EMBO/sd-character-level-ner huggingface dataset:
Lurbinectedin binds transcriptionally active regions in SCLC:(A-D) Chip-seq genome track of ASCL1/NEUROD1, RNAPII, and H3K27Ac on ASCL1, BCL2; INSM1 and MYB (hg19), in untreated (dark blue) and in lurbinectedin treated (green) conditions respectively; to be noticed the location of CGG rich motifs (lower part of each panel) which parallel the presence of lurbinectedin (red).Figure 3(A-D) Chip-seq genome track of ASCL1/NEUROD1, RNAPII, and H3K27Ac on ASCL1, BCL2; INSM1 and MYB (hg19), in untreated (dark blue) and in lurbinectedin treated (green) conditions respectively; to be noticed the location of CGG rich motifs (lower part of each panel) which parallel the presence of lurbinectedin (red). (E) Summary of the localization of lurbinectedin ASCL1-, NEUROD1-, RNAPII-, H3K27Ac-ChIP-seq, ATAC-seq and lurbinectedin-Chem-seq. (F, G) Histogram showing the distance (in bp) between TSS and either (F) lurbinectedin or (G) RNAPII bound to the DNA. Only peaks located in region between -2.0 and +2.0kb on each side of TSS were taken into consideration. The y axis on every Chip-seq profile represents the Coverage Score. (H) histogram showing the absolute distance between RNAPII and lurbinectedin peaks from nearest TSS.
Figure 7SIRLOIN is required for the function of an RNP, that includes hnRNPK, snRNP70, SLTM and potentially additional elements, which act together to enrich SIRLOIN-containing RNAs in the nucleus. Within the SIRLOIN sequence, the core part contains a single hnRNPK binding site with specific flanking sequences required for function, whereas an additional region upstream of the core mediates additional hnRNPK binding, but is not required for function.
Identification of Atg14 as a CbpC-interacting protein.Figure 2A Quantification of the band intensities following streptavidin-based pulldown assays using the in vitro-translated biotinylated CbpCT4 and FLAG-tagged autophagy-related proteins shown in Fig EV2A. B Quantification of the band intensities following GST-pulldown assays with 293T cell lysates expressing the autophagy-related proteins and recombinant GST-CbpFR6 shown in Fig EV2D. C Lysates from 293T cells transiently expressing 3Myc-Atg14 or Atg5-3Myc, and GFP-CbpCT4 or GFP were immunoprecipitated with GFP-Trap, and the bound proteins were analyzed by immunoblotting. D Lysates from 293T cells transiently expressing GFP-CbpCT4 or GFP, and 6Myc-Atg14 were immunoprecipitated with an anti-Myc antibody and Protein G PLUS-Agarose. The bound proteins were analyzed by immunoblotting. E Recombinant GST-CbpFR6 or GST, and 3Myc-Atg14 were used for GST-pulldown assays. The bound proteins were analyzed by immunoblotting using an anti-Myc antibody. F MEFs infected with R6 ΔcbpF expressing CbpFR6-FLAG for 2 h were subjected to IP and assayed using anti-HA agarose beads. The bound proteins were analyzed by immunoblotting using an anti-FLAG antibody.
Figure 5A. HeLa-Bclaf1-KO cells were transfected with the indicated siRNAs, and then transfected with CFLAR promoter and Bclaf1. The luciferase activity was measured as described before. B. CFLAR promoter were co-transfected with Bclaf1 or/and p50 into HeLa-Bclaf1-KO cells, and the luciferase activity was measured as described before. C. HeLa cells were transfected with siRNAs against Bclaf1 or p50. 24 hours post transfection, the cells were treated with TNF (10 ng/ml) for indicated hours. The total RNA was extracted and subjected to RT-PCR analysis. D. HeLa cells were transfected with siCtrl or siRNA against Bclaf1 or p50, treated with TNF (10 ng/ml) for 6 hours followed by western blotting analysis. E, HeLa cells were transfected with siCtrl or siRNA against Bclaf1 or p50 were pre-treated with DMSO or CHX (1 μg/ml) for 30 min, and then treated with TNF (10 ng/ml) for 12 hours. Cells were lysed and then analyzed by western blotting F. HeLa cells were transfected with siCtrl or siRNA against Bclaf1 or p50 were pre-treated with DMSO or CHX (1 μg/ml) for 30 min, and then treated with TNF (10 ng/ml) for 12 hours. Cells were lysed or collected and subjected to Annexin V/7AAD staining followed by flow cytometry analysis, all Annexin V positive cells were counted for analysis (F). G. CHIP analysis of the CFLAR promoter in HeLa-Flag-Bclaf1 cells. Immunoprecipitation was performed using anti-Flag and control IgG antibodies followed by RT-PCR analysis. Data information: Data are shown as mean ± SD. n=3 biological replicates. ns, not significant; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. One-way or Two-way ANOVA test.
Figure 3(A) Real-time qPCR analysis of ANXA1 expression using mRNA extracted from the non-cancerous SB, cancerous 10C, and human CRC cell lines HCT116, DLD1 and RKO, each grown to 50% or 100% confluency. All results were normalized to the ANXA1 mRNA levels in SB cells of 100% confluency, which was designated as 1. Data are mean values ± SEM. The experiment was performed in triplicates and repeated twice. **p<0.01 and ***p<0.001 (student's t-test). (B) Confocal microscopy analysis of SB and 10C cells grown to 20% (top panels) or 100% (bottom panels) confluency followed by immuno-fluorescence staining of Annexin A1 (green). The nuclei were stained with DAPI (blue). A series of 20-50 consecutive images in the z-axis were stacked together to generate the 3-D figure at 400x magnification. Annexin A1 is most abundantly expressed on the outer layer of 20% confluent 10C (pointed by arrows compared to 100% confluent 10C, or SB of either confluency. Scale bars, x = 1 μm, y = 1 μm, z = 1.6 μm. The experiment was repeated at least twice. (C) Cell proliferation assay of adenoma-derived non-cancerous SB and cancerous 10C and human CRC cell lines HCT116, DLD1, SW480, HT29 and RKO either untreated (black lines) or following treatment with control siRNA (gray lines) or ANXA1-specific siRNA (green lines). Data are mean values ± SEM. The experiment was performed in triplicates and repeated three times. *p<0.05, ** p<0.01 and *** p<0.001, compared to the untreated cells (two-way ANOVA). (D) Cell proliferation assay of SB (left panel) and RKO (right panel) cells transfected with ANXA1 (red line), as compared to the control cells (black lines). Data are mean values ± SEM. The experiment was performed in triplicates and repeated three times. *p<0.05, **p<0.01 and ***p<0.001 (two-way ANOVA). (E) Xenografted tumor growth in nude mice following subcutaneous and bilateral inoculation of HCT116 cells transfected with control siRNA or ANXA1-specific siRNA. The tumor volumes were measured after 7 days post injection (left panel, n=4). For each mouse, the tumor resulting from ANXA1-specific siRNA treated cells was normalized to that from control siRNA-treated cells, which was designated 100%. The line represents the average. *p<0.05 (paired t-test). The individual tumor pairs are shown on the right panel: top, tumors arising from control siRNA treated cells; bottom, tumors arising from ANXA1-specific siRNA treated cells.
Figure 2(A) Wild-type and EVL-/- P5 retinas were digested, labelled with CD31-, CD34- and EVL-specific antibodies and analyzed by flow cytometry. Endothelial cells were defined as CD31/CD34-double positive cells (gate indicated in the left panel) and analyzed for EVL expression (middle panel). Mean fluorescence intensity (MFI) of EVL in wild-type (magenta) and EVL-/- (black) cells is shown in the right panel. WT: n=3, EVL-/-: n=2; two retinas per animal. Error bars represent SEM. (B-C) EVL protein expression in sparse/migrating wild-type (WT) and EVL-/- mouse brain endothelial cells (MBEC) and mouse lung endothelial cells (MLEC). Actin was used as loading control. Western blots are representative of three independent experiments. (D) MLEC from wild-type (upper panel) and EVL-/- (lower panel) mice stimulated with 10 ng/ml VEGF were stained for actin (cyan) and EVL (magenta). Asterisks indicate focal adhesions, white arrows indicate filopodia and white arrowheads indicate the leading edge of lamellipodia. Representative images from four independent experiments are shown. Scale bar, 20 µm.
Figure 2Cell viability assays were performed as described in Materials and Methods after cells were treated with temozolomide (A), cisplatin (B), adriamycin (C), taxol (D), TRAIL (E), bafilomycin A1 (F), thapsigargin (G), and MG132 (H). Means ±S.D. from at least three independent experiments are shown as relative indexes after normalization to those of individual control cells treated with DMSO. (I) XO8 GSCs and DFCs were treated with 50 nM MG132 for 24 hrs. Cell viability was measured using trypan blue exclusion assay. Error bars represent the mean ± SD from three independent experiments. For statistical analysis, student t-test (two-sided, one type) were used. (*P < 0.05). (J) XO8 GSCs were treated with MG132 were cultured in 0.3% soft-agar as described in Materials and Methods. The colonies bigger than 100 µm in diameter in four randomly chosen fields were subjected to blind counting. Bars, means +S.D. from three independent experiments.
Genetic system of mitochondria.Figure 3(A, B) Selected proteins involved in DNA (A) and RNA (B) maintenance of control (143B, solid lines, grey bars) and ρ0 cells (dashed lines, colored bars). A, for POLG, UNG and TWNK, only abundances >1 MDa were quantified. Plots and gene names are in corresponding colors. Data are presented as mean ± SD (n=4). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 (Student's t-test for individual proteins and two-way ANOVA for changes of multiple proteins in a complex).
Analysis of the common morphology mutants of endocytic compartment phenotypes and the relationship to known protein complexes (see also Table EV5).Figure 3Matrix showing significant overlap of stringent specific phenotype mutants (p-value < 0.05; significance was determined using Fisher's exact tests). Circle size corresponds to the log value of the overlap odds ratio. Orange circles denote same-compartment phenotype pairs. Dark blue fill colour indicates phenotype pairs with at least one enriched protein complex in the overlapping set. LE: late endosome. Diagram illustrating co-occurrence of endocytic morphology phenotypes associated with protein complex perturbation. Shown are significant protein complexes from (A) with biological processes and linked phenotype pairs. # denotes a phenotype pair without significant enrichment. Phenotype names are color-coded by endocytic marker, using the colour key described in Figure 1 and indicated on the yeast cell diagram.
Figure 2(A) Scatter plot displaying log2 SILAC ratios of individual phosphorylation sites in the experiments setup SR on the y-axis and setup cdc55Δ on the x-axis. Black dots represent Hog1-dependent sites (ratio in setup SR ≥ 2, and affected by Hog1as-inhibition (ratio ≤ 0.5)) (Romanov et al., 2017). (B) Histogram displaying the SILAC ratios (log2) of stress-induced phosphorylation sites in setup cdc55Δ (upper panel) and setup rts1Δ (lower panel) (Hollenstein et al., 2020), stratified by their Hog1-dependence (grey area versus thick black line). (C) Schematic illustration of suggested model of Cdc55 regulation in the hyperosmotic stress response, with grey arrows signifying hypothetical relationships. (D) Line plot monitoring growth rate (y-axis) of indicated strains (colored lines) under different osmolarity conditions (x-axis). (E) Histogram displaying the SILAC ratios (log2) of stress-induced phosphorylation sites that are induced in setup cdc55Δ in two separate experiments. The blue histogram shows the distribution of fold-changes when IGO1 and IGO2 are deleted and cells are exposed to hyperosmotic stress (setup SR igo1∆igo2∆). The orange histogram shows the distribution of fold-changes after deletion of CDC55 and upon exposure to hyperosmotic stress (setup SR cdc55∆). The same set of sites was compared between the two experiments. (F) Scatter plot displaying log2 SILAC ratios of phosphorylation sites in the experiments setup SR on the x-axis and setup SR igo1∆igo2∆ on the y-axis. Phosphorylation sites that do not exhibit Cdc55-dependence are shown in grey, whereas Cdc55-dependent phosphorylation sites are highlighted in orange. We found an enrichment for Cdc55-dependent sites in the SR- and Igo1/Igo2-dependent set (Set1) (Fisher Exact test, p-value = 3.54 x 10-31, odds-ratio = 21.40). (G) Waffle plot showing the impact of different signaling mechanisms on the stress phosphoproteome. Each box corresponds to 1% of the entire set. Orange boxes represent the percentage of phosphosites regulated by PP2ACdc55, whereas grey boxes represent phosphosites regulated by Hog1 and downstream of the MAPK. Light purple boxes indicate the phosphosites that are neither regulated by Hog1 nor PP2ACdc55. Dependency was defined as log2 SILAC ratio below -1 or above 1. Rck2-dependency was extracted from (Hollenstein et al., 2020).
Figure 7(A) The confocal images, 3D-rendered images and orthogonal views of microglia engulfing vGluT2+ synapses in controls and Cdc50a-RNAi mice. Scale bar, 10 µm. (B) Quantification of engulfed vGluT2+ synapses in microglia. % Engulfment of vGluT2+ synapse = Volume of vGluT2+ synapse inside microglia / Volume of microglia. N = 5 for biological replicates. (C) Quantification of engulfed mCherry+ materials in microglia. N = 5 for biological replicates. (D) Quantification of colocalized vGluT2+/mCherry+ synapses within microglia. N = 5 for biological replicates.
Karyotypic characterization of the late passage ICL linesRepresentative karyotypes of late passage, large T antigen immortalized MEFs after exposure to Cre recombinase and serially sorted for control (hCD2 plus) and ICL (hCD2 Minus) cells for chromosome lines (A) Ch12, (B) Ch14, (C) Ch9 and (D) Ch10.Figure 3Representative karyotypes of late passage, large T antigen immortalized MEFs after exposure to Cre recombinase and serially sorted for control (hCD2 plus) and ICL (hCD2 Minus) cells for chromosome lines (A) Ch12 Boxed panels denote targeted chromosome loss; arrows and asterisks denote shared and unshared, non-targeted chromosomal copy number variations respectively. Representative karyotypes of late passage, large T antigen immortalized MEFs after exposure to Cre recombinase and serially sorted for control (hCD2 plus) and ICL (hCD2 Minus) cells for chromosome lines (B) Ch14. Boxed panels denote targeted chromosome loss; arrows and asterisks denote shared and unshared, non-targeted chromosomal copy number variations respectively. Representative karyotypes of late passage, large T antigen immortalized MEFs after exposure to Cre recombinase and serially sorted for control (hCD2 plus) and ICL (hCD2 Minus) cells for chromosome lines (C) Ch9 Boxed panels denote targeted chromosome loss; arrows and asterisks denote shared and unshared, non-targeted chromosomal copy number variations respectively. Representative karyotypes of late passage, large T antigen immortalized MEFs after exposure to Cre recombinase and serially sorted for control (hCD2 plus) and ICL (hCD2 Minus) cells for chromosome lines (D) Ch10. Boxed panels denote targeted chromosome loss; arrows and asterisks denote shared and unshared, non-targeted chromosomal copy number variations respectively.
MALAT1 subpopulations of rod photoreceptors is not due to donor variation.Figure 3A, B) (A) t-SNE visualization of human retinal cells colored by annotation of 13 transcriptionally distinct clusters (CCA0-CCA12) and (B) their distribution in 3 donor retina samples. C) Feature expression heatmap showing expression patterns of 7 rod photoreceptor markers across 12 retinal cell clusters. The size of each circle depicts the percentage of cells expressing the marker within the cluster. Brown colour indicates ≥50 nTrans (number of transcripts). D) t-SNE plots showing expression of MALAT1. E) Expression pattern of MALAT1 in the rod photoreceptor showing MALAT1-hi (CCA1, CCA10) and MALAT1-lo (CCA0) subpopulations. x-axis depicts normalized transcript levels. F) t-SNE plots showing expression of major ribosomal genes. G) Heatmap of differentially expressed genes between the two MALAT1-hi clusters CCA1 and CCA10. The rows correspond to top 10 genes most selectively upregulated in individual clusters (p< 0.01, Benjamini-Hochberg correction) and the columns show individual cells ordered in CCA1 and CCA10.
Figure 4(A) Representative confocal images taken at 60 min after admixture of 2 mM GTP and 10 µM Alexa-Fluor647- or Alexa-Fluor488-hGBP1F to formaldehyde fixed gram-negative and gram-positive bacteria expressing GFP (S. enterica Typhimurium [ST], L. monocytogenes [Lm]), or RFP (S. flexneri [Sf], uropathogenic E. coli [UPEC]), or dsRed (L. pneumophila [Lp], S. aureus [Sa]). (B) (Left panel) Confocal time-lapse microscopy frames of Alexa-Fluor488-LPS-O55:B5 after addition of 5 µM Alexa-Fluor647-hGBP1F supplemented with 2 mM GTP. (Right panel) Confocal images taken of 5 µM Alexa-Fluor488-LPS-O55:B5 20 min after addition of 5 µM Alexa-Fluor647-hGBP1F or -hGBP1FR48A supplemented with 2 mM GTP. Graphs depict average aggregate area of Alexa-Fluor488-LPS-O55:B5 supplemented with 2 mM GTP and 5 µM of the indicated hGBP1 variant. Mean area ± SEM of combined data from three independent experiments. Significance was determined by one-way ANOVA with Tukey's multiple comparison test.. n.s., not significant; ***, P ≤ 0.001; ****, P ≤ 0.0001. (C) Polymerization of 5 µM hGBP1F induced by addition of 2 mM GTP in the presence and absence of 5 µM LPS-O55:B5 was monitored by absorption spectroscopy at 350 nm. The absorbance signal was superimposed with nucleotide composition of the same solution analyzed at defined time points. Maximal hydrolysis rates (turnover numbers) were determined for hGBP1F variants in the presence and absence of 5 µM LPS-O55:B5. Graphs show mean turnover numbers ± SEM of combined data from at least three independent experiments. Significance was determined by two-way ANOVA with Tukey's multiple comparison test. n.s., not significant; ***, P ≤ 0.001; ****, P ≤ 0.0001. (D) Formaldehyde fixed GFP+ S. flexneri supplemented with varying LPS-O55:B5 concentrations were mixed with 5 µM hGBP1F and 2 mM GTP. After 60 min hGBP1F-enclosed S. flexneri were quantified. Combined data from three independent experiments are shown as mean ± SEM. Significance was determined by one-way ANOVA with Tukey's multiple comparison test. ****, P ≤ 0.0001. (E) Confocal images of formaldehyde fixed GFP-, RFP-, or dsRed-expressing gram-negative bacteria 60 min after addition of 5 µM Alexa-Fluor647- or Alexa-Fluor488-hGBP1F and 2 mM GTP in the presence and absence of 100 µM LPS-O55:B5. After 60 min hGBP1F-enclosed gram-negative bacteria were quantified. Combined data from three independent experiments are shown as mean ± SEM. Significance was determined by two-way ANOVA with Tukey's multiple comparison test. ****, P ≤ 0.0001.
Figure 6Representative images and analysis of Western blots for PAR, performed with lysates of HME control (vec) OAS single KO cells (KO1-3), treated with H2O2 for 15 or 40 min. β-actin is shown as a loading control. Signal intensity was measured using ImageJ software. Average lane densities, normalized to vector from three independent experiments, are plotted below as mean + SD, * p < 0.05 as assessed by Student's t-test. Same as (A), with lysates of HME control (vec) cells and cells expressing ectopic OAS1, OAS2 or OAS3 (OAS1-3hi). Experiments were repeated at least three times. Average lane densities normalized to vector are plotted below below as mean + SD, * p < 0.05 as assessed by Student's t-test. HME cells, pre-incubated with IFNβ for 2 days to increase OAS abundance, were treated with H2O2 for 0-60 min at 37oC. Western blots of PAR accumulation (top), shift in the position of PARylated PARP1 (middle), OAS2 induction by IFNβ, and β-actin as a loading control are shown. Experiments were repeated twice. HME control (vec) and KO1 cells were pretreated with 0-500 IU/ml IFNβ for 2 days, and then treated with 500 μM H2O2 for 10 or 30 min. Western blots of PAR accumulation (top, high and medium exposure; middle, low exposure), OAS1-3 induction by IFNβ, and β-actin as a loading control are shown. The experiment was repeated four times; ns, non-specific band.
Fig.Figure 3A. Representative pictures showing Peyer´s Patches in Casp8WT mice, and different severities of hemorrhage formation in Casp8ECko mice at an early disease stage. Scale bars: 1mm. Quantification of lymphatic vessel density (B) in Peyer's Patches in Casp8WT and Casp8ECko mice at an early disease stage (n = 9 WT, 7-10 ECko Peyer's Patches are pooled from 4-6 mice per genotype; one-way ANOVA with tukey's multiple comparison). Data information: Data shown as mean ± SEM. * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001, ns: not significant. representative pictures of CD31 (pan EC) and Lyve1 (lymphatic) staining (C) in Peyer's Patches in Casp8WT and Casp8ECko mice at an early disease stage (n = 9 WT, 7-10 ECko Peyer's Patches are pooled from 4-6 mice per genotype Scale bars: 200µm. D-F. Representative images (D) and quantifications (E, F) of stainings for CD31, Lyve1 and DAPI (nuclei) in small intestinal wholemounts at an early disease stage (blue arrowheads point to defective lacteals, white arrowheads indicate edema) in Casp8ECko mice (E; n= 7 WT, 6 ECko. F; n= 7 WT, 10 ECko; two tailed unpaired Student's t-test). Scale bars: 100µm. Data information: Data shown as mean ± SEM. * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001, ns: not significant. G. Representative images of small intestine sections of mice injected with a 70kDa fluorescently labeled dextran and stained for CD31 at an early disease stage. White arrowheads point to extravasated dextran in Casp8ECko mice (n= 5 WT, 5 ECko). Scale bars: 50µm. H. Quantification of extravasated dextran in small intestine and brain lysates in Casp8WT and Casp8ECko mice at an early disease stage (n= 4 WT, 4-5 ECko; two-way ANOVA with tukey's multiple comparison). RFU: relative fluorescent units. Data information: Data shown as mean ± SEM. * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001, ns: not significant. I. Representative images of CD31 and VE-Cadherin staining in intestinal wholemounts at an early disease stage (n= 5 WT, 5 ECko) in Casp8WT and Casp8ECko mice (red arrowheads point to VE-Cadherin 'empty' vessel patches). Scale bars: 25µm.