDA-MB-231 control cells are indicated by 231_P; RAD21-expressing MDA-MB-231 cells are indicated by 231_R. The data shown are the mean ± S.D. for three independent experiments. *P < 0.01, **P < 0.05. Statistical significance was validated by Student's t-tests. (B) Western blots for RAD21 in RAD21-overexpressing MDA-MB-231 cells. (C) A ChIP assay was performed for RAD21-overexpressing MDA-MB-231 and the empty vector expressing cells using antibodies specific for RAD21, Pol II, and H3K9ac, or IgG (as a control). Enrichment was measured by qPCR and expressed relative to the total input (4%). ChIP signals for H3K9ac were normalized to total H3. Results for at least three chromatin preparations are shown ± S.E.M. *P < 0.01, **P < 0.05. Statistical significance was validated by Student's t-tests. (D) Relative crosslinking frequencies among CTCF/RAD21 binding sites on the genes were measured with a 3C assay in the control (blue line) or RAD21-overexpressing (red line) MDA-MB-231 cells on day 10 after lentiviral transduction. BamH1 or XbaI restriction sites on the TGFB1 or ITGA5 gene, respectively, appear as gray shaded bars. Black shading indicates the anchor fragment. Each value was normalized to crosslinking frequency at the ERCC3 gene. The maximum crosslinking frequency was set at 1. (means ± S.E.M, n = 3). *P < 0.01, **P < 0.05. Statistical significance was validated by Student's t-tests. (E) A proposed model of cohesin-mediated dynamic chromatin architecture of the TGFB1 and ITGA5 genes associated with EMT plasticity. Tumor cells that have undergone EMT are characterized by proposed chromatin architectures. In epithelial cancer cells, cohesin tightly mediates distant intrachromosomal interactions in the TGFB1 and ITGA5 genes. When RAD21 mRNA stability is lost, RAD21 bound on the genes is reduced and intrachromosomal interactions in the genes are released, causing active recruitment of the transcriptional complex to the gene promoters. This enhances gene transcription and the cells acquire mesenchymal properties such as enhanced mobility and morphological changes.
Figure 5A Schematic view of the orthotopic transplantation strategy into the anterior prostate lobe of immunocompetent syngeneic C57BL/6J mice. B Frequency of engraftment (%) of mouse prostate organoid lines (C57#1 DLP and C57#3 DLP) expanded in complete medium (normal C57#1 n = 8 mice, normal C57#3 n = 2 mice) or adapted to the absence of A83-01 (adapted C57#1 n = 8 mice, adapted C57#3 n = 2 mice) (Binomial test, two-tailed). C Normalized mitotic (Ki67+) cell index in grafts of mouse prostate organoid lines (C57#1 DLP and C57#3 DLP) expanded incomplete medium conditions (normal) or adapted to the absence of A83-01 (non-parametric Mann-Whitney test). D Representative H&E, IF, and immunohistochemistry (IHC) analyses of host prostate tissue and engrafted donor organoid cells. Scale bar = 50 μm. E Model of the molecular pathway primed by Activin A to safeguard quiescence in adult prostate progenitors.
Weight loss, virus replication and specific IgG production in hACE2 mice post infection with SARS-CoV-2.Figure 1.SARS-CoV-2.For weigh loss record, hACE2 mice (n=7) and WT mice (n=3) were experimentally challenged intranasally with SARS-CoV-2, and the ACE2-Mock mice (n=3) were used as control. And then the weight loss was recorded over 14 days (a, ANOVA, ***p<0.001). To screen virus replication, 12 mice were infected in each group, and 3 mice per group were sacrificed and their major organs harvested for viral load and virus titer at 1 dpi, 3 dpi, 5 dpi and 7 dpi respectively. The distribution of SARS-CoV-2 in the primary organs of ACE2-HB-01 mice was detected by qRT-PCR (b). Virus titers of lungs were determined on Vero E6 cells (c, unpaired t-test, ***p<0.001) the virus isolated from lungs of ACE2-HB-01 mice at 3 dpi was observed by electron microscope (d). The specific IgG against SARS-CoV-2 was detected at 21 dpi by ELISA (e, unpaired t-test, ***p<0.001).
PrimPol suppresses R-loop formation in association with DNA secondary structure-forming sequences in BOBSC iPS cells.Figure 6A. Representative normalised RNA DIP-seq data in the SKI locus. Wild type in blue; primpol in red. The locations of H-DNA and G4 motifs are shown below the gene map. The corresponding RNAse H-treated samples are dashed. Since so little material was recovered following RNase H treatment, all samples were pooled prior to library generation. Data information: P values calculated with Mann-Whitney U test. In violin plots, bar = median; box = interquartile range (IQR); whiskers = upper and lower inner fences (1st / 3rd quartile + 1.5*IQR). B. Metagene analysis of RNA-DIP peak distribution in wild type and primpol BOBSC cells compared with the distribution of the indicated features in the genome. Data information: P values calculated with Mann-Whitney U test. In violin plots, bar = median; box = interquartile range (IQR); whiskers = upper and lower inner fences (1st / 3rd quartile + 1.5*IQR). C. RNA DIP-seq peak heights in wild type and primpol BOBSC cells normalised to a DT40 spike-in. n (wild type) = 32740; n (primpol) = 33721. D. Correlation of normalised RNA DIP-seq peak heights in the overlapping peaks between wild type and primpol. Blue line = 1:1 correlation; red line = linear regression through data. Data information: P values calculated with Mann-Whitney U test. In violin plots, bar = median; box = interquartile range (IQR); whiskers = upper and lower inner fences (1st / 3rd quartile + 1.5*IQR). E. Correlation between H-DNA forming sequences and all genes (white bar), and genes with DRIP peaks in wild type (blue) and primpol cells (red). Data information: P values calculated with Mann-Whitney U test. In violin plots, bar = median; box = interquartile range (IQR); whiskers = upper and lower inner fences (1st / 3rd quartile + 1.5*IQR). F. Correlation between G4 motifs ([G3-5N1-7]4) and all genes (white bar), and genes with DRIP peaks in wild type (blue) and primpol cells (red). Data information: P values calculated with Mann-Whitney U test. In violin plots, bar = median; box = interquartile range (IQR); whiskers = upper and lower inner fences (1st / 3rd quartile + 1.5*IQR). G. Normalised RNA DIP-seq peak heights in the genes identified as associating with H-DNA. Data information: P values calculated with Mann-Whitney U test. In violin plots, bar = median; box = interquartile range (IQR); whiskers = upper and lower inner fences (1st / 3rd quartile + 1.5*IQR). H. Normalised RNA DIP-seq peak heights in the genes identified as associating with G4 motifs ([G3-5N1-7]4). Data information: P values calculated with Mann-Whitney U test. In violin plots, bar = median; box = interquartile range (IQR); whiskers = upper and lower inner fences (1st / 3rd quartile + 1.5*IQR).
Figure 8Representative IF staining of TDP-43 and HDAC1 in the frontal cortices from normal individuals and patients with FTLD-TDP. Scale bar: 50 μm. The circled area is emphasized for showing the distribution of immunoreactivity in cell subregions. Scale bar: 15 μm. Quantification of cells with co-mislocalized HDAC1 and TDP-43 from each view of microscope. N = 5 per group. Data information: All data represent mean ± SEM, **** p < 0.0001 by t-test. Linear regression analysis of cells with HDAC1 mislocalization and TDP-43 proteinopathies. Total cell counts: 3000 per samples. P = 0.0001 by Pearson correlation analysis. Data information: All data represent mean ± SEM, **** p < 0.0001 by t-test. Representative IF staining of γH2AX and Ki67 in the frontal cortices from normal individuals and patients with FTLD-TDP from each view of microscope. Scale bar: 50 μm. The circled area is emphasized for showing the distribution of immunoreactivity in cell subregions. Scale bar: 15 μm. Quantification of cells with γH2AX and Ki67 immunoreactivity. N = 5 per group. Data information: All data represent mean ± SEM, **** p < 0.0001 by t-test. Linear regression analysis of cells with DNA damage and aberrant cell cycle activity. Cell counts: 40 -113 (normal) and 418-887 (FTLD) per samples. p < 0.0001 by Pearson correlation analysis. Data information: All data represent mean ± SEM, **** p < 0.0001 by t-test. Dot-blot of HDAC1 and TDP-43 in urea-soluble fractions from the frontal cortex of normal individuals and patients with FTLD-TDP. Quantification of HDAC1 and TDP-43 expression levels in urea-soluble fractions. N = 5 per group. Data information: All data represent mean ± SEM, **** p < 0.0001 by t-test.
p62 bodies are found both as membrane-free protein aggregates and as membrane-confined autophagosomal and lysosomal structures.Figure 3.(A) Localization of bodies containing endogenous p62 or transiently expressed GFP-p62 relative to EEA1-positive early endosomes. Endogenous p62 were stained green using p62 antibodies directly labeled with AlexaFluor488, and EEA1 was stained red with EEA1 mAbs directly labeled with AlexaFluor555. Alternatively, transiently expressed GFP-p62 was expressed in HeLa cells, and EEA1 was stained red with EEA1 mAb (bottom). The boxed area is shown to the right at a higher magnification. (B) Colocalization of GFP-p62 and CD63 (stained red using a mAb) in HeLa cells stably expressing GFP-p62. (C) Immunoelectron micrograph of S-GFP-p62 stained with a GFP pAb (10-nm gold particle, arrows) and monoclonal CD63 (15-nm gold particle, arrowheads). (D) Rapid detergent extraction of GFP-p62 from LysoTracker-positive acidic organelles. HeLa cells transiently expressing GFP-p62 were labeled with LysoTracker for 60 min. The detergent extractions were imaged in a time series with 15-s time intervals after adding 1% Triton X-100. The boxed area is shown in the bottom two images at a higher magnification before and after detergent extraction. Bars (A, B, and D), 20 μm.
Figure 4Flow cytometry analysis of H-2Kb surface expression on splenocytes derived from C57BL/6 wild type (left side) or LMP7-/- (right side) mice treated with vehicle (DMSO), ONX 0914 (300 nM), PRN1126 (300 nM), ML604440 (300 nM), LU-001i (300 nM), PRN1126+ML604440 (300 nM each), or PRN1126+LU-001i (300 nM each) overnight. Unstained splenocytes were used as negative control. Pooled data from three independent experiments (n=9) are shown as the means of median fluorescent intensity ± SEM. All data were statistically compared to the DMSO treated group. **P < 0.01, ***P < 0.001. One-way ANOVA.
Fig.Figure 4(a) Phenotypes of SA187-colonized or non-colonized wild type Col-0, hsfa2, hsfa1q and ein3-1 mutant plants upon long-term aquired thermotolerance treatment (LAT): 9 d old plants without and with SA187 (LAT, LAT+187) were treated at 37°C for 3 h, then returned to 22°C for 2 d. At d 11, plants were heat stressed at 44°C for 30 min and incubated for recovery at 22°C; or direct heat stress treatment (HS): 11 d old plants without and with SA187 (HS, HS+187) were treated at 44°C for 30 min and incubated for recovery at 22°C. (b,c) Fresh weight and percent survival of Col-0, hsfa2, hsfa1q and ein3-1 plants in HS, HS+187, LAT and LAT+187 treatments. Due to the dwarf size of hsfa1q mutants, LAT treatment was performed on d 18 and HS at d 20. All treatments are compared with plants upon 44°C HS. Data information: All plots represent the means of 3 biological replicates (n=36, 12 plants per biological repeat). Error bars represent SD of three biological repeats. Asterisks indicate a statistical difference based on the Student's t-test (* P≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001). (d) Fresh weight of 1µM 1-aminocyclopropane-1-carboxylic acid (ACC) treated wild type plants. 5 d old plants were transferred to 1µM ACC containing plates and 11 d old plants were HS treated at 44°C for 30 min before recovery at 22°C for 3 d (HS, HS+ACC). LAT: 9 d old plants without and with ACC treatment (LAT, LAT+ACC) were primed for 3 h at 37°C before incubation for 2 days at 22°C and further heat stress of 44°C was performed at day 11 for 30 min Data information: All plots represent the means of 3 biological replicates (n=36, 12 plants per biological repeat). Error bars represent SD of three biological repeats. Asterisks indicate a statistical difference based on the Student's t-test (* P≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001). (e) Percent survival of Col-0 plants with and without ACC under HS, HS+ACC, LAT and LAT+ACC conditions. Data information: All plots represent the means of 3 biological replicates (n=36, 12 plants per biological repeat). Error bars represent SD of three biological repeats. Asterisks indicate a statistical difference based on the Student's t-test (* P≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001). (f) Fresh weight of hsfa2 and control Col-0 plants treated with and without ACC under non-heat stress condition (NHS, NHS+ACC) of 22°C and heat stress (HS, HS+ACC) condition of 44°C for 30 min. Percent survival was scored at day 4 of recovery from 44°C heat stress. All treatments were compared with plants upon HS. Data information: All plots represent the means of 3 biological replicates (n=36, 12 plants per biological repeat). Error bars represent SD of three biological repeats. Asterisks indicate a statistical difference based on the Student's t-test (* P≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001).