EMBO/sd-character-level-ner huggingface dataset:
C oncogene upregulates Rho/ROCKI/pMLCII signaling pathway via SRC A.Figure 2A. Western blot analysis of Rho-GTP pull-down assay in MDA-MB-231 and MCF7 cell lines transiently transfected either with control or Flag-RASSF1C plasmid. B. Rho-GTP pull-down binding assay in MDA-MB-231 control or RASSF1C over-expressing cells and co‑transfected either with SRC plasmids, siRNA against SRC or siNT (non-targeting sequence). Western blot analysis shows Rho activity (Rho GTP-double band) and pMCLII and pRhoGDI binding. The ratio below Rho-GTP Western blot shows differences in Rho-GTP protein expression, compared to control condition. Data information: For both Rho-GTP pull-downs and immunoprecipitation assays total proteins were used as loading controls. All data are from n=3 independent experiments. Data are analyzed by Student T-test, represented as mean ± SEM. *** p ≤ 0.001. C. Western blot analysis of proteins in MDA-MB-231 cells transiently transfected with siRNA targeting a control sequence (NT) or targeting RASSF1. GAPDH was used as a loading control. D. Immunoprecipitation from MDA-MB-231 transiently transfected with control (pcDNA3) or HA‑RASSF1C plasmid. Pull‑down was performed using a SRC or same species IgG antibody and blotted with indicated antibodies. E. Schematic cartoon recapitulating the proposed RASSF1C/SRC‑driven mechanism. F. Top, schematic of RASSF1C domain structure indicating putative mutations in the RA domain affecting RhoA activity. Bottom, immunoprecipitation of MDA-MB-231 transiently transfected with either control (DsRed), DsRASSF1C plasmid, DsRASSF1C-R197W or DsRASSF1C-R199F mutants. Pull‑down was performed using a SRC or same species IgG antibody and blotted with indicated antibodies. Data information: For both Rho-GTP pull-downs and immunoprecipitation assays total proteins were used as loading controls. All data are from n=3 independent experiments. Data are analyzed by Student T-test, represented as mean ± SEM. *** p ≤ 0.001. G. Western blot analysis of Rho-GTP pull-down assay in MDA-MB-231 cells transiently transfected with RASSF1C or its mutants (R197W, R199F). The ratio under Rho-GTP Western blot shows differences in active Rho protein expression, compared to control condition. Data information: For both Rho-GTP pull-downs and immunoprecipitation assays total proteins were used as loading controls. All data are from n=3 independent experiments. Data are analyzed by Student T-test, represented as mean ± SEM. *** p ≤ 0.001. H. Quantification of rounded versus elongated cells in single cell morphology assay in 3D‑collagen indicating the degree of mesenchymal-amoeboid transition of MDA-MB-231 cells when RASSF1C or the described mutants are expressed. I. Representative confocal images show elongated or rounded cells per field of view of MDA‑MB-231 cells transfected with DsRed, DsRASSF1C, DsRASSF1C-R197W or DsRASSF1C-R199F, grown in 3D‑collagen and stained with Phalloidin-568 (red) for F‑actin and pMLCII/Alexa 633. Scale bars represent 10 μm.
Nuclear foci contain multiple viral RNAs and DNA genomes (A-C) vDNA foci are also foci of vRNA.Figure 2vDNA foci are also foci of vRNA. (A) Dual-color image of an infected ThP1 cell showing the viral DNA (red) and the RNA visualized by RNA-FISH (green). Arrows show foci containing both vDNA and vRNA. The bottom row shows the boxed region magnified, with vDNA and vRNA displayed separately and in combination. See also Appendix Fig. S6. (B) Venn diagram shows the number of vDNA foci and vRNA foci and the number of vDNA foci colocalizing with vRNA foci. The p-value indicates the significance of colocalization based on a jittering analysis (see Methods). (C) Scatter plot shows the intensities of vDNA and vRNA in colocalizing foci with the Spearman correlation r and associated p-value. In ThP1 cells co-infected with a HIV1-GFP virus and a HIV1-LUC virus, nuclear foci contain mixtures of both viruses. (D) Dual-color image shows RNA-FISH against GFP (red) and RNA-FISH against LUC (green). Arrows show foci containing both GFP vRNA and LUC vRNA. The bottom row shows the boxed region magnified, with GFP vRNA and LUC vRNA displayed separately and in combination. See Appendix Fig. S7. (E) Venn diagram shows the number of GFP RNA foci, the number of LUC RNA foci, and the number of GFP RNA foci colocalizing with LUC RNA foci. (F) Scatter plot shows intensities of GFP RNA and LUC RNA in colocalizing foci with the Spearman correlation r and associated p-value.
Pol λ exclusively stabilizes 8-oxo-dG in the syn-conformation upon initial binding.Figure 1(A) Substrate used for crystallization of the DNA binding complex. The oligo contains a templating 8-oxo-dG shown in green. (B) The 39-kDa catalytic domain (8-kDa subdomain not shown for clarity) is colored by subdomain: fingers (salmon), palm (yellow) and thumb (purple). The polymerase is in complex with a 1-nt gapped 16-mer oligo (grey) containing a templating 8-oxo-dG (green). Key residues involved in stabilizing the templating base (Y505 and R517) are shown in black. (C) (Left panel) 8-oxo-dG (green) preferentially adopts the pro-mutagenic syn-conformation. A simulated annealing Fo-Fc omit density map is shown for 8-oxo-dG (contoured at 3σ). (Right panel) 8-oxo-dG(anti) (black, modeled using 2PFO) was overlayed with the Fo-Fc omit density map. In the anti-conformation, the C8-carbonyl would directly clash with the 5′-phosphate (red-dotted line). (D) 8-oxo-dG(syn) (green) is stabilized by multiple interactions, including Tyr505 and Arg517 (grey).
Pyruvate metabolism modulation affects the behavior of rat neonatal cardiomyocytes in culture.Figure 4(a, b) Staining for Ki67, CTNI and DNA (DAPI) in control, PDHA1STA OE or PDK3 OE RNCMs cultured in growth (a) or non-growth (b) medium; arrowheads point to Ki67+ RNCMs; percentage of Ki67+ RNCMs shown on the right (n = 4 biological replicates for each condition). (c) Staining for CTNI and DNA (DAPI) in scratch assay using control, PDHA1STA OE or PDK3 OE RNCMs immediately or at 3 days after generating the scratch; magnified view of area in white boxes shown below; arrowheads point to membrane protrusions of RNCMs; quantification of CM membrane protrusions shown on the right (n = 4 biological replicates for each condition). (d) Reactome over-representation analysis of differentially regulated genes in PDK3 OE RNCMs compared to control. (e Heat map of gene expression for key regulators of cell cycle in PDK3 OE vs control RNCMs. f) Heat map of gene expression for key regulators of DNA replication in PDK3 OE vs control RNCMs.
Fig.Figure 3A Schematic representation of human SQLE genomic structure. Shown are the exon/intron organization and two potential p53 response elements (RE1 and RE2) and the corresponding mutant response elements. B p53+/+ and p53-/- HCT116 cells were analyzed by chromatin immunoprecipitation (ChIP) assay using normal IgG and anti-p53 antibody as indicated. C p53+/+ HCT116 cells transfected with control siRNA or SREBP2 siRNA for 72 hours were analyzed by ChIP assay with the indicated antibodies. D Control and SREBP2 knockout MCF-7 cells using sgRNA CRISPR/Cas9 were analyzed by ChIP assay using anti-p53 or normal mouse IgG antibodies. E The electrophoretic mobility shift assay (EMSA) of SQLE-RE2 or RE2 mut in the presence or absence of p53 antibodies as indicated. Blue box (shift band) means the binding between nuclear extracts and RE2, red box (super-shift band) means p53 as the protein presented in the EMSA band. F Luciferase reporter constructs containing RE1, RE2, RE1mut or RE2mut were transfected into p53-/- HCT116 cells together with control (PRK5-flag vector) or p53 expression vector (PRK5-flag p53) for 48 hours. Renilla vector pRL-CMV was used as a transfection internal control. Relative levels of luciferase are shown. Data represent three independent experiments. Protein expression is shown G Luciferase reporter constructs containing RE1, RE2, RE1mut or RE2mut were transfected into HEK293T cells together with control (PRK5-flag vector) or p53 expression vector (PRK5-flag p53) for 48 hours. Renilla vector pRL-CMV was used as a transfection internal control. Relative levels of luciferase are shown. Protein expression is shown. H Luciferase reporter constructs containing RE2 were transfected into HEK293T cells together with control, wild-type p53 or p53 R175H expression vector for 48 hours. Renilla vector pRL-CMV was used as a transfection internal control. Relative levels of luciferase are shown. I Luciferase reporter assay with indicated response element constructs in human HEK293T cells co-transfected with or without p53 expression vector. J Luciferase reporter constructs containing RE2 were transfected into HEK293T cells treated with or without 20μm PFTα for 24 hours. Renilla vector pRL-CMV was used as a transfection internal control. Relative levels of luciferase are shown.
Figure 6(A) Schematic regimen for AOM/DSS-induction of colon tumorigenesis. At 8~12 wk of age, AhR WT (H, n=10) and KO (HV, n=10) mice were treated with a single dose of AOM (10 mg/kg) by s.c injection, followed by 3 cycles of 2% DSS in the drinking water for 5 d. Mice were terminated 6 wk after the third cycle of DSS. (B) Representative colon images from AhR WT and AhR KO mice following AOM/DSS treatment. Scale bar, 9 mm. (C) H&E staining of representative colon tumor sections from AhR WT and KO mice, scale bar 200 μm. (D) Incidence of colon adenomas, adenocarcinomas and both combined (tumor) in AhR WT and KO mice, *p<0.05. (E, F) (E) Adenoma and (F) adenocarcinoma multiplicity in AhR WT and KO mice. (G) Tumor volume in AhR KO and WT mice. Grey line indicates mean. (H) FoxM1 mRNA expression in normal (uninvolved) crypts adjacent to colonic tumors. Images were quantified and expressed as FoxM1 positive area per crypt (n=5 mice per group). Scale 200 µm. Dashed line indicates median and dotted lines indicate 25th/75th quartiles. (I) FoxM1 mRNA expression in colon tumors. FoxM1 mRNA ISH area was averaged per tumor on each slide (n=5 mice per group). Scale 200 µm. Dashed line indicates median and dotted lines indicate 25th/75th quartiles.
Fig.Figure 6A Ferrets (n = 4 per group) were immunized with AAV-vectors or quadrivalent inactivated vaccine (QIV). Serum was obtained at indicated time points (red drops). After challenge with a Cal/7/9-like (H1N1) pdm strain clinical signs, temperature and weight loss were monitored. At 3 dpi, animals were sacrificed and necropsied. B Cal/7/9-specific serum antibody titers in individual sera at indicated time points. Black lines: individual animals, red lines: mean titer. Statistical significance between pre-immune and immune sera was determined using Friedman test with Dunn's multiple comparison testing (*p<0.05, **p<0.01). The lower table indicates Cal/7/9-specific MN50 and HAI mean titers. Assays were performed in technical duplicates. C to E Clinical score (C), body temperature (D), and relative weight (E) during challenge period. Statistical significance between pre-immune and immune sera was determined using Friedman test with Dunn's multiple comparison testing (AAV-HA ##p<0.01; AAV-cHA §§p<0.01; AAV-GFP $p<0.05; $$p<0.01; QIV *p<0.05;). Mean ± SD F Virus titers in nasal turbinates (NT), trachea or lung homogenates. Statistical significance between AAV-GFP and vaccine groups was determined using Kruskal-Wallis test with Dunn's multiple comparison testing (*p<0.05). Lines indicate the GMT. Assays were performed in technical duplicates. G and H IHC staining of submucosal glands (SMG) (G) or bronchi (H) with an anti-H1N1 polyclonal antibody (bar: 100 µm; red: influenza antigen (neufuchsin); blue: nuclei (haematoxylin)). I and J H&E stained histological sections of bronchi (I) and lung parenchyma with alveolar epithelial cells (AEC, J). Arrowheads: areas of suppurative bronchitis/bronchiolitis; arrows: interstitial pneumonia, necrosis of AEC (bar: (I) 100 µm; (J) 50 µm).
Figure 4 (a) mCherry-p62/GST-GFP-4xUb clusters were photobleached and fluorescence recovery in the bleached area was monitored. The mCherry-tagged low complexity domain (LCD) of hnRNPA1 (hnRNPA1LCD) was included as a reference for a liquid droplet-forming protein. Left: representative micrographs of bleached particles. White arrows indicate the position and orientation of the kymographs shown in Figure EV4a. Right: quantification of fluorescence recovery. Individual traces of all particles analyzed from three independent replicates are displayed (b) p62 puncta in STG-p62 cells were photobleached and the fluorescence recovery was monitored. Left: representative micrographs of bleached cells. White squares indicate the bleached puncta. Insert: magnification of the bleached puncta. Right: quantification of fluorescence recovery. Averages and SDs of 28 (unbleached) and 29 (bleached) puncta from three independent experiments are shown (c) Representative electron micrographs of negatively stained p62 filaments incubated in presence or absence of GST-4xUb for the indicated times
Figure 7(A) WT, Irgm2-/-, Irgm3-/-, Irgm2-/-Irgm3-/-, panIrgm-/-, and Casp11-/- BMMs were treated with LPS (1 µg/ml) or infected with E. coli K-12 (MOI 25) and IL-1β, and LDH release were assessed at 24 hpt (n = 3 independent experiments).(C) WT (n = 15), Irgm2-/- (n = 12), and panIrgm-/- (n = 13) mice were injected i.p. with LPS (8 mg/kg). Serum was collected 4 hpi and concentration of IL-1β and TNFα was measured via ELISA. Data shown are from 3 pooled experiments. (D) WT, Irgm2-/-, panIrgm-/- and Casp11-/- mice (n = 8 mice/genotype) were injected i.p. with LPS (8 mg/kg body weight). Morbidity and mortality were observed for 48 h at 3-h intervals.
Figure 5A BiFC analyses for the interactions between DJA6 and SUFE1 or SUFC in Arabidopsis mesophyll protoplasts. Yellow, YFP fluorescence signals; red, chloroplast autofluorescence signals. Scale bars = 10 μm. B Co-IP assays for the interactions of DJA6 with SUFE1 and SUFC. Total protein extracts of protoplasts co-transfected with designated constructs were subjected to immunoprecipitation, and then analyzed by immunoblotting with antibodies indicated on the right. Input (left lane) indicates that 100 μg total protein was loaded on the gel. C Pull-down assays of the interactions between DJA6 and chloroplast SUF components. GST-tagged mature DJA6 was used as the bait, MBP-tagged mature NFS2, SUFE1, SUFB, SUFC, SUFD and MBP were used as the prey. The recombinant proteins were incubated with glutathione-agarose beads, and the bound proteins were eluted and analyzed by staining. D Pull-down assay of the interactions between SUFC and apo- or iron-bound form of DJA6. GST-tagged mature iron-bound or apo-DJA6 was used as the bait, MBP and MBP-tagged mature SUFC were used as the prey. The bound proteins were eluted and analyzed by staining and immunoblotting. E Yeast two-hybrid analyses of the interactions between DJA6 and chloroplast SUF components. Positive control, the interaction between murine p53 fused with GAL4 BD and SV40 large T-antigen fused with GAL4 AD; negative control, the coexpression of Human Lamin C fused with GAL4 BD and SV40 large T-antigen fused with GAL4 AD. The dilution ratios are indicated on the left. F Schematic diagram of DJA6 with five different constructs: grey, TP (chloroplast transit peptide, residues 1-85); blue, J domain (residues 86-147); steel blue, GR domain (residues 148-223); cyan, CR domain (residues 224-287); green, C-terminal domain (residues 288-442). The different truncations of DJA6 are shown in detail below. G, H BiFC analyses for the interactions between truncated forms of DJA6 and SUFE1 or SUFC in Arabidopsis mesophyll protoplasts. Scale bars = 10 μm.
FOXP2 is a component of the FOXC2/NFATc1 pathway.Figure 6qRT-PCR analysis of FOXP2, FOXC2 and GJA4 expression in control (siCTRL) and FOXP2 or FOXC2 siRNA-treated HDLECs (n = 6 (FOXP2, FOXC2) or n = 3 (GJA4) independent experiments). Top panels: whole-mount immunofluorescence of E18 Foxc2flox/flox;Prox1-CreERT2 and littermate control mesenteries, showing downregulation of FOXP2 in Foxc2 deficient vessels. Bottom panels: extraction of nuclear FOXP2 staining (grey) using IMARIS surface mask generated based on PROX1 staining (green). Unmasked FOXP2 staining is shown in magenta. Quantification of FOXP2 levels in mesenteric lymphatic vessels of Foxc2flox/flox;Prox1-CreERT2 and control littermates based on nuclear FOXP2 signals as in (B) (n = vessels [mice], as indicated). Whole-mount immunofluorescence of P8 mesenteric lymphatic vessels of Foxp2flox/-;Tie2-Cre and littermate control mice showing loss of organized FOXC2high valve regions but unaltered expression of FOXC2 in Foxp2 deficient vessels. Whole-mount immunofluorescence of lymphatic valves in P7 mesenteries of control and Foxp2flox/-;Tie2-Cre mice showing NFATc1 subcellular localization. Note predominantly nuclear localization of NFATc1 in control valves (left panels), but cytoplasmic localization (middle panels, 3 out of 5 mice) or downregulation of NFATc1 (right panels, 2 out of 5 mice) in the mutant valves. Quantification of protein localization in NFATc1+ lymphatic valves from (D) (n = valves [mice], as indicated). Schematic of the molecular pathway involving FOXC2/FOXP2/NFATc1 in collecting vessel and valve morphogenesis.
Figure 2A Schematic diagram of the strategy to generate F9Y381D and F9Y381Smouse strains. Introduced mutant oligonucleotide is in red and the corresponding amino acids are in green. B Sanger sequencing showing the point mutations in F9 mutant strains. Mutated oligonucleotides are indicated by arrows and the premature stop codon is underlined. C Expression of F9 mRNA and protein in hepatic tissue of indicated mouse strains. Data represent means ± SE. M: Marker; arrowhead: FIX protein; Note: the bands of FIX in F9383STOP lanes are faint with small molecular weight smears. D Measurement of coagulation activity by aPTT in mice at 8 weeks of age. n=5 for each group, Data represent means ± SE. E Measurement of coagulation activity by PT in mice at 8 weeks of age. n=5 for each group, Data represent means ± SE. F Measurement of blood loss over a 5-minute period after tail transection in mice at 8 weeks of age. Wild-type, n=5; F9Y381S, n=6; F9Y381D, n=13; F9383STOP, n=5. G Survival rate of mice after the tail-clip challenge. The mice were monitored for two days after tail clipping. Wild-type, n=5; F9Y381S, n=6; F9Y381D, n=13; F9383STOP, n=5.
Figure 2A, Average trace of hippocampal neurons transfected with MtDsRed and SyGCaMP5 imaged after incubation in ACSF for 30 min. Neurons were stimulated using field stimulation for 10s at 10 Hz. Red trace = average of the terminals occupied with a mitochondrion, black trace = averaged response of terminals without a mitochondrion. B, as in A, but after 30 min treatment with 10 µM Ru360. C, Summary bar graph of the control neurons (corresponds to the traces shown in panel A). Average Ca2+ response (t=20-30s) following stimulation for terminals occupied by a mitochondrion (ΔF/F0 = 3.5 0.4) and unoccupied terminals (ΔF/F0 = 4.9 0.5, n=15 neurons, 96 terminals, ***p<0.001, paired t-test). D, Summary bar graph of the Ru360-treated neurons (corresponds to the traces shown in panel B). Average Ca2+ response (t=20-30s) following stimulation for terminals occupied by a mitochondrion (ΔF/F0 = 4.1 0.5) and unoccupied terminals (ΔF/F0 = 3.8 0.9, n=13 neurons, 79 terminals, p=0.53, paired t-test). Experiments performed in P0 rat hippocampal neuronal cultures at DIV 10-12. Error bars represent SEM.
mGluR and NMDAR alter global translation by regulating eEF2 phosphorylation with distinct kinetics.Figure 3(A) Schematic depicting the experimental workflow and the stimulation protocol to visualize and quantify newly synthesized proteins through metabolic labeling at various time points following DHPG or NMDA treatment in cortical neurons DIV 15 plated at low-density. (B) Representative images showing newly synthesized proteins on the basal condition visualized through FUNCAT metabolic labeling (pseudo-colored) in cortical neurons. MAP2B immunolabeling was used for identifying neurons and intensity was used for normalization. Scale bar 10μm. (C) Representative images showing newly synthesized proteins visualized through FUNCAT metabolic labeling (pseudo-colored) in cortical neurons at various time points following DHPG and NMDA treatment. MAP2B immunolabeling was used for identifying neurons and intensity was used for normalization. Scale bar 10μm. (D) Line graph showing the change in the average normalized FUNCAT intensity representing the quantity of newly synthesized proteins at various time points following DHPG and NMDA treatments. Data presented as mean +/- SEM. Data points in all groups were normalized to the average of the basal group. ***p<0.001 n= 21-54 neurons per group from 3 independent platings. (E) Representative images showing phospho-eEF2 immunolabeling (Pseudo-colored) in low-density cortical neurons on the basal condition, on DHPG treatment for 5 minutes and on NMDA treatment for 5 minutes. MAP2B immunolabeling was used for identifying neurons and intensity was used for normalization. Scale bar 10μm. (F) Box plot showing phospho-eEF2 normalized intensity distribution across multiple neurons on the basal condition, on DHPG treatment for 5 minutes and on NMDA treatment for 5 minutes. Data points in all groups were normalized to the average of the basal group. The box extends from 25th to 75th percentile with the middlemost line representing the median of the dataset. Whiskers range from minimum to maximum data point. *p<0.05, **p<0.01, n=31-49 cells per group from 3 independent platings. (G) Representative immunoblots describing changes in the phospho-eEF2 and total-eEF2 levels at various time points after DHPG and NMDA treatment in cortical synaptoneurosomes. Note in each case phospho and total eEF2 levels were normalized individually to their respective tubulin levels for calculating the phospho/total undefined ratio. (H) Line graph showing the average value of the synaptic phospho/total ratio of eEF2 at various time points after DHPG and NMDA treatment until 5 minutes of recovery. Data presented as mean +/- SEM, **p<0.01. Values in all other groups were normalized to the 0 min group for the corresponding experiment and all the data points are represented as a fraction of the initial time point. ***p<0.001, ****p<0.0001, n≥ 5 animals per group.