EMBO/sd-character-level-ner huggingface dataset:
Isolation of genetically corrected recessive dystrophic epidermolysis bullosa (RDEB) epidermal stem cells.Figure 3Single cells were isolated from a mass culture (passage V) of RDEB keratinocytes infected with SIN retroviruses bearing a COL7A1 cDNA. Clonal types were determined (Barrandon Green,1987) and listed in Supplementary Table S1. Growing clones were expanded for further characterisation.COLVII detection in clones by immunostaining. COLVII expression (green) was detectable in some clones (6, 17, 22, 58 and 61) and not in others (3, 24 and 54); nuclei were stained with Hoechst 33342 (blue). Dotted lines delimit the periphery of keratinocyte colonies from the surrounding irradiated 3T3-J2 feeder cells. Scale bar: 50 μm. Single cells were isolated from a mass culture (passage V) of RDEB keratinocytes infected with SIN retroviruses bearing a COL7A1 cDNA. Clonal types were determined (Barrandon Green,1987) and listed in Supplementary Table S1. Growing clones were expanded for further characterisation. Quantitative RT-PCR analysis of COL7A1 expression in transduced clones compared to untransduced RDEB keratinocytes. All clones shown in (A) were transduced but expressed different levels of COL7A1 transcripts. Clones 6, 17, 22, 54, 58 and 61 expressed higher levels of COL7A1 than control RDEB cells and keratinocytes obtained from healthy donors (YF29 and OR-CA, control 1 and 2, respectively). The level of COL7A1 expression in the RDEB untransduced cells was referenced as 1. Single cells were isolated from a mass culture (passage V) of RDEB keratinocytes infected with SIN retroviruses bearing a COL7A1 cDNA. Clonal types were determined (Barrandon Green,1987) and listed in Supplementary Table S1. Growing clones were expanded for further characterisation. Determination of proviral rearrangements in transduced clones. A Southern blot was performed using genomic DNA of RDEB cells, clones and the infected mass culture from which the clones were isolated. Genomic DNA was digested with EcoRV and SpeI that cut at the 3′ and 5′ end of the provirus (Supplementary Fig S2) and hybridised with a 907-bp COL7A1 probe radiolabelled with 32P isotope. The upper band corresponded to the endogenous signal. The retroviral producer line Flp293A-E1aColVII1 was used as a control for the digested 9.6-kb provirus (proviral signal). Smaller bands corresponded to rearranged proviruses marked with an asterisk. Single cells were isolated from a mass culture (passage V) of RDEB keratinocytes infected with SIN retroviruses bearing a COL7A1 cDNA. Clonal types were determined (Barrandon Green,1987) and listed in Supplementary Table S1. Growing clones were expanded for further characterisation. Identification of stem cells producing COLVII. Western blotting revealed that only clone 6 secreted COLVII in the culture supernatant, while clone 54 and surprisingly clone 22 did not (see A). RDEB cells were used as a negative control and healthy donor cells as a positive control. The secreted matrix metalloproteinase 2 (MMP2) was used as a loading control.
Figure 5(A) HeLa-GFP-Parkin cells were incubated with valinomycin for 0 or 2 h. Immunostaining was performed with anti-MTPAP and anti-Tom20 antibodies. Images are representative of two independent experiments. Scale bar, 10 µm (B) Mitochondrial-rich fraction prepared from HeLa-GFP-Parkin cells treated with or without valinomycin and epoxomicin for 2 h, was incubated with various concentrations of trypsin (0-1000 µg/ml) for 30 min on ice. Protease protection was analyzed by western blotting using antibodies for mitochondrial marker proteins (Tom20 for OMM, Tim23 for intermembrane space, UQCRC1 and TFAM for mitochondrial matrix). Data are representative of five independent experiments (C) HeLa-GFP-Parkin cells were incubated with 1 µM valinomycin and 100 nM bafilomycin A1 for 0-3 h in the presence or absence of 10 µM epoxomicin. Cell lysates were subjected to immunoprecipitation using anti-NDP52 antibody or control IgG. Immunoprecipitates were analyzed by western blotting. Data are representative of five independent experiments. (D) Quantification of MTPAP co-immunoprecipitated with NDP52 in (C). Results are a summary of five independent experiments. Values are the means ± SEM. **p < 0.01 compared to cells treated with valinomycin and bafilomycin A1 for 3 h, determined with one-way ANOVA followed by the Tukey-Kramer post hoc test (E) HeLa-GFP-Parkin cells were incubated with 100 µM DFO and 100 nM bafilomycin A1 for 0, 4 and 8 h in the presence or absence of epoxomicin. Cell lysates were subjected to immunoprecipitation using anti-NDP52 antibody. Immunoprecipitates were analyzed by western blotting. Data are representative of two independent experiments (F) HeLa cells and HEK293T cells were incubated with 1 µM valinomycin and 100 nM bafilomycin A1 for 3 h or 100 µM DFO and 100 nM bafilomycin A1 for 8 h. Cell lysates were subjected to immunoprecipitation using anti-NDP52 antibody. Immunoprecipitates were analyzed by western blotting. Data are representative of two independent experiments (G) Immunoelectron micrographs of HeLa-GFP-Parkin cells expressing FLAG-NDP52. Cells treated for 2 h with valinomycin and bafilomycin A1 with or without epoxomicin, were processed for immune-electron microscopy with anti-FLAG antibody. Images are representative of ten cells. Arrow, immunogold labeled FLAG-NDP52. Mito, mitochondrion; MP, mitophagosome. Scale bars, 200 nm (H) Selected frames from time-lapse images of HeLa GFP-Parkin cells expressing mCherry-NDP52 under valinomycin and bafilomycin A1 treatment. The images are representative of two independent experiments and correspond to the boxed regions of Movie EV 6. Cyan, GFP-Parkin; magenta, mCherry-NDP52. The white arrowheads indicate colocalization of GFP-Parkin and mCherry-NDP52. The yellow arrowheads indicate mCherry-NDP existing inside GFP-Parkin ring structures. Scale bars, 1 µm
Figure 6 Figure 6. Transitions between various modes of Min protein patterns. A. Time-lapse images showing the transition from a longitudinal pole-to-pole mode to the transverse mode. Scale bar, 5 μm. B. Time-lapse images showing the transition from a transverse mode to a longitudinal pole-to-pole mode. C. Bar plot showing the distribution of the number of transitions. Inset: Data from experiments carried out under nutrient-poor conditions in which growth rates are reduced. D. Representative time-course of a change in the mode of sfGFP-MinD oscillation. The black line is a sigmoidal fit. The dashed black lines indicate 15° and 75° and the dashed red line indicates 45°. E. Bar plot showing the time scale of the switch in the oscillations. Inset: Data from experiments carried out in nutrient-poor conditions. F. Bar plots showing the relative numbers of the indicated transitions that occur at different cell lengths. All cells have a width of 5 μm. G. Distribution of final patterns in cells of the indicated widths as indicates, and lengths of 9-10 μm. H. Time-lapse images of various modes of transitions between patterns. Cell sizes from top to bottom are respectively 10x2x1, 10x6x1, 9x5x1, 10x4x1 μm3. Note that the cells are scaled differently. On the right is an illustration showing Min pattern transitions through intermediate states.
for clinical PDAC outcome.Figure 7A: Kaplan-Meier plot was generated using the transcriptome data set of PDAC from TGCA and OncoLnc to visualize the survival of PDAC patients with low or high PRMT1 transcript levels and short-term (<500 days, left plot) and long-term survival (>500 days, right plot). B: Representative immunohistochemistry (IHC) stainings of PRMT1 (brown) are shown for normal human pancreatic tissue and three PDAC specimens deriving from the cohort of 75 patients. Based on the immunostaining intensity and the percentage of stained tumor cells, all specimens were divided into three PRMT1 expression scores: not elevated, moderately elevated and highly elevated PRMT1 expression in tumor cells in comparison to normal pancreas. Right images are magnifications of the left images, as indicated by the black rectangles. Scale bars in the left images: 100 μm. Scale bars in the right images: 50 μm. C: The pie chart depicts the percentage of patients of the PDAC cohort (n=75) belonging to the three PRMT1 expression scores: not elevated, moderately elevated and highly elevated PRMT1 expression. D: Patients of the PDAC cohort were grouped according to their survival time in months into three survival groups: short-term (≤12 months), medium-term (13-23 months) and long-term survivors (≥24 months). Subsequently, the percentage of patients belonging to the three different PRMT1 expression scores (legend as in (C)) are displayed for each survival group. E: Immunofluorescence (IF) stainings of the 75 PDAC specimens were performed using α-p14ARF (red), α-CK8/18 (green, staining ductal cells in normal pancreas and neoplastic cells in PDAC) antibodies and DAPI (blue, nuclei/DNA). Representative IF images are shown for normal human pancreatic tissue, two PDAC specimens displaying no p14ARF expression and three PDAC specimens displaying strong p14ARF expression in tumor cells. Left and corresponding right images show the same tissue section. Asterisks indicate nucleolar p14ARF localization in neoplastic cells of PDAC. The last image pair shows a magnification of the image pair above (indicated by the rectangle). Scale bars: 35 μm. F: p14ARF-positive patients of the PDAC cohort were grouped according to their survival time in month into two survival groups: short-term (≤ 12 months) and long-term survivors (≥ 24 month). Subsequently, the percentages of patients with either not/moderately elevated PRMT1 expression or highly elevated PRMT1 expression are displayed for the two survival groups.
Figure 8A Changes in ATAC-seq accessibility at H2A.B CUT&RUN peaks following H2A.B knockdown. The significance (-log10 (P value)) is plotted against the fold change in accessibility (log2 (shH2A.B/shNTC)). Red indicates H2A.B peaks that increased in accessibility (5,552). Blue indicates H2A.B peaks that decreased in accessibility (3,310). ATAC-seq data analyses were performed on two biological replicates. B Differential accessibility changes of H2A.B CUT&RUN peaks following H2A.B knockdown for the peaks that increased in accessibility (red) and peaks that decreased in accessibility (blue) in Panel A. The change in mean ATAC-seq coverage (RPM) is plotted against the centre of the H2A.B peaks. C Genomic distribution of H2A.B CUT&RUN peaks, shown as percentages, with increased (5,552 in total) or decreased (3,310 in total) accessibility following H2A.B knockdown. D, E Two examples show increased accessibility at the promoter peak (NXF1) (D) and decreased accessibility at the peak across the intron-exon boundary (KDM4A) (E), respectively.
Mature B cells deficient in Syk are not activated and do not proliferate when stimulated via the BCREx vivo activation assay of splenic mature B cells from Tam‐treated mb1‐CreERT2 (upper row) or Tam‐treated mb1‐CreERT2;Sykfl/fl mice (lower row).Figure 4Ex vivo activation assay of splenic mature B cells from Tam‐treated mb1‐CreERT2 (upper row) or Tam‐treated mb1‐CreERT2;Sykfl/flmice (lower row). Purified splenic B cells were either left unstimulated (medium) or were stimulated with the indicated stimuli. The cells were analyzed by flow cytometry after 24 h. Shown are CD19 versus CD86 dot plots. 7‐AAD was included to distinguish dead from viable cells. The numbers in the quadrants indicate the relative frequencies of cells in the gate. Ex vivo proliferation assay with mature B cells from Tam‐treated mb1‐CreERT2 (upper row) or Tam‐treated mb1‐CreERT2;Sykfl/flmice (lower row). The cells were incubated with the indicated stimuli for 90 h.
CYP27B1 induction in response to TLR8 ligands is CEBPB-dependent.Figure 4Macrophages transduced with non-specific scrambled shRNA (shNS) or CEBPB shRNA (shCEBPB) were treated for 24 h with CL097, ssRNA40, or ssRNA41. (A) Cells were lysed and immunoblot performed with antibodies to CEBPB and β-actin. B) Immunoblots of CYP27B1 expression using antibody to CYP27B1 or β-actin.
Figure 1DRAFTS uses crude cell lysate based cell-free expression systems to harness source host cell's endogenous gene expression machineries. Compared to conventional single-channel reporters with color or fluorescence readouts, multiplexed sequencing readouts from pooled reactions in DRAFTS scale up the throughput of measurement. Biological replicates of transcriptional profiles (Tx) measured in separately prepared cell free lysate batches of E. coli. Comparison of abundances of each library constructs in in vivo and in vitro measurements. Correlation between transcriptional profiles (Tx) for regulatory sequence libraries from in vitro and in vivo measurements in E. coli. The color scale indicates the density of data points in a given area of the plot. Correlation between primary TSS calls (in bp from ATG start codon) of regulatory sequences from in vitro and in vivo measurements in E. coli.
Figure 2A. Phosphorylated Y600 of LORE was identified by LC-MS/MS. MBP-tagged LORE-CD was used for in vitro kinase assays. Trypsin digested recombinant proteins were subjected to LC-MS/MS analysis. The doubly-charged peptide with m/z 476.24 matches GLLYLHR of LORE with Y600 phosphorylation. Fragment ions retaining charges in the N and C terminus were denoted by "b" and "y", respectively. pY600 indicated phosphorylated Y600. B. Y600 mutation reduced LORE kinase activity. MBP-tagged LORE-CD and LORE-Y600F-CD (Y600 tyrosine was substituted with phenylalanine, F) were subjected to kinase reactions. The kinase dead mutant LOREK516E served as a negative control. Autophosphorylated LORE was visualized by autoradiography following SDS-PAGE gel electrophoresis. Protein abundance was stained by CBB. C. LORE Y600 is phosphorylated in plants. Two-week-old pLORE:LORE-FLAG complementation plant leaves were subjected to immunoprecipitation (IP) with anti-FLAG agarose beads. Y600 specific phosphorylation antibody was used to detect Y600 phosphorylation in LORE. The experiment was repeated three times with similar results. D. 3-OH-C10:0 induced Y600 phosphorylation. Two-week-old pLORE:LORE-FLAG complementation plants were treated with 1 μM 3-OH-C10:0 at indicated times. Plant leaves were subjected to anti-FLAG IP. Phosphorylation level of Y600 was detected by anti-pY600 antibody. The experiment was repeated three times with similar results. E. Y600 mutation abolished the ROS burst triggered by 3-OH-C10:0. The transgenic plants were treated with 3-OH-C10:0 and ROS burst was measured. WT(6-1) and WT(10-3) are two independent complementation lines with LORE; Y600F(2-6) and Y600F(7-1) are two independent lines complemented with LOREY600F; and kinase-dead LOREK516E was transformed to lore mutant as a negative control. The 2-3 and 5-2 are two independent lines. Values are means ± SD (n=6 biological replicates). (ANOVA, P <0.01). F. Y600 is required for growth inhibition by 3-OH-C10:0. The Arabidopsis seedlings were grown on ½ MS containing 2.5 μM 3-OH-C10:0 for six days, and the root length was measured. The experiment was repeated three times with similar results. WT(6-1) and Y600 (2-6) lines were used for the assay. Scale bar=0.4 cm. Values are means ± SD (n=3 biological replicates). (ANOVA, P <0.01). G. Y600 mutation decreased the disease resistance to Pst DC3000. Plants were spray-inoculated with Pst DC3000 at a concentration of 1×109 cfu/ml, and were subjected to bacterial growth curve analysis at 3 dpi. Values are means ± SD (n=6 biological replicates). (ANOVA, P <0.01).
Analysis of γ-H2AX nuclear foci.Figure 2γ-H2AX nuclear foci in XRCC4wt and mutant fibroblasts (XRCC4m/m1 and XRCC4m/m2) were determined at different times after irradiation with 0.5 Gy of γ-rays. Values, subtracted of their non-irradiated control (1.3 foci/nucleus in XRCC4wt cells and 1.4 and 0.9 foci/nucleus in XRCC4m/m1 and XRCC4m/m2 cells, respectively), are the mean ± SD of three independent experiments.Percentages of γ-H2AX foci in XRCC4wt and XRCC4m/m fibroblasts (pooled values) remaining at the indicated time-points.Immunofluorescence micrographs of γ-H2AX foci in XRCC4wt and XRCC4m/m fibroblasts.Data information: ***P < 0.001; **P < 0.01; *P < 0.05 (XRCC4m/m vs. XRCC4wt cells), two-way ANOVA, Bonferroni post hoc test.
Figure 5(A) Flow cytometry data analysis. Bars represent the mCherry/YFP fluorescence ratio of the respective yeast mutants normalized to the signal values of the H3 wild type FPR strain. The error bars show SEM calculated from biological triplicates with three technical repeats. Unpaired two-tailed student's t-test analysis indicates the statistical significance of the mutant compared to the respective wild type. The asterisks *, ** and *** indicate p-values <0.05, <0.01 and <0.001, respectively. (B) Metagene profile of ChIP data (Data ref: Ha et al, 2019). The genomic intervals cover the first 500 bp of the coding gene (scaled to 100 bins), the first 1 kb of the DNC transcript (scaled to 200 bins), and the variable length gap between the coding TSS and the DNC TSS (scaled to 50 bins). The vertical lines represent transcription start site (TSS) of protein-coding gene (left) and the DNC transcript (0 bin). The H3ac ChIP-seq signal was normalized by the H3 signal in the same samples. (C) Working model of Hda1C repressing DNC. The coding (blue) and divergent (purple) transcript arise in opposing directions of a shared nucleosome-free region (black). Nucleosomes (grey) comprise histones capable of modification by histone deacetylase 1 complex (Hda1C). In the normal state, Hda1C limits the frequency of DNC by deacetylating histones. With the loss of Hda1C function, increased acetylation of H3 favors DNC.
Fig.Figure 5(a,b) Transcript levels of HS memory genes HSP18.2 and APX2 in ein3-1 non-heat stressed (NHS, NHS+187), 44°C heat stressed SA187-colonized (HS+187), thermoprimed (LAT) as well as thermoprimed SA187-colonized plants (LAT+187) at 1 h, 24 h, 48 h, 72 h and 96 h of recovery at 22°C . Data information: For transcript level analysis, the data were normalized to tubulin as reference gene and the respective 22°C NHS, NHS+187 control plants, that were harvested at the same time points. All treatments are compared with direct 44°C HS treatment for statistical significance. All plots represent the means of 3 biological replicates. Error bars indicate SE. Asterisks indicate a statistical difference based on the Student's t-test *P ≤ 0.05; **P ≤ 0.01; **P ≤ 0.001 for differences between NP in comparison to NP+187, P and P+187 treatments. (c) Relative enrichment of H3K4me3 at APX2 and HSP18.2 in control non-primed (NP), SA187-colonized non-primed plants (NP+187), 37°C-primed (P) and 37°C-primed SA187-colonized plants (P+187) at 24 and 72 h after priming as determined by chromatin immunoprecipitation-qPCR for the indicated regions of APX2 and HSP18.2 (Fig 3F). Data information: For H3K4me3 enrichment analysis, amplification values were normalized to input and H3 and region 1 of non-primed (NP) plants. All treatments are compared with direct 44°C HS treatment for statistical significance. All plots represent the means of 3 biological replicates. Error bars indicate SE. Asterisks indicate a statistical difference based on the Student's t-test *P ≤ 0.05; **P ≤ 0.01; **P ≤ 0.001 for differences between NP in comparison to NP+187, P and P+187 treatments. (d,e) Transcript levels of HS memory genes HSP18.2 and APX2 in hsfa2 mutant. Data information: For transcript level analysis, the data were normalized to tubulin as reference gene and the respective 22°C NHS, NHS+187 control plants, that were harvested at the same time points. All treatments are compared with direct 44°C HS treatment for statistical significance. All plots represent the means of 3 biological replicates. Error bars indicate SE. Asterisks indicate a statistical difference based on the Student's t-test *P ≤ 0.05; **P ≤ 0.01; **P ≤ 0.001 for differences between NP in comparison to NP+187, P and P+187 treatments. (f) Relative enrichment of H3K4me3 levels at APX2 and HSP18.2 memory genes in hsfa2 mutant. Data information: For H3K4me3 enrichment analysis, amplification values were normalized to input and H3 and region 1 of non-primed (NP) plants. All treatments are compared with direct 44°C HS treatment for statistical significance. All plots represent the means of 3 biological replicates. Error bars indicate SE. Asterisks indicate a statistical difference based on the Student's t-test *P ≤ 0.05; **P ≤ 0.01; **P ≤ 0.001 for differences between NP in comparison to NP+187, P and P+187 treatments.
Mif2 auto-inhibition is released upon binding to Cse4 nucleosomes.Figure 7Analytical SEC chromatograms and corresponding Coomassie stained SDS-PAGE gels of Cse4n (light blue), Mif2-wt (red), combination (green). Note that the same Cse4n (upper panel) and Mif2 (middle panel) elution profiles and SDS-PAGEs are also displayed as reference in B and C (Mif2) or C (Cse4n) to improve clarity. Analytical SEC chromatograms and corresponding Coomassie stained SDS-PAGE gels of Mtw1c (blue), Mif2-wt (red), combination (green). Note that the same Mtw1c (upper panel) elution profile and SDS-PAGE is also displayed as reference in C to improve clarity. Analytical SEC chromatograms and corresponding Coomassie stained SDS-PAGE gels of Mtw1c (blue), Cse4n (light blue), Mif2-wt (red), combination (green). Analytical SEC chromatograms and corresponding Coomassie stained SDS-PAGE gels of Cse4n (light blue), Mif2Ame-1N swap (red), combination (green). Note that the same Cse4n (upper panel) and Mif2Ame-1N swap (middle panel) elution profiles and SDS-PAGEs are also displayed as reference in E and F (Mif2Ame-1N swap) or F (Cse4n) to improve clarity. Analytical SEC chromatograms and corresponding Coomassie stained SDS-PAGE gels of Mtw1c (blue), Mif2Ame-1N swap (red), combination (green). Note that the same Mtw1c (upper panel) elution profile and SDS-PAGE is also displayed as reference in F to improve clarity. Analytical SEC chromatograms and corresponding Coomassie stained SDS-PAGE gels of Mtw1c (blue), Cse4n (light blue), Mif2Ame-1N swap (red), combination (green).
Figure 2Schematic depicting the experimental design of establishing clonal cell lines from the SVF isolated from inguinal and perigonadal clonal cell lines. Representative picture of preadipocyte clonal cell lines showing media color 48 hours after last media change. Cells in all wells were 100% confluent when media was changed. Hierarchical clustering of preadipocyte clonal cell line gene expression using Ward's agglomeration. Heatmap depicting the 50 most variable genes among all preadipocyte clonal cell lines. Red represents high expression of genes, while blue represents low expression of genes. Heatmap depicting the 50 most variable genes among Type 2 and 3 preadipocyte clonal cell lines. Red represents high expression of genes, while blue represents low expression of genes.
Figure 3 (A) LETM1 expression was suppressed in HeLa cells by transfection with targeted siRNAs (siR1, siR2 or siR3). A non-target dsRNA (NT) served as control. Cells were fixed and immunolabeled with anti-DNA antibody (green). A higher magnification of selected mtDNA foci is shown beside each picture. (B) Quantification of cells in (A) displaying mtDNA abnormalities. At least 50 cells per siRNA were counted from 4 (siR2) and 5 (siR1 or siR3) independent experiments. Data are expressed as means ± SEM. ***P<0.001 (One-way ANOVA)   (C) HeLa cells were labeled with bromouridine (BrU) for 60 mins, 144 h after transfection with NT or siR2 and stained with anti-BrdU (green) and anti-GRSF1 (red) antibodies. Cell nuclei were stained with DAPI (blue)   (D) HeLa cells were labeled with anti-DNA (green) and anti-GRSF1 (red) antibodies 144 h after transfection with NT or siR3. Cell nuclei were stained blue with DAPI