EMBO/sd-character-level-ner huggingface dataset:
JQ1 treatment preserves photoreceptor number and function in rd10 mice.Fig. 1JQ1 treatment preserves photoreceptor number and function in rd10 mice. Vehicle (DMSO) and JQ1 (0.1 mM) were intravitreally injected, respectively, into one eye and the contralateral eye of rd10 mice at PN14. WT (B6) mice were injected only with vehicle. At PN18, PN21, PN24, PN27, and PN30, mice were either subjected to ERG measurements, or sacrificed for preparation of retinal sections. a Representative H&E-stained retinal paraffin sections showing a protective effect of JQ1 on ONL thickness in rd10 mice. OS/IS outer/inner segment, ONL outer nuclear layer, INL inner nuclear layer, GCL ganglion cell layer. Scale bar 50 μm. For images of PN21 and PN27, see Additional file 1: Figure S1. b Quantification for a: photoreceptor cell number per 100 μm ONL length; mean ± SEM, n = 6 mice; **P < 0.01 compared to rd10 vehicle control. c Representative ERG traces (measured at PN24, in response to 0.3, 3, or 30 cd s/m2 light intensity of flashes) showing rescue of rd10 mouse retinal function by JQ1 treatment. d Quantification of a-wave and b-wave amplitudes. ERG a-wave is the downward deflected negative response; b-wave is from the a-wave peak to positive response peak. Oscillatory potential is visible superimposed on b-wave. Data are presented as mean ± SEM, n = 10 mice, *P < 0.05, **P < 0.01 compared to rd10 vehicle control
Figure 4(A) Western blotting of Drp1flox/floxOpa1flox/flox and Drp1Opa1-KO MEFs. The asterisk indicates non-specific bands of anti-Oma1 antibodies.(C) Drp1flox/floxOpa1flox/flox MEFs and Drp1Opa1-KO MEFs were transduced with the indicated Opa1 constructs. The cells were observed using laser scanning confocal microscopy for 30 min with 10-s intervals in the presence of TMRE. The percentage of cells that exhibited flickering is shown. Values are average ± SD (n = 3 experiments). In each experiment, 48-62 cells were analyzed. (D) Drp1flox/floxOpa1flox/flox MEFs and Drp1Opa1-KO MEFs carrying the indicated Opa1 constructs were subjected to confocal immunofluorescence microscopy using anti-PDH antibodies. Scale bar, 10 µm. (E) Quantification of mitochondrial morphology is shown (n = 3 experiments). In each experiment, 30 cells were analyzed. (F) L2-HA constructs carrying a mutation (K301A or K468D) in the GTPase domain. (G) Western blotting of Drp1flox/floxOpa1flox/flox MEFs and Drp1Opa1-KO MEFs, both of which carry the indicated Opa1 constructs. The asterisk indicates non-specific bands of anti-Oma1 antibodies. (H) Quantification of band intensity. Values are average ± SD (n = 3).
Radixin is the AKAP required for glucagon-induced PKA activation and hyperglycemia in vivo.Figure 4A. The expression of Prom1, radixin, AKAP7, AKAP8, AKAP8I, AKAP9, AKAP10, AKAP12 or AKAP13 was silenced in WT hepatocytes using siRNAs. Hepatocytes were then serum-starved for 18 hours and stimulated with glucagon (10 nM) for 10 minutes. The phosphorylation of PKA substrates was determined by immunoblotting. Prom1 and radixin expression were silenced in WT (Prom1+/+) and KO (Prom1-/-) hepatocytes by infection with an adenovirus harboring sh-control (sh-Con), sh-radixin (sh-Rdx) or sh-Prom1 for 24 hours. Hepatocytes were further serum-starved for 18 hours and stimulated with glucagon (10 nM) for 10 minutes. (B) The nuclear localization of p-CREB after glucagon stimulation was determined by immunofluorescence staining. Blue; DAPI, Red; p-CREB. The percentage of cells with nuclear p-CREB staining among more than 300 cells in each group was also statistically analyzed (Figure EV3G). Scale bar, 20 μm. Prom1 and radixin expression were silenced in WT (Prom1+/+) and KO (Prom1-/-) hepatocytes by infection with an adenovirus harboring sh-control (sh-Con), sh-radixin (sh-Rdx) or sh-Prom1 for 24 hours. Hepatocytes were further serum-starved for 18 hours and stimulated with glucagon (10 nM) for 10 minutes. (C) Levels of p-PKA substrates, p-CREB, CREB, p-IP3R, IP3R, Prom1, radixin and GAPDH after glucagon stimulation (10 minutes) were determined by immunoblotting. D. Immunofluorescence staining of Prom1 in mouse liver tranduced with adenoviral particles. Confocal microscope images of WT mouse liver infected with adenovirus harboring sh-Con or sh-Rdx linked with IRES-GFP. Scale bar, 20 μm. 12-week-old male wild type mice were infected with an adenovirus harboring sh-control (sh-Con) or sh-radixin (sh-Rdx) for 3 days, fasted for 4 hours and intraperitoneally injected with glucagon. (E) Blood glucose levels (mg/dL) were measured 0, 15, 30, 60 and 120 minutes after glucagon administration (200 µg/kg body weight, n = 10 mice/ group). 12-week-old male wild type mice were infected with an adenovirus harboring sh-control (sh-Con) or sh-radixin (sh-Rdx) for 3 days, fasted for 4 hours and intraperitoneally injected with glucagon. (F) Levels of p-PKA, p-CREB, CREB, radixin and GAPDH in the liver after a 10-minutes glucagon stimulation (2 mg/kg body weight) were determined by immunoblotting. 12-week-old male wild type mice were infected with an adenovirus harboring sh-control (sh-Con) or sh-radixin (sh-Rdx) for 3 days, fasted for 4 hours and intraperitoneally injected with glucagon. (G) Glucose disposal rates in sh-Con or sh-Rdx (radixin) mice were measured using glucose, insulin and pyruvate tolerance tests (n = 10 mice/group).
Figure 7(A-F) Immunofluorescence analysis of the old APPPS1 slice (14 DIV) treated with vehicle Ctr in A, anti-inflammatory factors IL-10 and TGF-β in B and C and pro-inflammatory factors IL-6, IL-12/p40 and GM-CSF in D, E and F, respectively and immunostained with CD68 (red) and M3.2 (green). In contrast to increased number of core-only plaques upon GM-CSF treatment, other factors tested did not induce amyloid plaque clearance, resulting in a better preservation of the M3.2 positive halos of Aβ surrounding dense core of amyloid plaques. Scale bar: 50 µm.
Figure 5Representative western blot images and quantification of phospho-mTOR S2448, total mTOR and actin in L6 cells after iron treatment (250 µM, 24 h) followed by withdrawal for 4 h. Representative western blot images of phospho-mTOR S2448, total mTOR phsophpo-TSC2 T1462, total TSC2, phospho-AKT S473, total AKT, actin in L6 cells after iron treatment (250 µM, 24 h) followed by withdrawal for 4 h with or without MK2206 (AKT inhibitor.) Representative western blot images of phospho-UVRAG S550 and total UVRAG in L6 cells transfected with FLAG-UVRAG and FLAG pulldown after iron treatment (250 µM, 4 h and 24 h) followed by withdrawal for 1 h and 3 h. Representative western blot images of endogenous phospho-UVRAG S550, total UVRAG expression in L6 cells after iron treatment (250 µM, 24 h) followed by withdrawal for 4 h with or without torin1 (200 nM). Live cell imaging analysis of L6 cells transfected with LAMP1-RFP from Movie EV2: lysosomal number in L6 cells after iron treatment (250 µM, 24 h) followed by withdrawal for 4 h with or without Torin1 (200 nM). Live cell imaging analysis of L6 cells transfected with LAMP1-RFP from Movie EV2: speed (F) in L6 cells after iron treatment (250 µM, 24 h) followed by withdrawal for 4 h with or without Torin1 (200 nM). Representative confocal images of time-lapse captures of lysosomes at 6 sec intervals for 30 sec. White and yellow arrows mark positions of two representative LAMP1 puncta over 30 seconds, and the traces of puncta were shown as lines in final panel. Schematic diagram of mTOR signaling regulation by IO.
Figure 5(A) Diagram of MDMX intramolecular interactions and communication between p53 and CK1α binding. (B) Purified FLAG-CK1α was incubated with ATP, GST-MDMX, GST-β-catenin-1-200, pDI peptide (LTFEHYWAQLTS, 5 µM), and pDI3A peptide (LTAEHYAAQATS, 5 µM). β-catenin pS45 level was determined by western blot. (C) H1299 cells were cotransfected with MDMX, CK1α and p53 mutants. MDMX-CK1α binding was determined by IP-western blot. (D) Endogenous p53 in A549 (wt) and Panc1 (R273H) cells were depleted by CRISPR/Cas9 knockout. β-catenin pS45 level was determined by western blot. (E) The β-catenin S45 kinase activity in A549 and Panc1 cells was determined
Fabs targeting the CC1 and OR of PrPC prevent prion-induced neurotoxicity in COCS.Figure 3(A) Fluorescence micrographs of tga20 slices chronically exposed to prions and cultured in the presence of either Fab3 (CC123‑50), Fab71 (OR51‑91) or Fab1 (GD) for 45 days. Scale bar: 500 μm. (B) Same as (A), but in the presence of the affinity-maturated antibodies. (C) NeuN immunofluorescence coverage of prion-infected slices cultured in the presence of the Fabs. Antibodies targeting the CC123‑50 and the OR51‑91 afforded protection against neurodegeneration. NBH: non-infectious brain homogenate. Each dot represents a cerebellar slice for all treatment groups. (D) Western blot analysis of COCS lysates. Prion-infected slices treated with Fab100, but not with Fab83 and Fab25 showed reduced levels of PrPSc. n = 3 biological replicates.
Figure 4(A) The GRFs of Neil3 recognizes PAR. The recombinant GST fusion proteins were incubated with PAR and the protein-PAR complex was pulled down by glutathione agarose beads. Samples were serially diluted three-fold, spotted onto the nitrocellulose membrane, and subjected to dot blotting using anti-PAR antibody (top panel). FL represents full length Neil3, N-ter represents N-terminal Neil3 (1-300 aa), Mid represents middle Neil3 (300-404 aa), and GRFs represents C-terminal Neil3 GRFs domain (480-606 aa). Recombinant GST protein was used as the negative control (NC). The GST fusion proteins were also examined by the SDS-PAGE followed with Coomassie blue staining (middle panel). The relative PAR-binding in the pull down assay was summarized in the graph (bottom panel). Values are mean ± SD of three assays. (B) Tandem co-immunoprecipitation and Western blotting assays demonstrate that Neil3 interacts with ADP-ribosylated H2AX but not the E141A mutant H2AX. (C,D) The GRFs of Neil3 is sufficient and required for the interaction with ADP-ribosylated H2AX. Tandem co-immunoprecipitation and Western blotting assays were used (E,F) The relocation kinetic of full-length Neil3 or the GRFs of Neil3 to DNA lesions was analyzed. GFP-tagged full length Neil3 or the GRFs of Neil3 was expressed in the indicated cells, and relocation kinetics was monitored in a time course following laser microirradiation (left panel). The laser stripe is indicated with yellow arrowheads. The relocation kinetics is shown in the right panel. Data represent mean ± SD from three biologically independent experiments (right panel). At least 20 cells were included in each experiment. (G,H) The recruitment of full-length Neil3 or the GRFs of Neil3 to the KR-induced oxidative lesions is mediated by H2AX ADP-ribosylation (left panel). Scale bars, 2 μm. The foci were indicated with white arrowheads. Scale bars, 0.5 μm. Right panel: the percentage of Neil3 or GRFs Neil3 foci co-localized with KR was quantified. Mean value with SD is from 20 cells.
Figure 4Schematic of the experimental plan for Plk1 and cMet inhibition/silencing for viability and apoptosis studies in mesenchymal and epithelial NSCLC cell lines. Cell viability was measured using CellTiter-Glo in NSCLC cell lines treated with volasertib, tepotinib, or a 1:2 ratio of both for 72 hours. Left: representative cell viability graph of cells treated with the indicated drugs. Right: heatmap depicting the calculated combination indices at Fa = 0.25 and Fa = 0.5. Apoptosis was measured using the Apo-BrdU assay in the indicated cell lines treated with the indicated drugs for 24 hours. Immunoblots from cells treated with indicated drugs for 24 hours (left) with densitometric quantification normalized with β-actin (right). All tested cell lines were treated as indicated for 24 hours and allowed to grow in drug-free medium for 15-20 days to form colonies, which were counted using ImageJ. Apoptosis was measured using the Apo-BrdU assay in the indicated cell lines transfected with 10nM siRNA as indicated for 48 hours. NT, non-targeting control. Immunoblots from cells transfected with siRNA as indicated for 48 hours (left) with densitometric quantification normalized with β-actin (right). Data are means ± standard error of the mean from three independent experiments. Significant differences using two-way analysis of variance with Bonferroni or Benjamini-Hochberg (BH) correction for multiple comparison are indicated (*p < 0.01). Mesenchymal and epithelial NSCLC cell lines are indicated in blue and red text, respectively.
Figure 3Each protein interacting with either of or both the CBL-OST and CBLB-OST baits is represented as a node and is linked by an edge to the corresponding bait. Proteins are identified by their BioGRID designations and color-coded according to their function or protein family; see key (right). As specified, a set of proteins interacts with both CBL and CBLB, and CBLB is found in the CBL signalosome.
Figure 2(A) Volcano plot of proteomic data reveals significant changes in proteins of the respiratory chain and factors involved in mitochondrial protein synthesis (FDR < 0.05). (B) Bar chart representing enriched Gene Ontology (GO) terms. FDR was calculated by Benjamini-Hochberg correction (n=4). (C) Respiratory chain enzyme activities. Relative enzyme activities of respiratory chain enzyme C I (NADH coenzyme Q reductase), C I/III (NADH cytochrome c reductase), C II (succinate dehydrogenase), C II/III (succinate cytochrome c reductase) and C IV (cytochrome c oxidase) in mitochondrial extracts from wild type and Clpp knockout hearts. (n=5). Bars present mean levels ± SD. Statistical difference was calculated by Student's t-test and changes are presented in the figure (n=5). (D) Substrate-dependent oxygen consumption rates in isolated heartmitochondria. Complex I substrates (pyruvate, malate, glutamate); complex II substrate (succinate). State 3 - ADP stimulated oxygen consumption rate; State 4 - respiration rate after addition of oligomycin; Uncoupled - oxygen consumption rate in the presence of FCCP. Bars present mean levels ± SD. Student's t-test was used to determine the level of statistical difference (n=7). (E) Decrease of steady-state levels of respiratory chain subunits in Clpp-knockout hearts relative to wild type (set to 1). VDAC or TOMM20 were used as loading controls. Bars present mean levels ± SD. Student's t-test was used to determine the level of statistical difference (n=4). (F) Blue Native Page Analysis (BN-PAGE) of the respiratory chain supercomplexes followed by Western blot analysis using antibodies against C I (anti-NDUFA9); C III (anti-UQCRC1) and C IV (anti-COX4A).
Loss of OGT in IECs causes intestinal damages in mice.Figure 2A Body weight of male wildtype and Vil-Ogt KO mice at 6 weeks of age (n = 6, P = 0.000015)  B Incidence of rectal prolapse in male wildtype and Vil-Ogt KO mice (WT n = 17, KO n = 9, P < 0.0001)   C Representative colonoscopy images of male wildtype and Vil-Ogt KO mice   D Body weight of female wildtype and Vil-Ogt KO mice at 9 weeks of age (WT n = 11, heterozygous KO n = 4, homozygous KO n = 5, P < 0.0001)   E Incidence of rectal prolapse in female wildtype and Vil-Ogt KO mice (WT n = 9, heterozygous KO n = 8, homozygous KO n = 6, P = 0.0439)  F H&E staining of duodenum, jejunum, ileum, colon, and rectum of 10-week-old male wildtype and Vil-Ogt KO mice. Scale bars = 100 μm
Figure 4CggR intracellular abundance in the wildtype (WT) strain on glucose strongly varies with the promoter used. The CggR intracellular levels were quantified by proteomics in steady-state cultures grown in minimal media with glucose as carbon source at a final concentration of 10 gL-1. Error bars represent the standard deviation of at least three replicate experiments. The relative abundance of CggR (normalized to the abundance measured on glucose and the same promoter) is almost constant with PTEFmut7 across multiple growth rates in WT and TM6 cells, but not with PTEFmut2 and PCMV. The CggR intracellular levels were quantified by proteomics in steady-state cultures grown in minimal media with glucose, galactose, maltose or pyruvate as carbon sources at a final concentration of 10 gL-1. WT data includes all three promoters, whereas TM6 only includes the PTEFmut2 and PTEFmut7 data. Error bars represent the standard deviation of at least three replicate experiments.
Figure 1Schemes illustrating distinct modes of cellular communication potentially contributing to DC activation. In the autocrine model, cells respond to their own cytokine production while in the paracrine model, cells respond to the cytokine produced by their close neighbors. Finally, during quorum sensing, the cellular response is collective but critically depends on the density of secreting cells. In this model, a low density of secreting cells may not achieve detectable activation while a high density of cytokine-producing cells triggers collective activation.
Fig.Figure 1A. Ncl and Pus7 mRNA levels (normalized to TBP) at different time points after LPS stimulation in c-mycwt/wt and c-myc∆/∆ cells. Data are presented as mean ± SD; n = 3. B. To quantify RNA synthesis, we exposed our cultures to a brief pulse of ethyl-uridine (EU) and measured its incorporation into RNA by light microscopy. The scatter plots show the nuclear Area as detected in the 2D plane (x axis, in pixels) versus the EU signal (y axis, as arbitrary units, AU) for each single cell identified by DAPI staining in c-mycwt/wt and c-myc∆/∆ populations at the indicated time points after LPS treatment. One representative experiment out of 5 is shown. C. Variation of each expressed mRNA at 4h of LPS treatment (log2FC, relative to time 0) for c-mycwt/wt (x-axis) and c-myc∆/∆ cells (y-axis), as measured by RNA-seq. Regulatory groups 1-6 and Grey Zone (GZ) are as defined in the text. D. Numbers of genes classified in the different regulatory categories at the indicated time points after LPS stimulation. E. Variations (log2FC) in mature and precursor RNAs (as measured by RNA-seq) and in the modeled rates (RNA synthesis, processing and degradation) for Myc dependent and independent genes. The grey scale represents the starting level for each parameter in unstimulated cells.
Figure 6Day 0: The TRs Rcan1, Taf1c, Tead4 and Tfap4 are expressed in naïve MEFS. Approximately 50% of naïve cells express all 4 factors. 18 hours: OSKM induce the expression of Cbfa2t3 in ~40% of the cells. Only a fraction of the population expresses all 5 TRs. Day 3: The expression of Gli2, Irf6, and Ovol1 has been induced by direct and indirect OSKM effects. Various interconnections between the TRs are established, thus stabilizing their expression. All eight TRs are expressed in a limited number of cells found exclusively in early-iPSCs colonies. Day 6: Nanog expression is induced in ~40% of the cells within the early-iPSCs colonies. All nine TRs are expressed only in ~6% of the cells found exclusively within these early formations. The nine TRs reconstruct the 9TR GRN, which is required to promote cellular reprogramming. iPSCs: The 9TR GRN GRN is dissolved in iPSCs because Ovol1 and Cbfa2t3 expression is dramatically reduced to undetectable levels.
Figure 3 (A+B) CD31 staining for endothelial cells revealed increased tumor vascularization in mice bearing human EGFL7 (hE7)- or murine EGFL7 (mE7)-positive tumors (n = 3; one-way ANOVA) (C+D) T2-weighted magnetic resonance imaging (MRI) analysis of these brains confirmed that all mice developed tumors of similar size. T1-weighted MRI images showed decreased vessel permeability as measured by Gadovist extravasation in tumors expressing hE7 or mE7 (red arrows; n = 6; one-way ANOVA). Scale bar represents 2.5 mm (C+D) T2-weighted magnetic resonance imaging (MRI) analysis of these brains confirmed that all mice developed tumors of similar size. T1-weighted MRI images showed decreased vessel permeability as measured by Gadovist extravasation in tumors expressing hE7 or mE7 (red arrows; n = 6; one-way ANOVA). Scale bar represents 2.5 mm Enhanced maturation of glioma vessels in the presence of hE7 or mE7 was verified by increased co-localization of (E+F) PDGFRβ (pericytes) with CD31 (n = 3; one-way ANOVA; quantifications normalized to CD31). Data presented as mean ± SEM, AU-arbitrary units. Scale bars represent 60 µm Enhanced maturation of glioma vessels in the presence of hE7 or mE7 was verified by increased co-localization o (G+H) SMA (smooth muscle cells) with CD31 (n = 3; one-way ANOVA; quantifications normalized to CD31). Data presented as mean ± SEM, AU-arbitrary units. Scale bars represent 60 µm Enhanced maturation of glioma vessels in the presence of hE7 or mE7 was verified by increased co-localization o (I+J) Col IV (basement membrane with CD31 (n = 3; one-way ANOVA; quantifications normalized to CD31). Data presented as mean ± SEM, AU-arbitrary units. Scale bars represent 60 µm
Figure 8 α-Syn is released from neurons, taken up into neighboring neurons and accumulated with endogenously expressed α-syn. In donor cells, c-src impairs autophagy, thus facilitating α-syn release. In recipient cells, c-src enhances the uptake of α-syn by regulating endocytosis. Accordingly, c-src plays a dual role in cell-to-cell transmission of α-syn. It regulates the uptake of α-syn via endocytosis. It also regulates the release and intracellular accumulation of α-syn via autophagy.
Figure 4(A) Effect of Dsl and COPII ts mutants on Ykt6 localization to autophagosomes. Wild-type and selected ts strains (sec12-1, tip20-5 and dsl3-2) expressing GFP-tagged Ykt6 and mCherry-tagged Atg8 were grown at 24°C in SGC for 8 h and then shifted to SG-N for 2 h at 37°C. Vacuoles were stained by CMAC and analyzed by fluorescence microscopy. Scale bar, 5 µm. Scale bar in inset, 0.7 µm. Arrows indicate dot colocalization of Ykt6 and Atg8 (A) Wild-type and selected ts strains (sec12-1, tip20-5 and dsl3-2) expressing GFP-tagged Ykt6 and mCherry-tagged Atg8 were grown at 24°C in SGC for 8 h and then shifted to SG-N for 2 h at 37°C. (B) Percentage of Ykt6 puncta co-localizing with Atg8. Cells (n≥150) and Atg8 dots (n≥100) were quantified. (C) Localization of Ykt6 relative to Sec63 and Sec13 during growth and nitrogen starvation conditions. tip20-5 cells expressing GFP-tagged Ykt6 and 3xmCherry-tagged Sec63 or Sec13 were grown at 24°C in SGC for 8 h and then shifted to SG-N for 2 h at 24°C or 37°C, and analyzed by fluorescence microscopy. Scale bar, 5 µm. Scale bar of inset, 0.7 µm. Arrows indicate dot colocalization of Ykt6 with Sec63 (C). (D) Effect of dsl mutation on autophagosome-vacuole fusion in vitro. The vam3∆ or dsl ts strain expressing Atg9-3xFLAG and GFP-Atg8 were starved for 3 h at 30°C before purifying autophagosomes, which was sufficient to induce the ts phenotype. Vacuoles were isolated from pep4∆ cells expressing Vac8-3xmCherry and then incubated with autophagosomes at 26°C for 1 h with or without ATP (E) Quantification of (D). Error bars represent standard deviation of 3 independent experiments. Fluorescence intensity of GFP-Atg8 in the vacuolar lumen was quantified by image J using the ROI (region of interest) manager tool. Vacuoles (n≥40) for each experiment were quantified.
Figure 5(A) Polarographic measurement of KCN-sensitive oxygen consumption, driven by NADH in isolated coupled mitochondria. Mitochondria were isolated from cells expressing the wild type form of Cor1 (Cor1WT) or the mutant Cor1N63A, N187A, D192A, V189A, Y65A, L238A, K240A (Cor1**). Thereby, strains either overexpressed cytochrome c (Cyt cOE) or harbored the empty plasmid as a control (Empty). Measurement was performed in the absence (basal respiration) or presence (phosphorylating condition) of ADP. Analysis of competitive fitness of strains described in (A). Cor1WT strains harbored a chromosomally-integrated clonNAT selection cassette, whereas Cor1** contained a hygromycin selection cassette. Strains were cultivated in CM media containing glucose (B) as carbon source. Analysis of competitive fitness of strains described in (A). Cor1WT strains harbored a chromosomally-integrated clonNAT selection cassette, whereas Cor1** contained a hygromycin selection cassette. Strains were cultivated in CM media containing galactose (C) or glycerol (D) as carbon source.