EMBO/sd-character-level-ner huggingface dataset:
Figure 1A) The first mouse experiment performed at LUMC was set up to compare gene expression profiles of healthy and dystrophic mice. It involved 40 mice and resulted in the collection of two datasets, one with cross-sectional RNA-sequencing in muscle (MoMus) and the other with longitudinal RNA-sequencing measurements in blood (MoLong). B) The second mouse experiment performed at LUMC aimed to assess the effect of dystrophin restoration by two antisense oligonucleotides on blood gene expression. It involved 15 dystrophic mice and led to the generation of a cross-sectional dataset (MoTreat) with blood RNA-sequencing data. C) The observational study carried out at SMDM involved 18 healthy subjects and 39 DMD patients. It resulted in the collection of a cross-sectional dataset (DMDMex) with blood RNA-sequencing data.
Figure 5Representative blots showing LATS and YAP protein levels in hOSEs at the fourth, ninth and thirteenth passage. β-Actin was used as a protein loading control. Representative gel photographs showing mRNA levels of LATS1 and LATS2 in hOSEs with or without LATS2 knockout. CRISPR/Cas-9 system was used to knock out LATS2 in hOSEs. LATS1/2 mRNA levels were analyzed using qRT-PCR at the fourth, ninth and thirteenth passage. Representative images showing the expression and location of YAP in control and LATS2 knockout hOSE cells. Cells were collected at the 4th passage and YAP expression was examined by fluorescent immunohistochemistry. YAP protein was visualized using an Alexa-488 (Green) conjugated secondary antibody. Nuclei were stained with DAPI. Representative images showing YAP protein expression in the 13th passage hOSE Cells with or without LATS2 knockout. YAP protein was examined by fluorescent immunohistochemistry and visualized using an Alexa-488 (Green) conjugated secondary antibody. Nuclei were stained with DAPI (blue). Representative images showing morphological change (E) in the 13th passage (P13) hOSE cells with (LATS2 KO) or without (CTRL) LATS2 knockout. Representative images showing SA-β-gal staining (F) in the 13th passage (P13) hOSE cells with (LATS2 KO) or without (CTRL) LATS2 knockout. Scale bar: 50 µm. Quantitative data showing the ratio of SA-β-gal positive cells in the control (CTRL) and LATS2-knockout (LATS2KO) P13 hOSE cells.
POMC neuronal autophagy is required for lipolysis.Figure 3(A) Percent loss of body fat and (B) lean mass in 24‐h‐fasted Con and KO mice on RD or HFD (n=6-8). (C) Histology for eWAT from fed or 6‐h‐fasted Con and KO mice on RD (n=3-4). (D) Serum fatty acids and (E) glycerol from fed or 6‐h‐fasted Con and KO mice on RD (n=7-9). (F) Serum fatty acids and (G) glycerol after intraperitoneal isoproterenol (Iso) in 6‐h‐fasted Con and KO mice on RD (n=5-6). (H) Immunoblot for oxidized proteins in eWAT from RD‐fed Con and KO mice (n=3). (I) FACS analysis for M1 and M2 macrophage proportions in adipose stromal vascular fractions from fed Con and KO mice on RD (n=3). Values are mean±s.e.m. P values are as compared with diet‐ and age‐matched controls. *P0.05, **P0.01, ***P0.001. Con, control; eWAT, epididymal fat; FACS, fluorescence‐activated cell sorting; HDF, high‐fat diet; Iso, isoproterenol; KO, knockout; POMC, proopiomelanocortin; RD, regular chow; Stv, fasted.
The expression of piRs-3-I2, piRs-3-I3, and pre-NPF1.Figure 7A U6 RNA was used as a nuclear RNA control (relative value set to 1 in the nuclear fraction), and glycine tRNA was used as the cytoplasmic RNA control (relative value set to 1 in the cytoplasmic fraction) (n=4 biological replicates), U6 and tRNA-Gly was used as an endogenous control. B Relative expressions of piRs-3-I2 and piRs-3-I3 determined by qPCR and high-throughput sequencing in the nucleus (n=3 biological replicates). The brain tissues were homogenized and then divided into two parts of equal volume; one was prepared for high-throughput sequencing and the other was used for piRNA quantification in nuclear and cytoplasmic fractions after subcellular RNA fractionation. The relative piRNA expression in nucleus was determined by multiplying the relative nuclear piRNA fraction estimate by qPCR quantification by the normalized count of piRNA in high-throughput sequencing libraries. Data information: The qPCR data are shown as the mean ± SEM (Student's t-test, *P<0.05, **P<0.01, ***P<0.001). C Expression level of pre-NPF1 in the nucleus after a mixture of inhibitors (piRs-3-I2 and piRs-3-I3) was injected (n=4 biological replicates). D qPCR expression analyses of pre-NPF1. The precursor mRNA expression level was measured by qPCR after the dsPiwi1 treatments (n=6 biological replicates). E Data information: The qPCR data are shown as the mean ± SEM (Student's t-test, *P<0.05, **P<0.01, ***P<0.001). E Expression levels of the mature NPF1 gene and pre-NPF1 in 293T cells transfected with piRs-3-I3 and mutated piRs-3-I3 mimics (n=5 biological replicates). F Analysis was performed to amplify pre-NPF1 and piRs-3-I3 in hnRNP F/H immunoprecipitates from extracts of brain tissue treated with piRs-3-I3 mimics compared to the negative controls (pre-NPF1 n=3 biological replicates; piRs-3-I3 n=4 biological replicates). G Analysis was performed to amplify pre-NPF1 in U2AF65 immunoprecipitates from extracts of brain tissue treated with piRs-3-I3 mimics compared to the negative controls (pre-NPF1 n=3 biological replicates). H H The predicted secondary structures of WT and mutated pre-NPF1. I Expression level of pre-NPF1 following ck and RNase III treatment (n=7 biological replicates). The center line of the boxplots represents median value, the bounds of the box represent 75th and 25th percentile, and the whiskers represent maximum and minimum value. J Expression level of the mature NPF1 gene and pre-NPF1 in 293T cells transfected with pre-NPF1 and mutated pre-NPF1 (n=5 biological replicates). NC means negative control. The center line of the boxplots represents median value, the bounds of the box represent 75th and 25th percentile, and the whiskers represent maximum and minimum value. Data information: The qPCR data are shown as the mean ± SEM (Student's t-test, *P<0.05, **P<0.01, ***P<0.001).
Figure 4Mouse fibrosarcoma MCA205 cells were stained with CellTracker Orange (CMTMR) and treated for 24 h with 1 µM dactinomycin (DACT) or 500 µM oxaliplatin (OXA) as a positive control. Then, untreated or dying MCA205 were co-cultured with differentiated bone marrow-derived dendritic cells (BMDCs) for 4 h at 37 °C or at 4 °C. Cells were collected and dendritic cells were stained with CD11c specific antibody before analysis by flow cytometry MCA205 cells were treated for 24 h with 500 µM oxaliplatin (OXA) or with 0.25, 0.5 or 1 µM dactinomycin (DACT). Then, untreated or dying MCA205 were co-cultured with BMDCs for 24 h at 37 °C. As a positive control, BMDCs were co-cultured with 1 μg/mL LPS and 100 ng/mL IFNγ. Cells were collected and stained with a marker of viability, as well as with CD11c, MHCII and CD86 specific conjugated antibodies and analyzed by flow cytometry Mouse fibrosarcoma MCA205 cells were stained with CellTracker Orange (CMTMR) and treated for 24 h with 1 µM dactinomycin (DACT) or 500 µM oxaliplatin (OXA) as a positive control. Then, untreated or dying MCA205 were co-cultured with differentiated bone marrow-derived dendritic cells (BMDCs) for 4 h at 37 °C or at 4 °C. Cells were collected and dendritic cells were stained with CD11c specific antibody before analysis by flow cytometry The percentage of CMTMR and CD11c double positive cells among all CD11c+ cells are indicated MCA205 cells were treated for 24 h with 500 µM oxaliplatin (OXA) or with 0.25, 0.5 or 1 µM dactinomycin (DACT). Then, untreated or dying MCA205 were co-cultured with BMDCs for 24 h at 37 °C. As a positive control, BMDCs were co-cultured with 1 μg/mL LPS and 100 ng/mL IFNγ. The increase in the percentages of MHCII+ and CD86+ cells among CD11c+ cells was quantified with respect to untreated controls. 1 x 106 mouse fibrosarcoma MCA205 cells were treated in vitro with 1 µM dactinomycin (DACT). Dying cells were harvested and injected subcutaneously (s.c.) into one flank of immunocompetent syngeneic C57Bl/6 mice (n=8 mice) to assess vaccination efficacy. PBS was injected in the control group (Ctr). Two weeks later, animals were rechallenged with 1x105 untreated MCA205 cells in the contralateral flank of the animals 1 x 106 mouse fibrosarcoma MCA205 cells were treated in vitro with 1 µM dactinomycin (DACT). Next, tumor size was measured regularly and individual tumor growths of DACT-vaccinated versus Ctr mice are depicted 1 x 106 mouse fibrosarcoma MCA205 cells were treated in vitro with 1 µM dactinomycin (DACT). Overall survival is depicted and p-values (*** p<0.001.) were calculated with a Log-Rank test
a) Cells untreated, or treated with chloroquine for 24 h, were fixed and stained for anti-pTyr-416-Src (red) and anti-c-Cbl (green).figf7(a) Cells untreated, or treated with chloroquine for 24 h, were fixed and stained for anti-pTyr-416-Src (red) and anti-c-Cbl (green). Solid arrows indicate co-localization at adhesions and dashed arrows indicate co-localization in autophagosomes. Scale bars, 20 μm. (b) c-Cbl was immunoprecipitated (IP) from FAK+/+ and FAK−/− cells and then immunoblotted with anti-Src, anti-pTyr-416-Src and anti-c-Cbl antibodies. (c,d) Cells were transfected with scrambled siRNA or with c-Cbl siRNA for 72 h and then immunoblotted with anti-c-Cbl and actin antibodies (c) or stained for anti-pTyr-416-Src (red) and anti-c-Cbl (green; d, left). Dashed arrows indicate co-localization and solid arrows indicate Src at focal adhesions. Scale bars, 20 μm. Quantification is shown (d, right). Data are presented as mean±s.d. and significance is P>0.5 for FAK+/+ cells and P0.001 for FAK−/− cells (n=3). (e) LC3B was immunoprecipitated from FAK−/− cells expressing either scrambled or c-Cbl siRNA and then immunoblotted with anti-Src and anti-LC3B antibodies. Uncropped images of blots are shown in Supplementary Fig. S9.
Figure 6(A) CHD1-depleted cells show hypersensitivity to Mitomycin (MMC) treatment. For colony formation assay shCont and shCHD1 PC3 cells were treated with the indicated MMC concentrations for 4 h and surviving fractions were measured by counting colonies after 3 weeks and mean values were represented (n=3, ± SD, p-value=0.0002, ANOVA, ***p ≤ 0.001). (B) CHD1-depleted cells show increased hypersensitivity to Irinotecan. For cell proliferation analysis, shCont and shCHD1 cells were treated with 1 µM Irinotecan and proliferation was measured by Celigo and the relative confluency (mean) was plotted in the graph (n=3, ± SD, p-value=0.0004, ANOVA, ***p ≤ 0.001). (C) Loss of CHD1 leads to increased sensitivity to PARP inhibition. Control or CHD1-depleted BPH1 cells were treated with the indicated concentrations of the PARP inhibitor olaparib and surviving fractions were measured by counting colonies after 3 weeks. Data represented as mean values (n=3, ± SD, p-value=0.0003, ANOVA, ***p ≤ 0.001). (D) CHD1 depletion render cells sensitive to PARP inhibition in combination with irradiation. shCont- or shCHD1-expressing BHP1 cells were treated with 1 µM of PARP inhibitor olaparib for 2 h before irradiation with indicated doses of X-rays and surviving fractions (normalized to the unirradiated condition) were measured by counting colonies after 3 weeks. Data is represented as mean ± SD (n=3, p-value=0.07, ANOVA, *p ≤ 0.1). (E) Frequency of CHD1 gene alterations in comparison with DSB repair genes in prostate cancer patients. Data obtained from cBioPortal for Cancer Genomics. (F) Frequency of CHD1 gene alterations from indicated data sets. See also Figure EV5A-EV5B.
Figure 5A time-lapse imaging of a representative MII oocyte with visible lagging chromatid at midzone during anaphase II expressing CENP-C- EGFP (centromeres, green) and H2B-mCherry (chromatin, red). Upper row shows maximum intensity z projection images from representative timepoints at metaphase II and anaphase II. A lagging chromatid is indicated by a blue arrowhead. Number next to the circles enclosing the segregating chromatids denotes number of chromatids at each pole. The bottom row depicts 3D positions for centromeres of normally segregating chromosomes as black dots, and centromeres of laggard-producing chromosome as red dots in a side view along the spindle equator. One of the centromeres is left behind at anaphase (blue arrowhead), but will segregate equationally. Tracks of individual centromeres are color-coded according to the speed, as indicated by the colour bar. Time is shown relative to anaphase II onset (min:sec). Scale bars, 5 μm.
Anti-MAPK therapy affects signaling networks and transcriptomes contingent on predictive mutations in organoids.Figure 5A Workflow of the experimental therapy experiment. In short, organoids were treated for 48h with inhibitors before disaggregation into single cells for CyTOF and scRNA-seq analysis. For scRNA-seq, organoids were labelled for 2h with 4sU. B Schematic representation of signaling network and transcriptional signatures associated with phenotypes or signaling pathways, as indicated. C Heatmap of key CyTOF data. Average activities of selected analytes are given as log fold-change after normalization to DMSO control condition. Range of color scale was adjusted for each analyte. For relative changes between all analytes, see Appendix Fig. S7). D Heatmap of signature gene expression, from scRNA seq data. Average activities of selected gene signatures are given as log fold-change after normalization to DMSO control condition. Range of color scale was adjusted for each signature. DNA repair and Apoptosis are Broad Institute Hallmark signatures. E Correlations between and within the CyTOF and scRNA-seq data sets. For each line, average protein analyte (CyTOF) or transcriptional signature (scRNA-seq) values were plotted for all six experimental conditions. Graphs give trend line and confidence intervals. F Upregulation of MAPK and YAP target genes in mouse intestinal organoids after induction of oncogenic BRAF. Wnt target genes is not significantly affected. For experimental details, see Riemer et al. (2015). Graph displays data points for signature gene expression values, mean and standard deviation. **** indicates p value of <0.0001 in two-tailed paired t test of gene expression ratios (luciferase control versus BRAF-induced). n.s.: not significant.
a) Targeting of autophagolysosomes to mitochondria in the absence or presence of C18-Pyr-Cer (C18PC) at 2 h in UM-SCC22A (22A) cells was examined by visualizing the colocalization of MTG (green) and LTR (red) using live-cell imaging and confocal microscopy.figf2(a) Targeting of autophagolysosomes to mitochondria in the absence or presence of C18-Pyr-Cer (C18PC) at 2 h in UM-SCC22A (22A) cells was examined by visualizing the colocalization of MTG (green) and LTR (red) using live-cell imaging and confocal microscopy. Scale bars, 10 μm. Enlarged images are shown in boxed areas. (b) Effects of C18-dihydro-Cer-14-piperidine (dihydro-C18PC) on OCR measured using the SeaHorse alongside those of C18PC- and vehicle-treated controls. Data shown are an average of at least two independent experiments performed in duplicate ± s.d. (*P 0.05).
Figure 7Yeast spot growth assays to monitor the ability of Rad54, Rdh54, Rad54NRdh54, and Rdh54NRad54 to complement MMS sensitivity in rad54∆ yeast strains.   Yeast spot growth assays to monitor the ability of Rad54, Rdh54, Rad54NRdh54, and Rdh54NRad54 to complement MMS sensitivity in rdh54∆ yeast strains.   Models depicting the relationship between binding sites and functional status for Rad54 and Rdh54 bound to a mitotic Rad51-ssDNA presynaptic complex. Speculative model for the distribution of Rad54 and Rdh54 within meiotic presynaptic complexes. Note, the Rad54 and Rdh54 binding sites are shown at the "top" and "bottom" of the Rad51-ssDNA filaments for convenience only to illustrate that they occupy different sites within the PSC; a more detailed understanding of their binding sites will require high resolution structural data. Additional details of the model are presented in the text.
The ATG16L1-binding motif present in TMEM59 recognizes the WD-repeat domain of ATG16L1.Figure 7(A-F) 293T cells were transfected with the indicated constructs, lysed and subjected to GST immunoprecipitation with agarose beads coupled to glutathione. Shown are WBs against the indicated molecules. (A) A deleted version of ATG16L1 lacking the WD domain (HA-ATG16L1-ΔWD) does not co‐precipitate with TMEM59-GST. (B) TMEM59 does not co‐precipitate with ATG16L1-ΔWD fused to GST. (C) The WD domain of ATG16L1 (HA-ATG16L1-WD) suffices to co‐precipitate with TMEM59-GST. (D) TMEM59 co‐precipitates with ATG16L1-WD fused to GST. (E) HA-ATG16L1-WD does not bind a 4M version of TMEM59-GST. (F) TMEM59-4M does not co‐precipitate with the ATG16L1-WD fused to GST.Source data for this figure is available on the online supplementary information page.
Figure 2Primary murine dermal fibroblasts, which had been treated with recombinant activin A at different concentrations, were analyzed for BrdU incorporation. N=3-4. Representative images of collagen gels and quantification of the extent of gel contraction (percentage of initial gel size) in the presence or absence of activin A (20 ng/ml) and/or TGF-β1 (1 ng/ml). N=3-4. qRT-PCR and Western blot analyses of ACTA2/αSMA expression using total RNA/lysates of fibroblasts treated with activin A or TGF-β1 for 6 h. N=3. The membrane was re-probed with GAPDH and mDia2 antibodies. Representative images of fibroblasts treated with activin A, TGF-β1 or vehicle (control) as in (C), stained for αSMA (green), counterstained with rhodamine-coupled phalloidin (red) and Hoechst (blue). Bar graph shows percentage of fibroblasts with stress fibers. N=3. epresentative images of control and activin A-treated fibroblasts stained with rhodamine-coupled phalloidin; scatter plot shows quantification of filopodium length in control, activin A- and TGF-β1-treated fibroblasts. N=27-70 filopodia. Scratch assay of fibroblasts treated with activin A or vehicle for 24 h. N=3. The area covered by cells in the original scratch was quantified. Migration in one control culture was set to 1. Chemotactic migration of primary mouse fibroblasts from wt mice for 24 h in a transwell assay towards increasing concentrations of activin A or vehicle. N=3. qRT-PCR analysis of CAF marker genes relative to Rps29 using RNA from fibroblasts treated with activin A for 6 h. N=3.
Figure 6A, B. Representative confocal images of mitochondrial fusion in Drp1-/- 293T polykaryons in cultures subjected to the PEG-based fusion assay in the presence or absence of exogenous hFis1. Drp1-/- cells stably expressing mitoGFP or mitoRFP were co-cultured, subsequently transfected with empty vector (control) or Myc-hFis1 as indicated and then stimulated with PEG treatment for cell fusion, followed by immunostaining with anti-Myc antibody (green). Mitochondrial fusion was indicated by co-localization of mitoGFP and mitoRFP (i.e. yellow mitochondria). Left panel: empty vector transfected; right panel: Myc-hFis1 transfected. Insets represent magnification of the boxed areas in upper panel (A). Quantitative analyses for measuring the extent of mitochondrial fusion in individual hybrid cells were performed by the Pearson's correlation coefficient (PCC) in three independent experiments and summarized in (B). C, D. Representative confocal images of mitochondrial fusion in Drp1-/- 293T cells assessed by the mito-PAGFP-based fusion assay in the presence or absence of exogenous hFis1. Drp1-/- cells co-transfected with mito-PAGFP, mito-DsRed, and either empty vector (the upper panel) or hFis1 (the lower panel) were photoactivated in a small region of interest (ROI) (white circle, 3 µm diameter) as indicated in preactivation images of mitochondria seen by mito-DsRed (red). After photoactivation, Z-stack images with photoactivated GFP (green) and mitochondrial marker (mito-DsRed) were collected at times 40 sec, 15, 30, 45 min as indicated (C). Mitochondrial fusion was quantified by analysis of changes in the fluorescence intensity of photoactivated mito-PAGFP in ROIs at 40 sec, 15, 30, 45 min after photoactivation. The dilution rates (percentage) of the GFP fluorescence intensity at different time points were normalized by the fluorescence intensity at 40 sec after photoactivation (D). E, F. Representative confocal images of mitochondrial fusion in Drp1-/- 293T polykaryons in cultures subjected to the PEG-based fusion assay in the presence or absence of endogenous hFis1. Drp1-/- cells stably expressing mitoGFP or mitoRFP were transfected with scrambled siRNA (control siRNA) or with hFis1-siRNA as indicated and then co-cultured and fused by PEG treatment. Left panel: control siRNA transfected; right panel: hFis1-siRNA transfected (E). Quantitative analysis of extent of mitochondrial fusion in individual hybrid cells was performed by the Pearson's correlation coefficient (PCC) in three independent experiments and summarized in (F). G, H. Representative confocal images of mitochondrial fusion in Drp1-/- 293T cells monitored by the mito-PAGFP-based fusion assay in the presence or absence of endogenous hFis1. Scrambled siRNA or hFis1 siRNA treated cells were co-transfected with mito-PAGFP and mito-DsRed and were subsequently photoactivated (G) as described in (C). Mitochondrial fusion was quantified by analysis of changes in the fluorescence intensity of photoactivated mito-PAGFP (H) as described in (D).
Model for functional redundancy of UPF3A and UPF3B An NMD activating mRNP composition will result in the activation of NMD in the presence of UPF3B (left part) or when UPF3B was depleted (middle part).Figure 8An NMD activating mRNP composition will result in the activation of NMD in the presence of UPF3B (left part) or when UPF3B was depleted (middle part). In the absence of UPF3A and UPF3B, NMD is inactive (right part). In normal cells, UPF3B can engage in multiple, potentially transient interactions (depicted by blue sphere), e.g. with the terminating ribosome via eRF3a, with UPF2 or with the EJC (left). When UPF3B is missing, UPF3A can at least partially compensate for it (depicted by pink sphere, middle). UPF3A interacts with UPF2 and possibly also with eRF3a. The interaction of UPF3A with the EJC is weaker than that of UPF3B, therefore UPF3A cannot establish a stable bridge between UPF2 and the EJC (middle).