EMBO/sd-character-level-ner huggingface dataset:
ScRNA-seq reveals cellular identity of ectopic cells in EML1-heKO cerebral organoids.Figure 3(A) scRNA-seq of 2,335 control and 3,358 EML1-heKO cells (3 pooled independent batches, 3 organoids each). Cells shown in UMAP plot and colored by annotated cell type. Data information: RG1: radial glia1, RG to bRG: radial glia to basal radial glia, RG2: radial glia to young neurons, IP: intermediate progenitors, YN: young neurons, MLC: mesenchymal-like cells. (B) Comparison of cell-type composition between control and EML1-heKO. Numbers indicate percentage of total cells belonging to the respective cell-type. Data information: RG1: radial glia1, RG to bRG: radial glia to basal radial glia, RG2: radial glia to young neurons, IP: intermediate progenitors, YN: young neurons, MLC: mesenchymal-like cells. (C) Normalized expression values of COL1A2, COL3A1, LUM and MEIS2 per cell-type (3 pooled batches, 3 organoids each). Data information: RG1: radial glia1, RG to bRG: radial glia to basal radial glia, RG2: radial glia to young neurons, IP: intermediate progenitors, YN: young neurons, MLC: mesenchymal-like cells. Asterisks indicate Bonferroni corrected p-values. Wilcoxon Rank Sum test: ***<0.001, *<0.05, ns: not significant. (D), Gene ontology (GO) term analysis of RG to bRG cells in control and EML1-heKO shows percentage of counts belonging to the set of genes associated with the respective GO term (3 pooled batches, 3 organoids each). Data information: Asterisks indicate Bonferroni corrected p-values. Wilcoxon Rank Sum test: ***<0.001, *<0.05, ns: not significant. (E) Control and EML1-heKO-derived cerebral organoid stained for COL1A2 and MEIS2. VZ areas are encircled, squares indicate areas enlarged in F. (F) Control and EML1-heKO-derived cerebral organoid stained for COL1A2, MEIS2 and MAPT. (G) Representative images showing EGFP-control and EGFP-EML1-heKO hybrid cerebral organoid stained for EGFP, FAM107A and NCAD. Yellow dotted lines indicate morphology of EGFP+ cells (4 independent batches each, 3 organoids per batch, at least 30 ectopic rosettes in total). Data information: Scale bars: (E) 50µm; (F) 10µm; (G) 50µm, enlarged 10µm.
Figure 7(A-D) Nuclear p-STAT3 (A,B) and p-ERK1/2 (C,D) in tumor CD31+ endothelial cells of mice treated for 14 days with SHP099 (200 mg/kg), AMG386 (5.6mg/kg) or the combination of SHP099 (200 mg/kg) + AMG386 (5.6mg/kg). Representative (3 tumors/group) immunostaining (A,C) and quantification of staining intensity/CD31+ cell (B, D); horizontal line: mean intensity/group; n=3 tumors/group; number of CD31+ cells counted in B: control (n=180), SHP099 (n=308), AMG386 (n=300), combined SHP099+AMG386 (n=322); number of CD31+ cells counted in D: control (n=1,733), SHP099 (n=821), AMG386 (n=1,399), combined SHP099+AMG386 (n=783). Scale bars: A, 50μm; C, 10μm. FOXO1 detection in tumor CD31+ endothelial cells after treatment with SHP099 (200 mg/kg), AMG386 (5.6mg/kg) or the combination of SHP099 (200 mg/kg) + AMG386 (5.6mg/kg). Representative (3 tumors/group) confocal images quantification (n=3 tumors/group) of FOXO1 fluorescence intensity/CD31+ cell. Number of CD31+ cells counted: control (n=2,285), SHP099 (n=2,262), AMG386 (n=2,300), combined SHP099+AMG386 (n=2,361). Data information: P values by analysis of variance with Dunnett's multiple comparison test; **P<0.01, ***P<0.001.
Peripheral RAB13 RNA promotes the local association of RAB13 with the exchange factor RABIF.Figure 7A. Immunoprecipitation and Western blot analysis to detect proteins bound to GFP-RAB13 expressed from constructs carrying wt or ∆PMO RAB13 3'UTR. GFP-RAB13 (T22N) expresses a nucleotide-free mutant which binds tightly to putative GEFs. RABIF panel is also shown with adjusted contrast to reveal lower binding to wtGFP-RAB13. B. Quantification of RABIF binding to RAB13 from experiments as in (A). n=6. Bars: mean ± s.e.m.. C. Active RAB13 pulldown assay from cells with CRISPR knockdown of RABIF using the indicated sgRNAs. n=3. Bars: mean ± s.e.m.. D. Quantification of RABIF-RAB13 PLA signal from cells transfected with the indicated siRNAs. Number of observed cells is indicated within each bar. Bars: mean ± s.e.m.. Similar results were observed in one additional independent experiment. E. Representative RABIF-RAB13 PLA images and quantitations from cells transfected with the indicated PMOs. N>70 cells. Bars: mean ± s.e.m.. Arrows indicate PLA signal at the cell periphery. Arrowheads indicate PLA signal within the cell body. Boxed regions are magnified in the insets. Scale bars: 15 μm; 5 μm in insets. F. Representative RABIF-GFP PLA images and quantitations from cells expressing GFP-RAB13(T22N) carrying wt or ∆PMO RAB13 3'UTR. N>70 cells. Bars: mean ± s.e.m.. Arrows indicate PLA signal at the cell periphery. Arrowheads indicate PLA signal within the cell body. Boxed regions are magnified in the insets. Scale bars: 10 μm; 4 μm in insets.
a) Knockdown of p62 expression attenuates the LC3–Dvl2 interaction.figf5(a) Knockdown of p62 expression attenuates the LC3-Dvl2 interaction. shRNA, short hairpin RNA. (b) Co-localization of GFP-LC3, p62 and Dvl in HeLa GFP-LC3 stable cells on starvation. Proteins visualized by immunofluorescence with anti-GFP (green), anti-p62 (blue) and anti-Dvl (red) antibodies. Scale bar, 10 μm. (c) p62 knockdown slows the degradation of Dvl2 on starvation. HEK-293T cells were transfected with p62 siRNA. At 48 h after transfection, cells were subjected to starvation for various times. The cell lysates were subjected to immunoblotting with the antibodies indicated. Uncropped images of blots are shown in Supplementary Information, Fig. S8.
CLEC12A Colocalizes with Autophagosome Proteins and Plays a Role in Pathogen Defense.Figure 5(A-C) Representative epifluorescent images of HeLa cells showing colocalization of (A) CLEC12A (green) and galectin 8 or galectin 3 (red) around intracellular Salmonella (blue), (B) GFP-CLEC12A (green) and NDP52 (red) around Salmonella (blue), and (C) endogenous CLEC12A (red) and GFP-LC3 (green) around Salmonella (blue). (D) CLEC12A or GFP-CLEC12A (green) and GABARAPL2 or ATG16L1 (red) around intracellular Salmonella (blue). Images are representative of three or more independent experiments. (E) Representative epifluorescent images of HeLa cells transfected with a control siRNA or a siRNA against CLEC12A and infected with DsRed-labeled Salmonella for 1 hr. (F) Quantitation of ubiquitin-bacteria colocalization at 1 hr post-infection in cells shown in (E). Data are shown as means ± SEM from three independent experiments. (G) Representative epifluorescent images of HeLa cells transfected and infected as in (E). (H) Quantitation of NDP52-bacteria colocalization at 1 hr post-infection in cells shown in (G). Data are shown as means ± SEM from three independent experiments. (I) Representative confocal micrographs of HeLa cells expressing GFP-CLEC12A. Cells were untreated or exposed to hypertonic media containing PEG 1000, followed by hypotonic shock.
The interplay of p53's C-terminal lysine acetylation and methylation regulates transiently expressed target genes in response to IR.Figure 5A schematic illustration of p53's C-terminal modifications and described functional implications, including key regulatory enzymes. Total p53, p53 acetylated at K382 and K370 as well as GAPDH were measured by Western Blot at indicated time points in the context of different p53 dynamics: pulsing p53 (10 Gy IR), transient p53 (10 Gy IR + BML-277, central lanes) and sustained p53 (10 Gy IR + Nutlin-3, right lanes). See Figure 3 and Methods section for details. The relative change in p53 acetylation at K370 (light green) and K382 (dark green) was quantified from Western Blot and normalized to the abundance 3 h post IR. Means and propagated standard errors from 3 independent experiments are indicated. Acetylation increased over time in the context of sustained p53. See also Appendix Fig S12. The p53-K370 methylase Smyd2 was down-regulated in a clonal stable A549 cell line expressing a corresponding shRNA. Transcript levels were measured in wild type and knockdown cells by qRT-PCR. Mean levels and standard deviation from technical triplicates are indicated. Promoter activity of CDKN1A (E) and MDM2 (F) were quantified in Smyd2 knockdown cells before (basal, grey) and 3 h (red), 6 h (blue) and 9 h (orange) after DNA damage (10 Gy IR). Left panel: The percentage of cells with active TSS, subdivided into populations with strong (> 75% of TSS, solid colors) and weak (<75% of TSS, shaded colors) activity, is shown as stacked bar graphs, the mean fraction of active promoters is indicated above each bar. Right panel: Distributions of calculated transcription rates at active TSS are presented for each time point as probability density estimates (PDF, see Data Visualization section). We measured a higher fraction of active promoters upon damage compared to A549 wild type cells (Fig 3), while transcription rates remained unchanged. The p53-K382 methylase Set8 was down-regulated in a clonal stable A549 cell line expressing a corresponding shRNA. Transcript levels were measured in wild type and knockdown cells by qRT-PCR. Mean levels and standard deviation from technical triplicates are indicated. Promoter activity of CDKN1A (E) and MDM2 (F) were quantified in Set8 knock-down cells before (basal, grey) and 3 h (red), 6 h (blue) and 9 h (orange) after DNA damage (10 Gy IR). Left panel: The percentage of cells with active TSS, subdivided into populations with strong (> 75% of TSS, solid colors) and weak (<75% of TSS, shaded colors) activity, is shown as stacked bar graphs, the mean fraction of active promoters is indicated above each bar. Right panel: Distributions of calculated transcription rates at active TSS are presented for each time point as probability density estimates (PDF, see Data Visualization section). We measured a higher fraction of active promoters upon damage compared to A549 wild type cells (Figure 3), while transcription rates remained unchanged.
Figure 6Schematic illustrations of the experiments in (C) and (D). Embryoid bodies were incubated with: (i) RAhi (1 µM) for 48 h; (ii) RAhi for 72 h; (iii) RAhi for 48 h then switched to RAlow (100 nM) for 24 h; (iv) RAhi for 48 h then switched to RAlow plus RAR inhibitor (1 µM) for a further 24 h; (v) RAlow for 48 h; (vi) RAlow for 72 h. Immunostaining (C) of EBs 96 h after ESC differentiation, and quantification (D) of Hoxa5on in MNs (Hb9on). Schematic illustration of the experiments in (F) and (G). Inducible ESC lines expressing eight repetitive mir-27b sponge sequences were inserted into the GFP 3' UTR. ESCs were differentiated by doxycycline treatment after 48 h of differentiation. A scrambled sequence was inserted as a control. Hoxa5 expression upon miR-27 knockdown, with exposure to RAhi for 72 h, or with exposure to RAhi for 48 h, and then switching to RAlow plus RAR inhibitor (1 µM) for a further 24 h, or with exposure to RAmed for 72 h. Bifurcation analysis of a mathematical model based on our mmi-3 Model. Steady states of the system in the presence of miR-27 (red, gray, and blue) or in the absence of miR-27 (purple) at various concentrations of RA are shown. Solid curve: stable steady state. Dashed curve: unstable steady state (See Appendix Supplementary Methods for parameter values).
Figure 2A. Schematic representation of the AOM/DSS procedure. To develop colitis-associated cancer (CAC), WT (n=11) and OMA1-/- (n=12) mice were injected intraperitoneally with AOM (10 mg/kg) on day 0. Then, three cycles of feeding water with 1.5% DSS treatment were administered. Mice were euthanized on day 105 and all intestinal tissues, tumors, and serum were collected. After mice were euthanized, intestines were removed and flushed with cold PBS. All experiments were repeated with three independent biological replicates. B. Body weight changes of WT (n=11) and OMA1-/- (n=12) mice during AOM/DSS treatment. Data are presented as mean ± SEM. Statistical significance was assessed by a two-way ANOVA, **p < 0.01. C. Typical intestine images on day 105 after mice were euthanized. D. Colorectal lengths (C) of WT (n=11 biological replicates) and OMA1-/- (n=12 biological replicates) mice were measured. Data with error bars are presented as mean ± SEM. Statistical significance was assessed by unpaired student's t-test, *p < 0.05. E. Representative images of colorectal tumors from WT (left) and OMA1-/- (right) mice. F. The number of tumors in WT (n=11) and OMA1-/- (n=12) mice was measured. Each dot represents the tumor number of one individual mouse. Data with error bars are presented as mean ± SEM. Statistical significance was assessed by unpaired student's t-test, ***p < 0.001. G, H. The number of tumors of different sizes (diameter) in each mouse (G) and the distribution of different tumor sizes (H) were shown (WT n=11 biological replicates, and OMA1-/- n=12 biological replicates). Results are shown as mean ± SEM. p-values were calculated by unpaired student's t-test, **p < 0.01, ***p < 0.001. I. Histological analysis of colorectal tumors of WT and OMA1-/- mice was shown by hematoxylin and eosin (H&E) staining. J. Relative mRNA expression levels of pro-inflammatory genes (TNFα, IL-6, COX-2, CCL2) in colorectal homogenates of WT and OMA1_KO mice treated with AOM/DSS (n =4-5 per genotype). The data represent the mean ± SEM., n = 3 independent experiments, and statistical significance was determined by a two-tailed student's t-test. *p < 0.05, **p < 0.01. K. The colorectal tissues of WT and OMA1-/- mice were stained with Ki67.
Figure 3A Semiquantitative RT-PCR of UPF1SL or UPF1LL transcript levels following transfection of HEK-293 cells with the indicated siRNAs. B RT-qPCR analysis of indicated transcripts following transfection of a NT siRNA or SRSF1-specific siRNA under conditions of CLIP-UPF1SL or CLIP-UPF1LL overexpression. Relative fold changes are in reference to the GFP expressing control line treated with a NT siRNA. Asterisk (*) indicates P < 0.05, as determined by unpaired Student's t-test. Black dots represent individual data points and error bars indicate mean±SD (n = 3 biological replicates). C Density plot of changes in relative mRNA abundance as determined by RNA-seq following SRSF1 overexpression Genes were categorized as up-regulated by siUPF1total only, siUPF1LL only, or both siUPF1total and siUPF1LL. Statistical significance was determined by K-W test, with Dunn's correction for multiple comparisons. D Density plot as in (C), with genes binned according to enrichment in the CLIP-UPF1LL or CLIP-UPF1SL affinity purifications.
Increased NF-κB binding in Brd4 KO macrophages (A) Heat maps of p65 binding centered at the TSS on BRD4 enriched promoters.Figure 6(A) Heat maps of p65 binding centered at the TSS on BRD4 enriched promoters. (B) Meta-gene profile of p65 ChIP-seq signals (rpm/bp) from the TSS to TES and +/- 5kb on BRD4 independent genes (top) and BRD4 dependent genes (bottom). (C) Gene tracks of BRD4 and p65 ChIP-seq peaks and RNA-seq profiles on a BRD4 independent gene (Il1b), a gene upregulated in Brd4KO macrophages (Ccl7), and a BRD4 dependent gene (Ptgs2). (D) Enhancers ranked by increasing p65 signals in WT (left) and Brd4 KO macrophages (right). (E) Meta-gene analysis of p65 occupancy on enhancers in WT (red) and KO macrophages (Blue).
Biochemical properties of PHD ‘closed’ and ‘open’ states.Figure 4(A) Design of dynamin mutants to restrict the PHD in the closed (Dyn1Closed) or open (Dyn1ΔΔ) state. (B) Histogram shows basal and assembly-stimulated GTPase activities of non-crosslinked Dyn1CC vs crosslinked Dyn1Closed and Dyn1WT. Inset shows crosslinking of the PHD and stalk domains in Dyn1Y354C/S607C (Dyn1CC) with the bifunctional crosslinker, MTS-2-MTS, to yield Dyn1Closed, which migrates more slowly on SDS-PAGE, as well as liposome binding of Dyn1CC compared to Dyn1Closed (Bound, B; Unbound, UB). (C) Ability of Dyn1WT, Dyn1CC and Dyn1Closed to catalyze membrane fission and vesicle release from SUPER templates. (D) Size exclusion chromatographic profiles of Dyn1CC, Dyn1Closed at room temperature or after incubation for 10 min at 37°C. (E) Oligomeric state of Dyn1ΔΔ at room temperature. (F) Negative-stain electron micrographs of Dyn1WT, Dyn1ΔΔ and Dyn1Closed assembled onto lipid nanotubes. Scale bar = 200 nm (G) Curvature dependent liposome-stimulated GTPase activity of Dyn1WT and Dyn1ΔΔ. (H) Ability of Dyn1WT and Dyn1ΔΔ to catalyze membrane fission and vesicle release from SUPER templates.
Figure 3Genotypes are indicated as WT for S100a4cre-/- x Fstl1flox+/flox+ mice and cfKO for S100a4cre+/- x Fstl1flox+/flox+. (A) S100a4 expression is induced in the ischemic area (IA). qPCR analysis of mRNA expression of S100a4 in ischemic heart and sham operated heart of WT mice and cfKO mice. Heart samples were harvested at 7 days after the surgery. Statistical analysis was performed by two-way ANOVA. Post Hoc test was performed by Tukey test. Error bars represent mean ± SEM (n=16 and 15 for WT and cfKO sham group, n=15 and 14 for WT and cfKO MI group, respectively). Genotypes are indicated as WT for S100a4cre-/- x Fstl1flox+/flox+ mice and cfKO for S100a4cre+/- x Fstl1flox+/flox+. (B) qPCR analysis of mRNA expression of Fstl1 and Tgfβ1 in sham and post-MI heart. Error bars represent mean ± SEM (n=16 and 15 for WT and cfKO sham group, n=15 and 14 for WT and cfKO MI group, respectively). Statistical analysis was performed by two-way ANOVA. Post Hoc test was performed by Tukey test. Genotypes are indicated as WT for S100a4cre-/- x Fstl1flox+/flox+ mice and cfKO for S100a4cre+/- x Fstl1flox+/flox+. (C) Western blot analysis of Fstl1 protein expression in ischemic and sham-operated hearts at day 7 after the surgery. Quantified values of Fstl1 protein in WT and cfKO mouse hearts normalized by GAPDH band intensity are shown. Statistical analysis was performed by two-way ANOVA. Post Hoc test was performed by Tukey test. Error bars represent mean ± SEM (n=5 for each sham group and n=6 for each MI group). Genotypes are indicated as WT for S100a4cre-/- x Fstl1flox+/flox+ mice and cfKO for S100a4cre+/- x Fstl1flox+/flox+. (D) Fstl1 protein expression in isolated cardiac fibroblasts from cfKO and littermate WT neonatal mice. Cell lysate and its media from cells cultured for 24 hours without FBS were assessed by western blotting. Error bars represent mean ± SEM (n=3 per group). Statistical analysis was performed by unpaired t-test (2-tailed). The experiments were performed twice independently.
Figure 5A. Cell proliferation of DAOY cells upon Fbxl17 depletion using a non-targeting siRNA (Control), two siRNAs against Fbxl17 (1) and (2) or upon reintroduction of a Myc-tagged Fbxl17 construct in Fbxl17-depleted DAOY cells using siRNA Fbxl17 (2) (mean ± SEM from 3 independent experiments, **p<0.005; ***p<0.0005). B. Quantification of Fbxl17 mRNA levels in DAOY cells transfected with a non-targeting siRNA (Control) or two siRNAs against Fbxl17 (1) and (2) (mean ± SEM from 3 independent experiments, **p<0.005). C. Sufu protein levels in DAOY cells treated as in A. Representative image of three independent experiments is shown. D. Cell proliferation of DAOY cells transfected with non-targeting siRNA (Control), siRNA against Fbxl17 or a combination of siRNA targeting Fbxl17 and Sufu. (mean ± SEM from 3 independent experiments, **p<0.005; ***p<0.0005). E. Quantification of Fbxl17 mRNA levels in DAOY cells treated as in D mean ± SEM from 3 independent experiments, ***p<0.0005). F. Sufu protein levels in DAOY cells treated as in D. Representative image of three independent experiments is shown.
Figure 2Scores of principal components 1 (PC1) of each sample from the transcriptome (control, n=14; mild, n=179; severe, n=37), proteome (control, n=12; mild, n=80; sever, n=57), metabolome (control, n=12; mild, n=42; sever, n=20), and exRNA-seq (control, n=14; mild, n=179; sever, n=37) principal component analyses. Differences between groups were estimated using Kruskal-Wallis test. The horizontal box lines in the boxplots represent the first quartile, the median, and the third quartile. Whiskers denote the range of points within the first quartile − 1.5× the interquartile range and the third quartile + 1.5× the interquartile range. Circos plots showing the significant correlations between clinical parameters and the multi-omics data.
a) Ratios from proteins with quantifications from all samples were log2 transformed and z-score normalized.f3(a) Ratios from proteins with quantifications from all samples were log2 transformed and z-score normalized. Columns containing data from the different samples were clustered hierarchically and rows that contained protein entries were clustered by k-means. The proteins in each k-means cluster were tested for enrichment of GO CC (indicated in red) and GO BP (indicated in blue) terms. (b) Proteins associated with different cellular compartments were sequentially extracted and the average of their ratios were compared with averages of remaining proteins. (c) The temporal dynamics of the protein ratios in each mutant was clustered by Fuzzy c-means. (d) GO BP enrichment analysis of proteins in the different clusters compared with the remaining proteins.
DRP-1 and DAPk dominant negative mutants reduce membrane blebbing resulting from p55/TNFR1 transfections in 293 cells.Figure 6.(A) 293 cells were cotransfected with p55/TNFR1 in combination with DAPk death domain fused to GFP or DRP-1 K42A and pEGFP-N1 vector. A control luciferase vector was also used. GFP-positive cells were visualized by fluorescent microscopy and scored for the appearance of membrane blebbing 24 h after transfection. (B) Photographs of the various categories of p55/TNFR1-transfected 293 cells: spread cells (1), blebbed cells (2), condensed cells (3), cells displaying a fragmented cytoplasm (4), and cells carrying fragmented nuclei within a noncondensed cytoplasm (5). (C) Quantification of each death morphology observed 24 h after transfection with GFP, p55/TNFR1, and either luciferase or DRP-1 K42A.
Figure 7(A) Comparison of mEPSCs in cortical layer 2/3 pyramidal neurons from P30 - P40 WT and CA7 KO mice. Sample traces of mEPSC recordings from WT and CA7 KO neurons, low-pas filtered at 1 kHz (left). The data are summarized in the bar diagrams (right). mEPSC frequency (P = 0.63) and amplitude (P = 0.90) were not significantly different between WT and CA7 KO and neurons (n = 7 and 5 neurons, respectively, Student's independent samples t-test). (B) Representative confocal images of apical dendrites from Lucifer Yellow injected cortical layer 2/3 pyramidal neurons from WT and CA7 KO mice. The dendritic spine density and spine head size were examined in fixed slice preparations from P34 - P37 mice. Scale bar 2 µm. (C) Summary of the spine density analysis done from the Lucifer Yellow injected neurons. Spine density was a higher in CA7 KO neurons both in apical and basal dendrites (n = 28 neurons for both) compared to WT (n= 29 neurons for apical and n=30 for basal dendrite analysis) (P = 0.000002 for apical dendrites, analyzed with Mann-Whitney test, and P = 6,8 x 10-8 for basal dendrites, Student's t-test with Welch-correction.) A total of 8279 spines were analyzed from four CA7 KO mice and 8730 spines from two WT control mice. (D) The spine head width distribution7differed significantly between the genotypes (n= 467 spines from WT and n = 421 spines from CA7 KO animals, 15 neurons analyzed from both genotypes, Wilcoxon rank sum test with continuity correction, W = 134540, P < 0.001).