EMBO/sd-character-level-ner huggingface dataset:
Figure 2Unsupervised hierarchical clustering (Ward linkage with Euclidean distance) of CNA profiles generated from patient primary tumors, the vaginal metastasis, isolated single and pooled CTCs from the primary blood collection and CTC-ITB-01 (early and later passage) and leukocytes. "CL" represents the cell line, followed by the passage analyzed (p04/p30). "CTC" marks circulating tumor cells isolated from the initial blood draw, followed by 01/05/10 indicating single (01) or pooled cells (5 or 10, respectively). Light and dark grey bars to the left signify chromosomes, starting from the top with chromosome 1. Red color represents chromosome loss, blue represents gain. All copy number aberrations were calculated using the patient's own germline DNA extracted from leukocytes as reference to eliminate copy number variations (CNVs).
Membrane topology of MPC proteins A, B Determination of MPC membrane topology by IASD labeling of isolated mitochondria expressing the indicated single cysteine variants of Mpc1 (A) and Mpc3 (B).Figure 3A, B Determination of MPC membrane topology by IASD labeling of isolated mitochondria expressing the indicated single cysteine variants of Mpc1 (A) and Mpc3 (B). Mitochondria were treated with IASD as indicated. Negative [quenching by pretreatment with DTT (pre‐DTT)] and positive [lysis with 0.5% Triton X‐100 (T‐X100)] controls for the labeling reaction are shown. As a further control, also translocase of the inner membrane subunit Tim23 is detected by Western blot, which contains three endogenous cysteines, all of which are located in membrane‐embedded regions. Labeling of an IMS‐exposed cysteine with IASD is indicated by a mobility shift on SDS-PAGE corresponding to the molecular weight of IASD (˜500 Da). C Protease protection assay in mitochondria expressing Mpc1‐GFP, Mpc2‐GFP, or Mpc3‐GFP. Intact mitochondria (M), mitoplasts with a ruptured outer membrane after hypo‐osmotic swelling (Sw), or mitochondrial lysates with 0.5% Triton X‐100 (Tr) were treated with proteinase K (PK). Loading controls are Tom70 (outer membrane), Tim23 (inner membrane), and Mdh1 (matrix). GFP‐fused Mpc2 and Mpc3 are degraded in mitoplasts, whereas Mpc1‐GFP is not. D Model showing the proposed membrane topology of Mpc1 and Mpc3. Whereas Mpc1 has only two transmembrane helices, Mpc3 has three.
Yap deficient adults are small and exhibit liver hypoplasia.Figure 3Fluorescent and histological analysis of liver morphology in dissected WT and yap-/- mutant adults on a lf:CFP background. The blue colour overlay represents the fluorescent liver tissue. Scale bar: 2mm (brightfield, BF) and 50μm (histology, H+E) Quantitative determination of body mass in WT and yap-/- adults. n=8, *p<0.05, two-sided Student's t-test; values represent the mean ± s.e.m Quantitative determination of liver size in WT and yap-/- adults as determined by measuring liver mass as a percentage of body mass. n=12, *p<0.05, two-sided Student's t-test; values represent the mean ± s.e.m qPCR analysis of the hhex expression in adult liver tissue isolated from WT and yap-/- fish. n>3, **p<0.01, two-sided Student's t-test; values represent the mean ± s.e.m qPCR analysis of the prox1 expression in adult liver tissue isolated from WT and yap-/- mutant fish. n>3, *p<0.05, two-sided Student's t-test; values represent the mean ± s.e.m
null embryos exhibit altered expression of neural crest genes (A-B) Sox10 in situ hybridization in (A) wildtype and (B) Slc7a5-null E9.Figure 3(A-B) Sox10 in situ hybridization in (A) wildtype and (B) Slc7a5-null E9.5 embryos (n=4 each) with TSs of (a1, b1) the hindbrain at level of trigeminal ganglion V, (a2, b2) otic vesicles and the neural tube at more posterior levels (a3, a4, b3, b4). Scale bars 200 µm, except sections a1 - b4, 100 µm. Closed white arrowheads in (a3, a4) indicate neural crest, open white arrowheads indicate depleted neural crest in (b3) and position where neural crest should be in (b4). (C-G) Immunofluorescence on TSs caudal spinal cord of E9.5 wildtype littermates and Slc7a5-null embryos; Pax3 and FoxA2 were used as indicators for dorso-ventral organisation in (C, F) wildtype, n=2 (for FoxA2) and n= 4 (For Pax3) and (D, G) Slc7a5-null, n=3 (for FoxA2 ) and n = 3 for Pax3) neural tube. Arrowheads indicate border of Pax3 expression domain. Scale bars 50 µm. (E) Percentage of Pax3 expressing cells was determined by counting these cells and all DAPI labelled nuclei in the neural tube and comparison made between wildtype (4 embryos, 6 sections each) and Slc7a5-null (3 embryos, 6 sections each), each dot represents the average for one embryo, unpaired t-test, *p = 0.018 (see original source data). Error bars indicate SEM.
Shank2-/- mice ignore and abandon their pups after delivery (A) In contrast to the immediate care of Shank2+/+ dams shortly after delivery Shank2-/- dams neglect the offspring.Figure 1(A) In contrast to the immediate care of Shank2+/+ dams shortly after delivery Shank2-/- dams neglect the offspring. No proper nest building is observed, and pups are lying randomly scattered within the bedding. Right diagram: Average percentage of pups surviving until weaning per pregnancy. None of the litters of Shank2-/- mice survived after delivery, Mann-Whitney test, ***P<0.001, Shank2+/+ n = 12, Shank2-/- n = 9. (B, a-h) Series of pictures displaying the neglected appearance of pups delivered by Shank2-/- dams C), the percentage of pups gathered in the nest location is significantly reduced in Shank2-/- mice. Mann-Whitney test, ***P<0.001. Data information: All data are presented as mean ± SEM. D) Shank2-/- mothers fail to remove extra-embryonical tissue after delivery, Mann-Whitney test, *P=0.036. Data information: All data are presented as mean ± SEM. E) Shank2-/- dams showed impaired placentophagia (arrowheads), Mann-Whitney test, ***P<0.001. Data information: All data are presented as mean ± SEM. , F) Shank2-/- dams attack their pups inducing injury in the head and body region (arrowheads), Mann-Whitney test, *P=0.013, Shank2+/+, n = 12, Shank2-/- n = 9. Data information: All data are presented as mean ± SEM.
WASH inhibits autophagy.Figure 2(A) WASH deficiency enhances autophagy induction. WASH+/+ or WASH−/− MEFs were treated with EBSS for the indicated times in the presence or absence of 20 nM bafilomycin A1 (BafA1), and harvested for immunoblotting. Ratios of LC3‐II/β‐actin were calculated and shown at the right panel. (B) WASH knockout accelerates LC3 conversion by confocal microscopy. Endogenous LC3 puncta were visualized by staining with antibody against LC3 (left panel), and calculated as shown in the right panel. Nuclei were stained with DAPI. Scale bar, 10 μm. (C) Poly‐ubiquitinated proteins are reduced in WASH−/− MEFs. WASH+/+ and WASH−/− MEFs were starved in EBSS for 2 h treated with or without 20 nM BafA1 or 10 μM MG132. Cells were harvested for immunoblotting with anti‐poly‐ubiquitin antibody (Enzo, clone FK1) that only recognizes poly‐ubiquitinated ubiquitin chains. (D) Autophagosome‐like structures in WASH+/+ and WASH−/− MEFs were visualized after starvation for 1 h by immuno‐electron microscopy with antibody against LC3 (left panel). Black arrowhead indicates autophagosome. Scale bar, 500 nm. *, Cellular reference. Fifty cells were quantified from three independent experiments (right panel). Data were shown as means±s.d. *P0.05 and **P0.01. Experiments were repeated for three independent times with similar results.Source data for this figure is available on the online supplementary information page.
Compound effects of the patient mutation confer a neomorphic function for Dfmrp in sLNV neurons.Figure 6Overexpression of wild-type dfmr1 (UAS-dfmr1wt) using a LNv neuron-specific driver line (Pdf-Gal4) causes reduction of branching area-"axonal collapse"-in the sLNv axonal termini. dfmr1ΔCt and dfmr1wt+NLS overexpression phenocopies overexpression of dfmr1wt, suggesting that the truncation of the C-terminus or the presence of the patient NLS alone does not impair or alter the functional efficacy of dfmr1 in this context. However, overexpression of dfmr1ΔCt+NLS fails to cause collapse of sLNv termini, indicating a significant loss or change of Dfmrp protein function. Axonal morphology is visualized by expressing CD8-GFP under the control of the same neuronal driver. Scale bar represents 10 μm.Quantification of axonal termini branching in LNv neurons overexpressing various dfmr1 alleles. Overexpression of dfmr1wt, dfmr1ΔCt, or dfmr1wt+NLS significantly reduced axonal branching area [****P < 0.0001; control 101.3 ± 29.76 SD, n = 30; UAS-dfmrwt 45.41 ± 21.69 SD, n = 29; UAS-dfmrΔCt 52.31 ± 14.67 SD, n = 32; UAS-dfmrwt+NLS 72.45 ± 21.83 SD, n = 29; UAS-dfmrΔCt+NLS 117.8 ± 36.96 SD, n = 28]. dfmr1ΔCt+NLS overexpression does not result in a significant change of axonal branching area compared to the background control [Pdf-Gal4, UAS-GFP] (P = 0.672). For quantification purposes, each branching area (n) was manually outlined, starting from the first point of defasculation of the axonal bundle. These measurements were normalized for variability in brain size across samples using LNv commissure length measurements for each brain. Two-tailed t-tests using Welch's correction were then used to compare controls with mutant phenotypes (GraphPad). Error bars represent mean values with SD. Dots visible for box plots of UAS-dfmrwt and UAS-dfmrΔCt represent samples that outlier the whiskers. Overexpression of dfmr1ΔCt+NLS results in aberrant axonal termini morphology. Axonal guidance defects were frequently observed (arrowheads) and scored manually for bifurcations of the axon bundle (9/48), tangling of axons in the termini (13/48), and apparent looping of axons (7/48).
Figure 6Relative intracellular Fe2+ levels in normoxic B16-F10 cells (n=4). Representative pictures of FerroOrange intensity are shown on the left and analyzed using a RM one-way ANOVA (Geisser-Greenhouse correction) with Holm-Sidak's multiple comparisons test. Scale bars represent 50µm. Intracellular total iron content (left) and Fe2+ to Fe3+ ratio (right) measured by CE-ICP-MS in normoxic B16-F10 cells (n=3). Data were analyzed using a one-way ANOVA with Tukey's multiple comparisons test. Immunoblot detection of NCOA4, FTL and ACTIN levels from normoxic lysates of B16-F10 cells cultured in the presence or absence of BafA 5nM for 24h (n=3). Immunoblot detection of NCOA4, PHD2 and BNIP3 levels after immunoprecipitation of endogenous BNIP3 protein from normoxic lysates of B16-F10 shCntl cells (representative of n=3 immunoprecipitations). A lysate from B16-F10 shCntl cells cultured in the presence of BafA was used for band reference. Lad. stands for the reference ladder. Intracellular Fe2+ to Fe3+ ratio measured by CE-ICP-MS in normoxic B16-F10 cells transfected in presence of non-targeting siRNA sequences (Scr) or siRNA against NCOA4 (n=4). Data were analyzed using a one-way ANOVA with Tukey's multiple comparisons test. Immunoblot detection of Hydroxylated HIF-1α (HIF-1α-OH), total HIF-1α, LDHA, NCOA4 and ACTIN levels from normoxic lysates of B16-F10 cells transfected in presence of non-targeting siRNA sequences (Scr) or siRNA against NCOA4 (n=4).
Figure 6A-E B1-8 B cells, 3046SM cells, 3046M cells, 3046SD cells, or 3046D cells were stimulated with 1NIP-pep (80 nM), or anti-CD19 antibody (62 nM), or costimulated with 1NIP-pep and anti-CD19 antibody. The calcium fluxes were measured by FACScan. F-J B1-8 B cells, 3046SM cells, 3046M cells, 3046SD cells, or 3046D cells were stimulated with 1NIP-pep (80 nM), or anti-CD20 antibody (62 nM), or costimulated with 1NIP-pep and anti-CD20 antibody. The calcium fluxes were measured by FACScan.
Figure 2Thioperamide treatment of tissue culture cells. BHK, CHO and HeLa cells were treated with thioperamide and processed as in Fig 1C. Low magnification views are shown after staining with anti-LBPA antibodies (green) and DAPI (blue). n=3 independent experiments with > 200 images acquired and analysed automatically. Cholesterol quantification by mass spectrometry. A431 cells were treated with DMSO alone, pitolisant 10µM (Pito), thioperamide 10µM (thio) or U18666A 10µM (U18) for 18h at 37°C. After extraction, free cholesterol, cholesteryl esters and total cholesterol were quantified by mass spectrometry, and normalized to the DMSO controls. (n=3 independent experiments, error bars = SD, one-way ANOVA, ****=p<0.0001 Screen data plot of the cholesterol content of LBPA-endosomes. Automated unbiased quantification of the filipin integrated fluorescence signal (cholesterol content) in LBPA-containing endosomes, after treatment with each compound of the Prestwick library. As in Fig 1D, each dot is the average of replicates in the duplicate plates, and ≥600 cells were analysed per compound - only compounds that showed less than 20% toxicity are plotted. The fluorescence signal of LBPA-endosomes was used to segment the imaged and generate a mask, which was then applied on the micrographs to quantify the integrated intensity of filipin staining. The samples treated with thioperamide (pink), trimeprazine (light blue), DMSO (red) and U18666A (green) are indicated.
USP22-regulated modification of RIPK3 lysine residue 518 affects necroptosis in RIPK3-reconstituted HeLa cells A HeLa cells expressing Dox-inducible RIPK3 WT, K518R or 3xKR were treated with 1 µg/ml Dox and/or 10 µM NSA and 20 µM Dab overnight.Figure 6A HeLa cells expressing Dox-inducible RIPK3 WT, K518R or 3xKR were treated with 1 µg/ml Dox and/or 10 µM NSA and 20 µM Dab overnight. Protein levels of inducible RIPK3 expression and phosphorylated MLKL were examined by Western blotting. β-Actin was used as a loading control. B HeLa cells expressing Dox-inducible RIPK3 WT or 3xKR were treated with 1 µg/ml Dox overnight before pre-treatment with 20 µM zVAD.fmk, 5 µM BV6 for 1 h. After pre-treatment, 10 ng/ml TNFα were added for 1, 2, 3 and 4 h. Protein levels of inducible RIPK3 expression and phosphorylated MLKL were analyzed by Western blotting. β-Actin served as loading control. C HeLa cells expressing Dox-inducible RIPK3 WT, 2xKR or 3xKR were treated with 1 µg/ml Dox overnight before pre-treatment with 20 µM zVAD.fmk, 5 µM BV6 for 1 h. After pre-treatment, 10 ng/ml TNFα were added for 2 h. Protein levels of inducible RIPK3 expression were analyzed by Western blotting. β-Actin served as loading control. D HeLa cells expressing Dox-inducible RIPK3 WT, K518R or 3xKR were treated with 1 µg/ml Dox overnight before pre-treatment with 20 µM zVAD.fmk, 5 µM BV6 for 1 h. After pre-treatment, 10 ng/ml TNFα were added for 2 h. Whole cell lysates were generated using RIPA lysis buffer containing 2% SDS. Protein levels of inducible RIPK3 expression were analyzed by Western blotting. β-Actin served as loading control. E HeLa cells expressing Dox-inducible RIPK3 WT, D160N, K518R or 3xKR were incubated overnight with 1 µg/ml Dox and pre-treated with 20 µM zVAD.fmk, 5 µM BV6 for 1 h. After pre-treatment, 10 ng/ml TNFα were added for 1 and 2 h. Strep-RIPK3 was immunoprecipitated by using anti-Strep-beads. Co-immunoprecipitated phosphorylated MLKL and RIPK1, as well as protein expression of indicated proteins were analyzed by Western blotting. β-Actin served as a loading control. F HeLa cells expressing Dox-inducible RIPK3 WT, K518R or 3xKR were incubated with 1 µg/ml Dox and transfected with HA-ubiquitin for 24 h, as indicated. Cells were pre-treated with 20 µM zVAD.fmk, 5 µM BV6 for 1 h. After pre-treatment, 10 ng/ml TNFα were added for 2 h. HA-ubiquitin was immunoprecipitated using anti-HA-beads and detection of indicated proteins was performed by Western blotting. β-Actin served as loading control for the input, whereas HA-ubiquitin levels served as loading control for immunoprecipitated ubiquitin.
5-AzadC + Panobinostat and 5-AzadC + Romidepsin treatments induce a significant decrease in the cell surface CD4 receptor expression.Figure 10One night after cell purification, HLA DR-CD69-CD25-CD4+T cells from 4 HIV negative donors were mock-treated or treated with 5-AzadC. Three days post-treatment, 1/3 of medium was replaced and HDACIs were added in the cultures. The median fluorescence intensity of CD4 receptor of viable CD4+T cell subset was assessed 6 days after 5-AzadC treatment by flow cytometry analysis relative to mock treatment before 5-AzadC stimulation corresponding to day 0. Means are represented.
Apg9p is an unglycosylated, integral membrane protein.Figure 3Apg9p is an unglycosylated, integral membrane protein. A, Wild-type (WT; SEY6210), apg9Δ (JKY007), and the apg9Δ strain transformed with single copy (CEN) or multicopy (2μ) plasmids encoding APG9 were grown to log phase in SMD. Protein extracts were prepared and analyzed by immunoblot using antiserum to Apg9p. Apg9p is detected as an ∼125-kD protein. . B, Hydrophobicity analysis of Apg9p based on the Kyte-Doolittle algorithm (Kyte and Doolittle 1982) predicts six to eight transmembrane domains. C, Cells of wild-type strain YW5-1B were grown in YPD, converted to spheroplasts, and osmotically lysed as described in Materials and Methods. After a preclearing step to remove cell debris, the lysates were incubated with or without 1% Triton X-100 on ice for 30 min, as indicated. Centrifugation at 10,000 g for 1 h separated the lysate into supernatant (S) and pellet (P) fractions, which were analyzed by immunoblots with antiserum to Apg9p. Apg9p is found in the low-speed pellet in the absence of Triton X-100. Detergent extraction completely solubilizes Apg9p and causes it to appear in the supernatant fraction D, Strain CTD1 harboring a multicopy 3×HA APG9 plasmid was grown in YPD and converted to spheroplasts as described in Materials and Methods. A crude lysate was prepared and treated with or without endo H as indicated overnight at 37°C and analyzed by immunoblots with anti-HA antibodies or anti-CPY serum. Endo H treatment removes the N-linked glycosyl residues of CPY, causing a shift in its electrophoretic mobility. In contrast, Apg9p is not glycosylated and remains unchanged in the presence or absence of endo H treatment.
Telomere dysfunction leads to preferential depletion of position 1 and 2 cells at the crypt bottom in between Paneth cells A–I Staining of small intestinal tissue sections of 12‐ to 16‐month‐old G3 mTerc−/− mice and age‐matched mTerc+/+ mice (n = 4–5 mice per group).Figure 4A-I Staining of small intestinal tissue sections of 12‐ to 16‐month‐old G3 mTerc−/− mice and age‐matched mTerc+/+ mice (n = 4-5 mice per group). Representative pictures of (A, B) H&amp;amp;E staining, (C, F, I) Quantification of the absolute numbers of ISPCs at the indicated positions in the crypt base per 50 crypts as determined by counting of (C) spindle‐shaped cells in between and adjacent to the Paneth cells. Mean values ± SEM are given. Unpaired two‐tailed Student's t‐test. A-I Staining of small intestinal tissue sections of 12‐ to 16‐month‐old G3 mTerc−/−mice and age‐matched mTerc+/+mice (n = 4-5 mice per group). Representative pictures of (D, E) PCNA staining. Arrowheads and numbers indicate the position of ISPCs in the basal crypts. Scale bars: 20 μm. (C, F, I) Quantification of the absolute numbers of ISPCs at the indicated positions in the crypt base per 50 crypts as determined by counting of cells staining positive for (F) PCNA . Mean values ± SEM are given. Unpaired two‐tailed Student's t‐test. A-I Staining of small intestinal tissue sections of 12‐ to 16‐month‐old G3 mTerc−/− mice and age‐matched mTerc+/+ mice (n = 4-5 mice per group). Representative pictures of ((G, H) Msi1 staining are given. Arrowheads and numbers indicate the position of ISPCs in the basal crypts. Scale bars: 20 μm. (C, F, I) Quantification of the absolute numbers of ISPCs at the indicated positions in the crypt base per 50 crypts as determined by counting of (I) Msi1. Mean values ± SEM are given. Unpaired two‐tailed Student's t‐test. J, K Representative pictures of Olfm4 in situ hybridization on small intestinal sections of 9‐month‐old G3 mTerc−/− mice (K) and age‐matched mTerc+/+ (J) mice. Arrowheads point to positive cells. Dashed lines outline the crypts. Note that the remaining ISPCs in G3 mTerc−/− mice were mostly located in position 4 above the Paneth cells. Scale bar: 20 μm.
A) Detection of protein–protein interactions using the two‐hybrid system.Figure 5(A) Detection of protein-protein interactions using the two‐hybrid system. Intense staining indicates an interaction between Aut2p and Aut7p, Aut2p and Tub1p, and Aut2p and Tub2p. AUT7, TUB1 and TUB2 are cloned in‐frame with the GAL4‐activator domain. AUT2 and AUT7 were fused to the GAL4‐binding domain (see Materials and methods). (B) Affinity chromatography confirms protein-protein interaction between Aut2p and Aut7p. A fusion protein of Aut2p with the 26 kDa glutathione‐S‐transferase (GST) domain from Schistosoma japonicum (see Materials and methods) was bound to glutathione-Sepharose 4B. This GST-Aut2p column was incubated with a crude extract from aut7Δ cen Met25::AUT7-GFP cells. The non‐bound supernatant (S) fraction (lane 1) was taken away. Gel beads were successively washed with NaCl solutions of increasing concentrations to remove non‐specifically bound proteins. After the final 500 mM NaCl wash (W, lane 2), bound proteins were eluted with Laemmli buffer (E, lane 3). Samples were analyzed in immunoblots with monoclonal antibodies directed against GFP. Using Aut2p-GST, a clear band corresponding to Aut7p-GFP is seen in the eluate fraction (lane 3, arrowhead). As a control, GST alone was attached to the column and no band was detectable (lane 6). (C) As described in (B), bovine tubulin binds to GST-Aut2p glutathione-Sepharose 4B (lane 12), but not to GST glutathione-Sepharose 4B (lane 9). Protein staining with Coomassie is shown in lanes 1-6; in lanes 7-12 tubulin was detected using monoclonal antibodies (Boehringer Mannheim). Further details are given in Materials and methods. (D) Similar to (C), bovine tubulin does not bind to GST-Aut7p glutathione-Sepharose 4B (lane 12) and to GST glutathione-Sepharose 4B (lane 9). Lanes 1-6, protein staining with Coomassie; lanes 7-12 probed with antibodies directed against tubulin.
Figure 5(A) NSC-34 and SH-SY5Y cells were infected with EV-A71 S41 at MOI 30 and 0.01, respectively. At 48 h.p.i., cells were fixed and permeabilized prior to immunostaining. (B) Total infected cell lysates were pulled down with anti-PRPH or IgG isotype control antibody prior to immunoblotting. Uninfected cells were included as negative control. IP: immunoprecipitation; IB: immunoblotting. C) Proximity ligation assay. NSC-34 and SH-SY5Y cells were infected with EV-A71 S41 at MOI 10 and 0.01, respectively. At 48 h.p.i., the cells were fixed and permeabilized prior to PLA staining using antibodies specific to PPRH, VP0, 3C and 3D.