EMBO/sd-character-level-ner huggingface dataset:
Figure 10Experiments were performed to assess possible physical interaction, colocalization at the cell plate, and functional interplay of PI4Kβ1 and MPK4. PI4Kβ1 interacts with MPK4 in yeast two hybrid tests. MPK4 was used as a bait, while PI4Kβ1 was divided into two fragments to be used as prey. The results are representative for three independent experiments. Experiments were performed to assess possible physical interaction, colocalization at the cell plate, and functional interplay of PI4Kβ1 and MPK4. In vivo co-immunoprecipitation of MPK4 and PI4Kβ1 from 14-day-old plants. PI4Kβ1 was specifically co-precipitated with MPK4-myc using anti-myc antibodies. Arrowhead, Migration of PI4Kβ1. The results are representative for three independent experiments. Experiments were performed to assess possible physical interaction, colocalization at the cell plate, and functional interplay of PI4Kβ1 and MPK4. Coordination of MPK4-YFP and mCherry-PI4Kβ1 during cytokinesis in five-day-old root meristem cells. MPK4 co-localized with PI4Kβ1 from early to late cytokinesis and gradually concentrated at the leading edges of cell plates. Images were obtained with an LSM880 in Airyscan super-resolution (SR) mode. Scale bars, 10 µm. Live cell time lapse of MPK4-YFP and mRFPFAPP1-PH during cytokinesis, obtained with an LSM880 in Airyscan resolution versus sensitivity (R-S) mode. The time series is representative for 6 cells from 5 roots. Arrowheads indicate the initiation and leading edges of the growing cell plate. Scale bar, 10 µm. Left panels, The distribution of mRFPFAPP1-PH was observed at the cell plate at the end of cytokinesis in root meristem cells of wild type controls (top) and mpk4-2 mutants (bottom). Intensity profiles were recorded as indicated by the dashed lines. Scale bars, 10 µm. Right top, intensity of mRFPFAPP1-PH at the cell plate normalized vs. the intensity at the apical plasma membrane (wild type, n = 43 cells, 34 roots; mpk4-2, n = 32 cells, 28 roots). Right bottom, intensity of mRFPFAPP1-PH at the TGN normalized vs. the intensity at the apical plasma membrane (wild type, n = 40 cells, 29 roots ; mpk4-2, n = 26 cells, 23 roots). *, a significant difference (p<0.05) according to a two-tailed Student's test.
Figure 4A, PTPN3 interacts with TβRI at endogenous levels. HaCaT cells were harvested after TGF-β treatment (2 ng/mL, 1 h) and then subjected to IP by PTPN3 antibody or control IgG. PTPN3 and TβRI were detected by Western blotting. B, Both PTPN3 and PTPN3 (D811A) interact with TβRI. FLAG-TβRI was co-transfected with HA-PTPN3 or HA-PTPN3 (D811A) into HEK293T cells. Cell lysates were harvested and subjected to IP with FLAG antibody. PTPN3, PTPN3 (D811A) and TβRI were detected by Western blotting with appropriate antibodies. C, The FERM domain of PTPN3 is necessary for its binding to TβRI. FLAG-TβRI and HA-PTPN3 (wildtype or truncations) were co-transfected into HEK293T cells. Cells were harvested after 24 h and subjected to IP by HA antibody. TβRI, PTPN3 and PTPN3 truncations were detected by Western blotting with appropriate antibodies. D, Schematic representation of TβRI deletion mutants and their interactions with PTPN3 or Smad7. EC, extracellular domain; TM, transmembrane domain; GS, Gly-Ser-rich motif; +, positive interaction; -, no interaction. E, Knockdown of endogenous PTPN3 promotes the Smad7-TβRI association. HEK293T cells were transfected with the indicated plasmids and treated with MG132 (20 µM) for 4 h before harvested. SFB-Smad7 proteins were retrieved with the Streptavidin pulldown. Levels of TβRI and other proteins were examined by Western blotting with the indicated antibodies. F, PTPN3 and PTPN3 (D811A) abolish the interaction between TβRI and Smurf2 in HEK293T cells. HEK293T cells were co-transfected with plasmids as indicated. 20 h later, cells were treated with MG132 (20 μM) for 4 h, followed by IP and Western blotting.
A subset of Scl‐bound enhancers can be accessed by cardiac transcription factors A Scl DNA binding motif analysis shows enrichment of Tal1_Gata, Gata and Ets motifs in both Scl activated and repressed genes.Figure 5A Scl DNA binding motif analysis shows enrichment of Tal1_Gata, Gata and Ets motifs in both Scl activated and repressed genes. B Venn diagram showing overlaps between Scl, Hand1 and Gata4 (Oda et al, ) binding sites in Flk1+mesoderm documents that some of SclMES binding sites are also bound by cardiac TFs. C Gata4, Hand1 and SclChIP‐seq tracks with cardiac and hematopoietic gene regions show examples of three subgroups of enhancers-those bound by Scl, Hand1 and Gata4 (gray), Scl and Gata4 (orange) and those bound only by Scl (blue). D-H Scl binding sites that overlap with both Hand1 and Gata4 (gray) or with Hand1 (green) or Gata 4 (orange) alone show higher evolutionary conservation (D) and higher average enrichment of Scl (E), H3K4me1 (F), H3K27ac (G) and Lsd1 (H) in mesoderm compared to sites bound by Scl alone (blue). I Average expression changes between Scl‐expressing and Scl‐deficient mesoderm shows that genes that are regulated by enhancers bound by all three (Scl, Hand1 and Gata4) factors or two factors (Scl and Hand1 or Scl and Gata4) show higher absolute expression changes compared to those bound by Scl alone. J Analysis of Scl‐regulated key hematopoietic and cardiac genes shows that many of them can be bound also by cardiac factors, Scl, Gata4 and Hand1 (orange); Scl and Hand1 binding (green); Scl and Gata4 binding (orange); and Scl binding alone (blue).
Enhanced thermogenesis in QKI-deficient mice.Figure 3O2 consumption rates were measured via indirect calorimetry using CLAMS under chow-fed conditions (n = 8, per genotype). And average light and dark O2 consumption rates are shown on the right. Heat production rates in WT fl/fl and QKI KO fl/fl:AP2-Cre mice (n = 8, per genotype). And average light and dark heat production rates are shown on the right. Heatmaps of log2 (fold change QKI KO fl/fl:AP2-Cre BAT vs. WT fl/fl BAT) for genes involved in thermogenesis and oxidation. (3 samples, per group) Photograph of WT fl/fl and QKI KO fl/fl:AP2-Cre classical BAT in 4% paraformaldehyde solution, representative transmission electron micrographs, and UCP1 staining of BAT from WT fl/fl and QKI KO fl/fl:AP2-Cre mice. The average diameter of lipid droplets and the number of mitochondria per field from WT fl/fl and QKI KO fl/fl:AP2-Cre BAT. (n = 3 biological replicates) The relative mRNA levels of genes related to thermogenesis in BAT analyzed by RT-PCR. (n = 4 biological replicates) Total oxygen consumption rates (OCR) per gram in BAT from WT fl/fl and QKI KO fl/fl:AP2-Cre mice under basal conditions were measured using a Clark electrode. (n = 3 biological replicates) The relative mRNA levels of genes related to thermogenesis in ingWAT analyzed by RT-PCR. (n = 4 biological replicates) Total oxygen consumption rates (OCR) per gram in ingWAT from WT fl/fl and QKI KO fl/fl:AP2-Cre mice under basal conditions. (n = 3 biological replicates) Representative recording of Continuous measurement of oxygen consumption rate (OCR) in isolated mitochondria from BAT of WT fl/fl and QKI KO fl/fl:AP2-Cre mice on NCD; Oxygen consumption was performed under basal conditions, following the addition of 0. 25 mg mitochondria (Mit), 5 mM substrate glycerol-3-phosphate (G3P), 2 mM GDP, and 450 mM ADP, 2 μg/ml oligomycin, and 1.5 μM FCCP. Quantification of experimental data. (n=3 biological replicates, per group) UCP1-dependent oxygen consumption rates estimated as GDP-inhabitable rates, based on the data shown in (K). (n=3 biological replicates)
Figure 4A Domain structure of NLRP1B. The LT cleavage site and the FIIND auto-cleavage site are marked. B Effect of LT treatment on NLRP1B protein level. 293T cells were transfected with Flag-NLRP1B expression plasmid for 24 h, and cells were then treated with WT LT (+) or its E687C mutant (-) for 4 h. Shown is the anti-Flag immunoblot of the total cell lysates. C RFP-ASC specks formation in 293T cells expressing an indicated NLRP1B variant. Cells were treated with LT for 3 h. Scale bar, 20 μm. 1-983 and 984-C (residues 984 to the C terminus) are the FIIND-mediated autocleaved fragments of NLRP1B. Effect of MG132 on LT-induced NLRP1B degradation. 293T cells expressing NLRP1B (1-983)-HA were treated with LT and MG132 for 8 h. Shown are the anti-HA and anti-tubulin immunoblots of the total cell lysates. E Effect of MG132 and Leu on LT-induced NLRP1B degradation. 293T cells expressing NLRP1B (1-983)-HA were treated with LT and MG132 for 8 h. 10 mM Leu was added to cells 30 min prior to LT treatment. Shown are the anti-HA and anti-tubulin immunoblots of the total cell lysates. F LT-induced ubiquitination of NLRP1B. 293T cells expressing NLRP1B (1-983)-HA were treated with LT and/or MG132 for 6 h. Cells were then harvested for anti-HA immunoprecipitation and subjected to immunoblotting analyses using indicated antibodies.
Figure 3(A) 1 μg of purified long mouse DNGR-1 ECD was transferred into 10mM MES pH 6.1 and 0.5 μg into PBS. Half of the MES sample and the whole PBS sample were dialyzed against 2 l of PBS overnight at 4°C. After dialysis, all samples were prepared for reducing SDS-PAGE and Western blot. (B) Supernatants after production of indicated proteins in 293F cells were harvested and diluted into indicated buffers at a 2:3 ratio and analyzed using reducing SDS-PAGE and Western blot. HRP-conjugated anti-FLAG antibody was used for detection of all proteins. Numbers on the side of blots indicate positions of molecular weight markers. Reduction-resistant dimers are indicated with an * and reduction-sensitive protein with a +.
Overexpression of CEP55 rescue cell death during perturbed mitosis.Figure 4(A) Box and whiskers plot showing average time spent in mitosis and (B) mitotic outcomes in control and CEP55 knockdown MDA-MB-231 cells following treatment with the BI2536 (5 nM). Time taken to complete mitosis was defined as the time from nuclear envelope breakdown until two daughter cells were observed whereas mitotic slippage or death was defined as cells that prematurely exited mitosis with a flattened and a multinucleated phenotype or died during mitosis, characterized by membrane blebbing. Graph represents the mean±SEM of two independent experiments. For each experiments n=50 mitotic cells were counted per condition using Olympus Xcellence IX81 time-lapse microscopy. (C, D) Cells were pre-treated for two hours with 50 µM of the pan Caspase inhibitor Z-VAD-FMK, followed by treatment with B12536 (5 nM), before assessment of cell fate (C) and an average time spent in mitosis (D) Graph represents the mean±SEM of two independent experiments. (E) Cells were synchronized using double thymidine then released into nocodazole (0.5 µM), and protein lysates were collected at the indicated time points. Immunoblot analysis was then performed to determine the expression and activity of mitotic regulators as indicated. Levels of phospho-MEKT286 and dephosphorylation of phospho-CDK1Y15 served as markers of Cdk1 activation/mitotic entry. COX-IV served as a loading control. (F) Schematic representation of the mCerulean-CDK1-FRET biosensor. CDK1 activity is low when FRET signaling is low (high lifetime reading) vs. CDK1 activity is high when the FRET signaling is high (low lifetime reading). Adapted from (46), reprinted with permission from AAAS. (G) Representative mCerulean lifetimes maps of Hs578T control and CEP55 knockdown cells upon nocodazole (0.5 µM) treatment at indicated time points. Cells were synchronized using double thymidine for 16 hours prior to nocodazole treatment. (H) Quantification of mCerulean lifetimes of control and Hs578T CEP55 knockdow ncells and in response to treatment with nocodazole at indicated time points as shown in G. Blue: 1 ns; red: 3 ns. Graph represents the mean±SEM of three independent experiments. (I) Control and CEP55 knockdown MDA-MB-231 cells were synchronized as above, released into culture medium for six hours and then blocked with nocodazole (0.5 µM) for an additional four hours prior to treatment with the Cdk1 inhibitor RO-3306 for an additional 16 hours. Sub-G1 cells were then identified by propidium iodide staining and quantified by FACS. Graph represents the mean±SEM of two independent experiments. (J) Control and CEP55 knockdown MDA-MB-231 cells were transfected with MAD2 siRNA (5 nM) for 48 hours followed by nocodazole (0.5 µM) treatment for an additional 12 hours. Cells were collected, fixed and sub-G1 analysis was performed as described in methods. Graph represents the mean±SEM of two independent experiments.
FGMYB genes are phylogenetically closely related to each other and expressed in female gametophytes A Phylogenetic tree of R2R3-MYB proteins of clades 11, 12, and 14-16, as described by Bowman et al.Figure 2A Phylogenetic tree of R2R3-MYB proteins of clades 11, 12, and 14-16, as described by Bowman et al. from representative land plant species constructed using the maximum likelihood method based on conserved MYB domain sequences. Numbers at nodes indicate bootstrap values calculated from 1,000 replicates. The tree is drawn to scale, with branch lengths reflecting the number of substitutions per site. Scale bar, 0.5 substitutions per site. Arrows indicate FGMYB orthologues involved in embryo sac development in A. thaliana, MpFGMYB of M. polymorpha (this study), and the most similar P. patens genes The FGMYB clade is shaded in red, and a distantly related FOUR LIPS (FLP) clade is in blue. B Schematic representations of the FGMYB polypeptide structures. R2R3 MYB domains are shown in red and a conserved amino-terminal motif of PpFGMYBs and MpFGMYB in orange. C Genomic PCR analysis indicating the existence of MpFGMYB in both male [Y] and female [X] genomes of M. polymorpha. Two biological replicates were analyzed. Constitutively expressed MpEF1α was used as a control. D Real-time RT-PCR analyses indicating preferential accumulation of MpFGMYB transcripts in female sexual organs and the sporophytes. MpEF1α was used for normalization. Measurements of six biological replicates for thalli and sporophyte, and three biological replicates for gametangiophore are plotted. Bars represent mean ± SD. Symbols above the bars indicate grouping by p <0.05 in a Tukey-Kramer test. E A transcriptional reporter with 5′- and 3′-flanking sequences revealed transcription of MpFGMYB throughout mature archegonia. Scale bar, 10 µm. Magenta, chlorophyll autofluorescence; green, Citrine fluorescence. F MpFGMYB-Citrine fusion proteins expressed using the 5′- and 3′-flanking sequences rescued the Mpfgmybge-1 mutant and accumulated in the nuclei of the egg and the ventral canal cell (VCC) Scale bar, 10 µm. Magenta, chlorophyll autofluorescence; green, Citrine fluorescence. G A transcriptional AtMYB64 reporter (AtMYB64-NYG) is specifically expressed in all four cell types of the A. thaliana embryo sac Scale bar: 25 µm. Green, YFP fluorescence; white, cell walls. H Expression of AtMYB64-Citrine fusion proteins under the AtMYB64 promoter was detected in the central cells (CC) and egg cells (EC) of the A. thaliana embryo sac Scale bar: 25 µm. Green, Citrine fluorescence; white, cell walls.
Figure 5(A) Structure of ScPrx1 (PDB ID: 5YKJ). The positions of relevant amino acids are indicated and the applied modifications in Prx1-P233stop and Prx1-C91A are shown. The C-terminal region absent in the Prx1-P233stop mutant is highlighted in green. (B) The response of a mitochondrial matrix-localized roGFP2-Prx1-P233stop probe expressed in wild-type cells to the indicated concentrations of exogenous H2O2. Cells were grown in SGal (-Leu) medium and harvested at early exponential phase. Error bars represent the standard deviation (n = 3 biological replicates, with cells obtained from 3 independent cultures for every strain and probe combinations. For each biological replicate three technical replicates were performed). (C) Maleimide-based gel-shift assay to assess Prx1 cysteine redox state. Wild-type cells transformed with an empty plasmid or ∆prx1 cells with either an empty plasmid or a plasmid encoding wild-type Prx1, the hyperoxidation-resistant Prx1-P233stop or the inactive Prx1-C91A variants, were grown to exponential phase in SGal (-Ura) medium. Cells were subsequently treated with the indicated concentrations of H2O2 for 10 mins and then directly treated with the alkylating agent mmPEG24. (D) The response of a mitochondrial matrix-localized Grx1-roGFP2 probe, expressed in Δprx1 cells containing a plasmid encoding either wild-type Prx1, Prx1-P233stop or Prx1C91A to the addition of 1 mM exogenous H2O2. Cells had been pre-treated with either 10 mM H2O2 or with water as a control. For these experiments, cells were grown in SGal (-Leu, -Ura) medium and harvested at early exponential phase. OxD refers to the degree of sensor oxidation. Error bars represent the standard deviation (n = 3 biological replicates, with cells obtained from 3 independent cultures for every strain and probe combinations. For each biological replicate three technical replicates were performed). (E) Hydrogen peroxide 'acute stress' assay. Δprx1 cells co-transformed with empty vector or with a vector encoding either wild-type Prx1, the P233stop or the C91A variants were grown in SD (-Ura) and treated with the indicated concentrations of H2O2 for 30 mins. Afterwards, the cells were diluted and a fixed volume plated on YPD plates. The number of viable colonies was counted after 2 days growth at 30°C, here represented as a percentage relative to the 0 mM treatment. Error bars represent standard deviation (n = 5-8 biological replicates, with cells taken from independent cultures for each individual biological replicate).
Figure 2U87, T98G and LN229 cells were treated with 1 µM ABT263, 20 µM LXR623 or the combination of both for 72h. Mewo melanoma and HCT116 colon carcinoma cells were treated with 1 µM ABT263, 10 µM LXR623 or the combination of both for 72h (MeWo) or for 48h (HCT116). Thereafter, cells were stained with annexin V/propidium iodide and analyzed by multi-parametric flow cytometry. , Stem-like GBM cells, NCH644, NCH421k and NCH690 were treated with 1 µM ABT263, 20 µM LXR623 or the combination of both for 48h. Thereafter, cells were stained with annexin V/propidium iodide and analyzed by multi-parametric flow cytometry. U87 (72h treatment) and HCT116 (48h treatment) were treated with 1 µM ABT263, 20 µM (U87) or 10 µM (HCT116) LXR623 or the combination of both, stained with TMRE and analyzed by flow cytometric analysis for dissipation of mitochondrial membrane potential. HCT116 colonic carcinoma cells were treated with ABT263, LXR623 or the combination of both. Whole protein lysates were collected and subjected to capillary electrophoresis for the expression/cleavage of PARP, total caspase-9 (CP9) and Vinculin. CF: cleaved fragment, FL: full length. HCT116 colonic carcinoma or NCH644 glioblastoma stem cells were treated with selective BH3-mimetics, WEHI-539 (Bcl-xL inhibitor), ABT199 (Bcl-2 inhibitor) or A1210477 (Mcl-1 inhibitor) in the presence or absence of LXR623 for 48h. Thereafter, cells were labeled with annexin V/propidium iodide and analyzed by multi-parametric flow cytometry.
Figure 5A ICC staining of CTC-ITB-01 for ß-catenin (green), E-cadherin (orange), and DAPI (blue). The scale bar corresponds to 20 µm. Two representative panels are shown for CTC-ITB-01. B ICC staining of CTC-ITB-01 for α-catenin (green), visualization of actin filaments by phalloidin (orange), and nuclei by DAPI (blue). The scale bar corresponds to 20 µm. Two representative panels are shown for CTC-ITB-01. C Generic EMT score of the adherent and non-adherently growing CTC-ITB-01 fractions in relation to tumor samples from different cancer entities. Scaling ranges from 1 (completely epithelial) to -1 (entirely mesenchymal). The adherent fraction is shown with a black dot, the non-adherent fraction with a black triangle, and the mean score per cancer entity by a black line. D Generic EMT score of the adherent and non-adherently growing CTC-ITB-01 fractions in relation to established cancer cell lines. Scaling ranges from 1 (completely epithelial) to -1 (entirely mesenchymal). The adherent fraction is shown with a black dot, the non-adherent fraction with a black triangle, and the mean score per cancer entity by a black line.
Figure 3(A-D) Succinate dehydrogenase staining was performed in WT and MCK-KO soleus at sedentary (A, B) and after a bout of low-intensity exercise for 50 min (C, D) (10X, 20X magnifications), and the staining intensity was quantified using ImageJ (n = 6, means ± SEM, * p < 0.05, two-tailed student's t-test). (E-H) The analogous set of data is shown with WT and MCK-KO GA muscles. (n = 6, means ± SEM, * p < 0.05, two-tailed student's t-test). (I-L) The analogous set of data is shown with WT and MCK-KO EDL muscles. (n = 6, means ± SEM, two-tailed student's t-test. EDL: Extensor digitorum longus) (M, N) Mitochondrial respiration was measured in WT and MCK-KO soleus tissue after a bout of low-intensity exercise for 50 min under basal conditions and in response to 4 mM oligomycin (complex V inhibitor), 4 mM FCCP (uncoupler), or 4 mM each of rotenone and antimycin A (complex I inhibitor) (n = 8, means ± SEM, * p < 0.05, two-tailed student's t-test and two-way ANOVA followed by Bonferroni post-hoc testing). (O, P) Mitochondrial respiration was measured in Dnmt3a knocked down L6 myotubes which were transduced with lentiviral under basal conditions and in response to 4 mM oligomycin (complex V inhibitor), 4 mM FCCP (uncoupler), or 4 mM rotenone and antimycin A (complex I inhibitor) (n = 5, means ± SEM, * p < 0.05, two-tailed student's t-test and two-way ANOVA followed by Bonferroni post-hoc testing).
Figure 2A Lytic growth of intracellular wild type Toxoplasma gondii tachyzoite stages over a period of 8 days results in plaques within mononolayers of host cells. Destabilisation of PKAr-TyDD leads to the absence of plaque formation. B A drop in PKAr-TyDD expression is detected 5 hours after Shld-1 removal and a significant parasite growth defect is observed as early as 12 hours later. Profilin (αPRF) served as loading control. C IFA analysis showed that the down regulation of PKAr-TyDD leads to the abnormal segregation of the apicoplast-associated thioredoxin family protein (ATrx1) 24 hours after Shld-1withdrawal. D Replication assay of PKAr-TyDD parasites grown for 24 hours +/- Shld-1 indicates that no more than four parasites are observed per vacuole upon PKAr destabilisation. (100 parasites were counted in three independent replicates). E, F Stabilisation of a second copy of DDmyc-PKAc1 leads to the complete block of cell growth indicated by plaque assay (E) E, F Stabilisation of a second copy of DDmyc-PKAc1 leads to the complete block of cell growth indicated by western blot analysis (F). G, H Stabilisation of a second copy of DDmyc-PKAc1 leads to the observation of mainly one parasite per vacuole in cells treated with Shld-1 (100 parasites were counted in three independent replicates).Data information: In C and G, data are presented as mean ± SEM. Scale bars = 2 μm (C and G), 3 mm (A and E). G, H Stabilisation of a second copy of DDmyc-PKAc1 leads to the observation of mainly one parasite per vacuole in cells treated with Shld-1 (100 parasites were counted in three independent replicates).