EMBO/sd-character-level-ner huggingface dataset:
PGE2 activates MEF2 through PAK2 in NRVMs.Figure 5 A After serum starvation NRVMs were pretreated with or without 50 μM NSC23766 for 2 hours and stimulated with 1 μM PGE2 for 1 hour. Total and phosphorylated PKD levels were analyzed by immunoblot. PKD was activated by PGE2, but it was not affected by NSC Data information: In (A-C) representative images from 3 independent experiments are shown  B After infection with Flag-HDAC5 and serum starvation, NRVMs were treated with 50 μM NSC23766 for 2 hours prior to stimulation. The cells were stimulated with 1 μM PGE2 for 1 hour. HDAC5 phosphorylation at Ser-498 was detected by immunoblot. The phosphorylation of HDAC5 at this site was not prevented by NSC Data information: In (A-C) representative images from 3 independent experiments are shown  C NRVMs and neonatal cardiac fibroblasts (CFs) were serum-starved, pretreated with vehicle or 200 nM L798106 (EP3 receptor antagonist) for 20 minutes, and were stimulated with 1 μM PGE2 for 10 minutes. PAK2 and PKD phosphorylation was assessed by immunoblot. Calsequestrin was used as a myocyte-marker Data information: In (A-C) representative images from 3 independent experiments are shown  D MEF2 activity was determined in cells infected with the 3xMEF2-Luc reporter after serum starvation. Cells were stimulated with vehicle or 1 μM PGE2 for 24 hours after 1-hour of 30 μM IPA-3 pretreatment. IPA-3 inhibited the MEF2 activation by PGE2 Data information In (D) values are mean±s.e.m., n=3, biological replicates, *,P<0.05 (two-way ANOVA, P<0.0001). The exact n and P values can be also found in the Source Data excel file for Figure 5
Model for the different expression characteristics for paused versus TATA promoters.Figure 5Paused promoters have high levels of paused Pol II throughout the embryo, even in tissues where the genes are not expressed; they show high promoter accessibility in all tissues, low gene expression noise when active, but also high background expression when inactive. TATA promoters mediate highly tissue-restricted expression and only show Pol II occupancy when active; they have lower chromatin accessibility and background expression but show higher expression noise when active.
a, Percentage of ciliated cells in control (Ctrl) and IFT20− MEFs (*P = 0.f1a, Percentage of ciliated cells in control (Ctrl) and IFT20− MEFs (*P = 0.030, n = 3) and wild-type (WT) and Ift88−/− KECs (**P = 0.003, 25 cells each, n = 3) after 24 h of serum removal. Arrows, cilia. b, LC3 immunoblot (left) for autophagic flux quantification (right) in Ctrl and IFT20− MEFs (top, **P = 0.0003, n = 7), and in WT and Ift88−/− KECs (bottom, *P = 0.035, n = 9). PI, protease inhibitors; S, serum. c c, Autophagic flux in WT and Ift88−/−KECs by mCherry-GFP-LC3 puncta quantification. Puncta: yellow, autophagosomes; red only, autophagolysosomes; total, both together (**P = 0.008, *P = 0.025). . d, LC3 immunoblot (left) for quantification of autophagosome formation (right). (*P = 0.048, n = 4). e, Electron microscopy images (left) and morphometric quantification of autophagic vacuoles (AV, right) in Ctrl and IFT20− MEFs (top, **P = 0.001, 10 fields in 2 experiments) and KECs WT and Ift88−/− (bottom, *P = 0.037, 5 fields). Arrows, autophagic vacuoles (AV, black) and endosomal compartments (yellow). f, Percentage of ciliated MEFs after induction of cilia resorption with 50 ng ml−1 platelet-derived growth factor (PDGF) treatment. (**P = 0.001, ***P = 0.0009, 121, 230 and 162 cells, n = 5). g, LC3immunoblot (top) for autophagic flux quantification (bottom) upon PDGF treatment (*P = 0.005, n = 3). Scale bars in a, 10 µm. n.s., statistically non-significant. Mean ± s.d. in b, d and mean ± s.e.m. in other panels.
High protein synthesis in CAFs mediates chemoprotective effect on pancreatic cancer cells—reversion through inhibition of protein synthesis with SOM230.Figure 2Immunoblotting of protein extracts from PaSCs or CAFs treated (+) or not with SOM230 (10−7 M) for 48 h, using the anti-puromycin antibody (representative of n = 3). Polysomes profiles of CAFs treated or not with SOM230 for 48 h (representative of n = 3). Immunoblotting using an anti-P-Akt, anti-P-S6 or anti-β-actin (loading control) antibody of protein extracts from PaSCs or CAFs treated (+) with SOM230 for 30 min (representative of n = 3). Immunoblotting of protein extracts from CAFs treated (+) or not with SOM230 (10−7 M) for the indicated times, using anti-P-Akt, anti-P-S6 or anti-4E-BP1 antibody (representative of n = 3). Protein concentration in untreated or SOM230-treated CAF-CM or extracts normalized per 1 × 106 cells. Results (mean ± SD) are presented as a percentage of the untreated CAFs (= 100%) (n = 3; from left to right: ##P = 0.008, ##P = 0.007). Immunoblotting using an anti-4E-BP1 or anti-GAPDH (loading control) antibody of protein extracts from siCTR- or si4E-BP1-transfected CAFs (representative of n = 3). Immunoblotting of equal amounts of protein from siCTR- or si4E-BP1-transfected CAFs treated (+) or not with SOM230 for 48 h, using the anti-puromycin antibody (representative of n = 3). Panc-1 cell viability was assessed by MTT. Panc-1 cells were incubated with gemcitabine in the presence of CM from untreated or SOM230-treated CAFs transfected with the siCTR or si4E-BP-1. Results (mean ± SD) are presented as a percentage of the untreated CAFs (= 100%) (n = 3; **P = 0.003, §§P = 0.002). Caspase-3 and PARP cleavage induced by gemcitabine in Panc-1 was evaluated by Western blot using the respective antibodies (representative of n = 3). Arrow indicates cleaved forms of PARP.
Figure 1(A) SDS-PAGE gels stained with coomassie showing the purity of the Chlamydomonas IFT-B2 components, over-expressed in E. coli or insect cells, after the last purification step by SEC. IFT54/20 and IFT57/38 were purified as hetero-dimeric complexes whereas IFT80, IFT172 and an N-terminal IFT172(1-968) (IFT172ΔC) constructs were purified as single components. A cartoon representation of the predicted domain architecture of IFT-B2 components using HHPRED (Söding et al, 2005) is shown at the center. (B)Pull-down analysis for the hexameric IFT-B2 complex. A mixture of IFT172ΔC, IFT80, and the IFT57/38 complex ('input') shows only weak non-specific binding to GSH-resin ('empty'), but all proteins are pulled down by IFT54/20-GST complex. Note that the IFT20-GST and IFT38 proteins co-migrate on the gel and cannot be properly resolved. However, the presence of the IFT57 protein in the pull-down sample implies that its binding partner IFT38 is also there. (C) (left) SEC elution profiles of individual IFT-B2 components and of the assembled hexameric IFT-B2 complex. The elution peak of IFT-B2 is shifted compared to all of the individual components. Note that the peak heights were adjusted for better visualization of the result. (right) Coomassie stained SDS PAGE gel of the six main fractions of the IFT-B2 SEC elution peak demonstrating the presence of all six IFT-B2 subunits. Weak contaminating bands are marked with asterisks.
Figure 6Maximum growth rates of NUP58 mutant clone C5 during long-term culture. Each dot represents a technical replicate (n=22). Improvement in active nuclear import upon cell passaging. Dots represent analysed cells (WT P3 n=16, WT P19 n=25, C5 P1 n=24, C5 P3 n=27, C5 P19 n=23) of one independent experimental replicate performed. Representative images of analysed C5 P1 (left) and C5 P19 (right) NLS-3xmCherry stable cells. Mean intensity of fluorescence for cytoplasm and nucleoplasm for each cell are labelled. Scale bar, 20 μm. Cytoscape visualization of protein abundance changes between C5 P19 and C5 P1. Red nodes indicate proteins that were upregulated upon passaging; blue nodes indicate proteins that were downregulated upon passaging. White nodes display proteins with comparable expression levels at both early and late passage. NUP58 protein expression in C5 at P1 and P19. Blots were performed using Antibody 1 (Ab1, top) or Antibody 2 (Ab2, bottom). Actin was used as loading control. The band corresponding to the full-length NUP58 is indicated by a black arrow with possible alternative isoform by an asterisk. Alternative NUP58 isoform localizes to the NPC. Representative images of HCT116 cell line showing localization of WT NUP58 protein (top WT isoform, z-stack = 10) and of an isoform lacking exon 4 and ending at exon 13 (bottom mutant isoform, z-stack = 11). Scale bar, 20 μm Cellular viability following Cas9-induced gene editing of native NUP58 (left) or NUP85 (right) in WT cell line (black circles) and in a cell line overexpressing NUP58 mutant isoform (grey circles).
Figure 7(A) Schemes of glucose metabolism alternation detected by targeted metabolomics. The fold change of metabolites in mitochondria and cytoplasm between TFAM knockdown and WT SNU-368 cells were shown (n =6 per group). Increased and decreased metabolites are indicated by red and green circles, respectively. (B) The fold change of intracellular acetyl-CoA, malonyl-CoA and lactate in SNU-368 cells with TFAM knockdown was evaluated by ELISA or enzymatic reaction (n = 3 independent experiments). Data information: Graphs show mean ± s.e.m., two-tailed unpaired t-test for (B *P < 0.05; **P < 0.01. (C) Extracellular acidification rate (ECAR) was measured in TFAM knockdown and WT SNU-368 cells with serial addition of glucose, oligomycin and 2-DG (n = 3 independent experiments). The panel shows the calculated glycolytic parameters, including glycolysis and maximal glycolysis. Glycolysis was calculated as maximum rate measurement before Oligomycin injection minus last rate measurement before glucose injection. While glycolytic capacity was calculated as maximum rate measurement after Oligomycin injection minus last rate measurement before glucose injection. Data information: Graphs show mean ± s.e.m., two-tailed unpaired t-test for , C *P < 0.05; **P < 0.01. (D) Western blot analysis of anti-mDia2 IPs and whole-cell lysates (WCL) derived from HCC cells and mouse tumor tissues (n = 3 independent experiments). NT, non-tumor liver tissues; MT, metastatic tumor tissues; NMT, non-metastatic tumor tissues. (E) The level of malonylated mDia2. Equal amount of immunoprecipitated mDia2 either from TFAM knockdown or control cells was loaded in Western blot assay. (F) Western blot analysis for the distribution of mDia2 in SNU-368 cells incubated with malonyl-CoA (5 μM) or ND-630 (10 μM) (n = 3 independent experiments). (G) Bioluminescent images for lung metastasis in mice inoculated with HCC cells. n =10. Ten days after orthotopic implantation of SNU-368 shTFAM cells, ND-630 (3 mg/kg) was intravenously injected in mice every 2 days until euthanasia was performed. (H) Representative H&E images of lung metastasis in mice used in (G). Scale bars, 100μm. (I) Number of metastatic nodules per lung in mice used in (G). Data information: Graphs show mean ± s.e.m., two-tailed unpaired t-test for , I). *P < 0.05; **P < 0.01. (J) Kaplan-Meier survival curves for mice inoculated with orthotopic HCC cells as indicated. n=10 per group. (K and L) Representative merged images of mDia2-GFP (Green) and DAPI staining (blue) (K) and Phalloidin (Red) and DAPI staining (blue) (L) in HCC cells transfected with different mutant mDia2-GFP constructs as indicated (n = 3 independent experiments). Scale bars, 20μm. K12E and K27E are malonylation-mimetic mutants at lysine residues 12 and 27. K12Q and K27Q are acetylation-mimetic mutants at lysine residues 12 and 27. (M) Statistical analysis of transwell migration and invasion assays for HCC cells with treatment as indicated (n = 3 independent experiments). Data information: Graphs show mean ± s.e.m., One-way ANOVA for (M). *P < 0.05; **P < 0.01.
Figure 7EBV-LMP1 inhibits the ANT1 conformation during cisplatin chemotherapy alone to keep it in the m-state and prevent the opening of mPTP to resist cell death (A). CATR, a conformational inhibitor of ANT1, used in combination with cisplatin increases the chemosensitivity in EBV-LMP1-positive NPC (B).
Figure 3Morphology of four-week-old soil-grown plants of Col-0, mekk1-5 ndr1, and two independent transgenic lines of SNIPER1 OE into mekk1-5 ndr1 background. Scale bar = 1 cm. The mekk1 ndr1 double mutant was used instead of the mekk1-5 single mutant is to overcome the sterility. SNIPER1 gene expression in the indicated plants as determined by RT-PCR. Error bars represent mean ± SD (n = 3). Two independent experiments were carried out with similar results. Quantification of H.a. Noco2 sporulation in the indicated genotypes 7 days post inoculation (dpi) with 105 spores per ml water. Error bars represent mean ± SD (n=4). Three independent experiments were carried out with similar results. Growth of P.s.m. ES4326 on four-week-old leaves of the indicated genotypes at 0 and 3 dpi with bacterial inoculum of OD600 = 0.001. mn stands for mekk1-5 ndr1 double mutant. Error bars represent mean ± SD (n=4). Three independent experiments were carried out with similar results. In vitro ubiquitination assay using E. coli-expressed SNIPER1-myc (E3), AtUBC8 (E2), AtUBA1 (E1) and/or HIS-FLAG-Ubiquitin. The molecular mass markers are indicated on the left (kDa). Asterisk indicates SNIPER1-myc. Arrow points to His-FLAG-Ub and straight lines on the right indicate self-ubiquitinated SNIPER1.
Figure 2A-L Wild type cultured in C- (A), cho2opi3 cultured in C+ (B ), coS#4 (C-D), coS#3 (E-G), and coS#2 (I-K) cultured in C- were analyzed by electron microscopy. In addition, coS#3 (H) and coS#2 (L) are shown after culture in C+ for 3 generations. The arrow heads (F, I, J) point to protrusions in the nuclear envelope. CW, cell wall; ER, endoplasmic reticulum; PM, plasma membrane; M, mitochondria; N, nucleus; V, vacuole; *, lipid droplet. Scale bars correspond to 200 nm (A, B, D, F, G) or 500 nm (C, E, H, I, J, K, L).
Figure 3(A) A significant delay in RPA foci formation in the absence of Snf1. The percentage of cells with RPA foci was calculated and plotted against the incubation time in 200 mM HU. Error bars represent SD from three biological repeats. At least 200 cells were analyzed for each experiment of three independent experiments. Statistical significance was evaluated based on Student's t-test (*0.01 < P-values < 0.05; **0.001 < P-values < 0.01). (B) The experimental scheme for the Rfa1-13MYC ChIP in the indicated yeast strains. (C) Snap shot of Rfa1 ChIP-Seq at Chromosome VI. Cells were synchronized in G1 and released into the fresh medium containing 200 mM HU for 30 min. The sequencing reads were mapped to the yeast reference genome. The red dashed box indicated the relative read intensity of ARS607. (D) The average Rfa1 ChIP-seq read density from cells released into HU medium around ACS sites. ACS, ARS consensus sequence. (E, F) SNF1 is required for efficient recruitment of Rfa1, but not Psf2, to HU-stalled replication forks. Cells were grown and synchronized in G1 by α-factor before release into the fresh medium supplemented with 200 mM HU for the indicated time. Cell extracts were prepared and subjected to MYC-ChIP of Rfa1-13MYC (E) or Psf2-13MYC (F). The amounts of DNA in the precipitates were quantified by qPCR. Error bars represent SD from three biological repeats.
Figure 6Structural model of βarr1 in complex with phospho-peptides corresponding to the V2R, B2R and B2RL370T were generated based on previously determined structure of V2Rpp-βarr1 complex (middle panel). A bifurcated salt bridge links the V2R and B2RL370T peptides to K294 in the lariat loop of βarr1 via T359/T360 and T369/T370, respectively, but not in B2RWT peptide (upper panel). The frequency of salt bridge formation between the phospho-sites on the receptor and K294 in βarr1 was computed over 4 μs accumulated simulation time per complex using a distance threshold of 3.2 Å between the oxygen of the phosphate group of phosphorylated threonines and the protonated nitrogen of K294. B2RL370T mutant establishes a bifurcated interaction with K294 in the lariat loop, analogous to that in V2R peptide while B2R peptide forms only a single salt bridge (Figure 6, lower panel). Differential lariat loop stabilization correlates with distinct conformational states. Simulations of 4 μs per complex were clustered with a rmsd cutoff of 2.2 Å yielding "Cluster 1" and "Cluster 2" while "None" reflects conformations that did not fulfill this condition. The phospho-peptides corresponding to V2R and B2RL370T with a conserved bifurcated salt bridge adopt preferential conformational states belonging to "Cluster1" the B2RWT peptide with a single salt bridge favors the conformations of "Cluster 2"
The role of CK1α in FAM83D phosphorylation: A: Schematic representation of the anti-GFP nanobody (aGFP.Figure 5A: Schematic representation of the anti-GFP nanobody (aGFP.16)-based targeting strategy used to deliver CK1α to the CK1-binding-deficient FAM83D(F283A)-GFP mutant. B: STLC-synchronised mitotic FAM83DGFP/GFP knockin (KI), FAM83DGFP/GFP (F283A) (FA), and FAM83DGFP/GFP(F283A) stably expressing aGFP.16-CK1α (FA + aGFP.16-CK1α) U2OS cells were subjected to GFP TRAP immunoprecipitation (IP), followed by immunoblotting (IB) with the indicated antibodies. Asynchronous (AS) cells were used as controls. Data information: All blots are representative of at least 3 independent experiments. C: FA cells were infected with retroviruses encoding wild-type aGFP.16-CK1α (WT), or aGFP.16-CK1α with one of two distinct CK1α kinase-inactive mutants (D136N or K46D). Uninfected cells (-) were included as a control. Cells were lysed, subjected to anti-GFP IP and IB with the indicated antibodies. Data information: All blots are representative of at least 3 independent experiments. D: Immunofluorescence analysis for the cells described in (B) following synchronisation with STLC. Cells were stained using anti-CK1α antibody, and DNA is stained with DAPI. Scale bars, 20 µm. E: Schematic depicting the IP kinase assay strategy used to test whether FAM83D-bound CK1α was catalytically active. F: FAM83D-/- knockout (KO), KI, and FA cells were synchronised in mitosis (M) using STLC. Lysed extracts were subjected to anti-GFP IPs, followed by ATP [γ-32P] kinase assays, using an optimised CK1 substrate peptide (CK1tide). AS cells were used as controls. Error bars, SEM; P<0.01; ANOVA. n=3. Input and IP samples were analysed by IB with the indicated antibodies. Data information: All blots are representative of at least 3 independent experiments.
Fig.Figure 3Internalization of bacteria is reduced in Tpcn-/- BMDM, as assessed by single-cell imaging of BMDM allowed to internalise M. smegmatis tagged with mCherry (in red) for 1 h. Internalization of bacteria assessed discontinuously in BMDM populations: (B) The resultant growth of bacteria colonies on agar plates following lysis of BMDM infected with M. smegmatis and (C) quantification of colony forming units (CFU) expressed as a percentage of WT control (n = 12); Real-time internalization of fluorescent bacteria into BMDM populations: (D) Relative Fluorescence Units (RFU) of internalised mCherry-tagged M. smegmatis (E) and mCherry-tagged BCG over time, read using a plate reader (n = 8).
Figure 6 Cultured hippocampal neurons were co-transfected with mCherry and EGFP-tagged PSD-95 wild-type (WT), E17R, or T19K at 10-11 DIV and treated with BIC (50 μM) or left untreated at 17 DIV for 24-48 h before fixation. (A) Representative confocal microscopic images of PSD-95-EGFP (green) and mCherry (red) used as "fill" to visualize the dendrite with spine heads (scale bar: 2 µm) after control (top) and BIC treatment (bottom). Individual channels are shown in grayscale. (B) BIC treatment reduced spine enrichment of WT but not E17R or T19K PSD-95 as quantified as the ratio of EGFP intensity in spine heads relative to that in the adjacent dendritic shaft. Values were normalized to control treatments, which were set to equal 100% for each PSD-95 construct. (C) mCherry showed no enrichment in spines and BIC had no effect on its localization. (D-F) Spine enrichment and puncta size and intensity of PSD-95 were comparable under basal conditions for WT, E17R, and T19K PSD-95 (A.U., arbitrary unit). (G-I) Spine density, head diameter, and length were comparable under basal conditions for neurons expressing WT, E17R, and T19K PSD-95.
Recovery of HIF-1α levels in BNIP3-silenced cells restores melanoma glycolytic phenotype and growth in vivo.Figure 7Immunoblot detection of Hydroxylated HIF-1α (HIF-1α-OH), total HIF-1α, Luc, LDHA, BNIP3 and ACTIN levels from normoxic lysates of shCntl and shBNIP3 cells expressing either Luc or HIF-1α-AA and collected 48h after plating (n= 4). Densitometric quantifications relative to ACTIN levels are shown below each corresponding band and they were analyzed with a one-sample T-test against shCntlLuc except for LDHA in shBNIP3 Luc, which was analyzed with Wilcoxon rank test. Glut1, Hk2, Pkm1, Pkm2, Pdk1, Ldha, Ldhb, Mct4 and Vegfa transcript levels measured by qPCR in B16-F10 cell lysates collected 48h after plating (n= 5). Transcript expression is represented as the fold-change relative to their corresponding shCntl value. Heatmap shows the average per condition and values were analyzed with a one-sample T-test against shCntl Luc except for Pdk1 in shCntl HIF-1a-AA that was analyzed with Wilcoxon rank test. OCR (C) and ECAR (D) per µg of protein of B16-F10 cells assayed in medium containing either 25mM galactose (C) or 25mM glucose (D) medium using the Seahorse technology (n=4). Immunoblot detection of NCOA4 and ACTIN levels from normoxic lysates of shCntl and shBNIP3 cells expressing either Luc or HIF-1α-AA, collected and treated for 24h in the absence or presence of 5nM BafA (n=3). Densitometric quantifications relative to ACTIN levels are shown below each corresponding band and they were analyzed with a one-sample T-test against shCntl Luc. Scheme explaining the feedforward loop between HIF-1α and BNIP3 in the different tumor conditions (G). Growth curves of B16-F10 tumors generated by shCntl or shBNIP3 cells expressing Luciferase (Luc) or an undegradable HIF-1α (HIF-1α-AA) construct (n=7 for CntlKD Luc/BNIP3KD HIF-1α-AA, n=8 CntlKD HIF-1α-AA and n=5 for BNIP3KD Luc) and analyzed with a two-way ANOVA with Tukey's multiple comparisons test. Representative double immunostaining for CD31 and αSMA in B16-F10 tumors (n=5 per condition). Scale bars represent 50µm.
Fig.Figure 6Graphical summary of the alterations in the dynorphinergic system between healthy (top panel) and epileptic (middle panel) hippocampi and their consequences on neuronal signaling. The bottom panel indicates the proposed mode of action of AAV-pDyn gene-therapy. The incoming burst stimulus triggers the release of dynorphins in a "release on demand" mode.