EMBO/sd-character-level-ner huggingface dataset:
Figure 1A Schematic illustration of the working principle of the ATP sensor AT1.03NL. Binding of ATP to the sensing domain (ε subunit of ATP synthase) leads to a conformational change that brings the donor (mse-CFP) and the acceptor (cp173-Venus) closer, resulting in an increase in FRET efficiency (Tsuyama et al., 2013). B Cartoon to illustrate the experimental setup for inhibiting oxidative phosphorylation (OxPhos) in late third-instar wing discs expressing AT1.03NL. To inhibit OxPhos, dissected wing discs are treated with Antimycin A for 2 h. A reduction in ATP is revealed by a reduced FRET efficiency. The gray frame marks the region of the wing disc shown in FRET efficiency and immunofluorescence (IF) images. C Cartoon of late third-instar wing disc showing the expression pattern of ap-GAL4, tub-GAL80ts (apGalts) driver. Dashed yellow and green lines indicate the anterior-posterior (A/P) and dorsal-ventral (D/V) compartment boundaries, respectively. The expression pattern of apGalts driver in the dorsal compartment is shown in light red. D, E Images of FRET efficiency in mock-treated (- ant.A, D) and Antimycin A-treated (+ ant. A., E) ubi-AT1.03NL wing discs at time point 2 h. A rainbow colormap is used to indicate the FRET efficiency levels. Scale bars= 50 µm. F Line graphs showing the FRET efficiency in individual mock-treated (- ant. A, n=26) and ant. A-treated (+ ant. A., n=19) ubi-AT1.03NL wing discs before (0 h) and after 2 h of treatment. Paired t-test, ns= not significant, ***p≤0.001. G-J Time-controlled knock-down of metabolic enzymes in the dorsal compartment of ubi-AT1.03NL wing discs using the apGalts driver. Images of FRET efficiency in control (G), apGalts>PfkRNAi (H), apGalts>GapdhRNAi (I), and apGalts>Glo1RNAi (J) wing discs after RNAi induction for 48 h (G), 120 h (H, J), and 93 hours (I). A rainbow colormap is used to indicate the FRET efficiency levels. Scale bars = 50 µm. K Line graphs showing the FRET efficiency in the ventral and dorsal compartment of individual control (n=27), apGalts>PfkRNAi (n=7), apGalts>GapdhRNAi (n=10), and apGalts>Glo1RNAi (n=6) wing discs. Loss of Pfk, Gapdh or Glo1 in the dorsal compartment of the wing disc reduces the levels of ATP. Paired t-test, ns= not significant, **p≤0.01, ***p≤0.001. L Time-controlled knock-down of ecdysoneless (ecd) in the dorsal compartment of ubi-AT1.03NL wing discs using apGalts>ecdRNAi. Image of FRET efficiency in apGalts>ecdRNAi wing disc 48 h after RNAi induction. A rainbow colormap is used to indicate the FRET efficiency levels. Scale bars = 50 µm. M Line graph showing the FRET in the dorsal and ventral compartment of apGalts>ecdRNAi (n=28) wing discs. Loss of Ecd in the dorsal compartment of the wing disc reduces the levels of ATP. Paired t-test, ***p≤0.001.
Figure 10Figure 10. Higher-order SPOP oligomers localize to nuclear speckles. We propose a model in which the ability of SPOP to form higher-order oligomers promotes its localization to nuclear speckles. Smaller oligomeric SPOP species are diffusely distributed, but higher-order oligomers preferentially localize to speckles. Dynamic BACK domain association mediates exchange between higher-order/punctate structures and the diffuse pool. SPOP interacts with a number of binding partners, including other components of the CRL and substrates, and can likely recruit them to punctate structures (Chen et al, 2009; Kwon et al, 2006). We speculate that nuclear speckles serve as hotspots of SPOP-mediated ubiquitination by concentrating SPOP oligomers, substrates, and other components locally.
Figure 4(A) qPCR levels of collagen in PSCs, normalized to RPLP0 and relative to control (B) Western blot analysis of collagen normalised to total protein and relative to control (C) Quantification of time-lapse collagen synthesis and deposition by PSCs (D) Representative immunofluorescent images used for the quantification in C, scale bar 20 μm, collagen-I was assessed with a specific primary antibody staining (E) Relative values of protein levels for collagen in PDAC tumors assessed by proteomic analysis (6 mice per condition and samples were analysed in duplicates) (F) qPCR levels of MMP-2 in PSCs, normalized to RPLP0 and relative to contro (G) MMP-2 activity on control and tamoxifen treated PSCs assayed by gelatin zymography; above signal intensity of the representative bands used for the quantification presented in the plot below quantification of MMP-2 levels in PDAC tissues from KPC mice treated with vehicle (control), and 2mg and 5mg of tamoxifen, scale bar 100 μm (n=4 animals per condition and n≥ 5 sections per animal) Immunohistochemistry image of MMP-2 levels in PDAC tissues from KPC mice treated with vehicle (control), and 2mg and 5mg of tamoxife (J) Relative values of protein levels for MMP-2 in PDAC tumors assessed by proteomic analysis (6 mice per condition and samples were analysed in duplicates) (K) qPCR levels of MMP-2 in PSCs, normalized to RPLP0 and relative to 1 kPa (L) qPCR levels of MMP-2 in PSCs, normalized to RPLP0 and relative to control. Histogram bars represent mean±s.e.m.
PLTs constrain the WOX5 expression domain.Figure 2A-F, Representative FM4-64-stained Arabidopsis roots (grey) expressing pWOX5::mVenus-NLS (green) in Col, plt2, plt3 and plt2, plt3 double mutant background in longitudinal (A-D), or transversal (E-F) optical sections. E', F', Analysis of representative images in (E) and (F) in Imaris to detect and count individual expressing nuclei. E'', F'', Overlay of 10 roots (biological replicates) showing the area of detected fluorescence (high levels in red, low levels in blue) in Col and plt2, plt3 double mutant roots. G, Number of nuclei (biological replicates) expressing pWOX5::mVenus-NLS in Col and plt2, plt3 double mutant roots summarized in box and scatter plots. H, Area of WOX5 expression in µm2 in Col and plt2, plt3 double mutant roots summarized in box and scatter plots. G, H Box = 25-75 % of percentile, whisker = 1.5 interquartile range, − = median, □ = mean value, = minimum/maximum. (G, H) Kruskal-Wallis ANOVA analysis with subsequent Dunns test (G) or one-way ANOVA and post-hoc Holm-Sidak multiple comparisons test was used to test for statistical significance (H). Asterisks indicate statistically significant differences (α = 0.01). Number of analysed roots (n) (biological replicates) is indicated for each genotype and results from three technical replicates per genotype. Scale bars represent 10 µm; NLS = nuclear localisation signal.
DDX3 expression and knockdown phenotype in lung cancer cell lines and in lung cancer patient samples.Figure 1A Immunoblot of DDX3 expression in lung cancer cell lines. B, C Colony-forming assays in H1299 (B) and A549 (C) lung cancer cells after knockdown by shRNA lentiviral constructs designed against DDX3 or vector control. Corresponding immunoblots displaying knockdown levels of DDX3. Mean from 3 replicates with SD. D Proliferation of A549 and H1299 cells after knockdown of DDX3. Mean from 3 replicates with SD. (A549 P = 0.011, H1299 P = 0.014; exponential curve fit, extra sum of squares F-test). E β-galactosidase staining in parental A549 cells and A549 DDX3 knockdown cells displaying senescent cells identified by the blue color. F Expression of DDX3 by immunohistochemistry in normal lung tissue.G DDX3 expression in squamous cell carcinoma.H DDX3 expression in adenocarcinoma.I DDX3 expression in small cell carcinoma.J Expression of DDX3 in different histological types of lung cancer. All data sets were compared against each other (chi-square test, P = 0.481). K Survival analysis of lung cancer patients in low and high DDX3 expressing tumors (Kaplan-Meier curve and log-rank test, P = 0.016).
a) Comparison of AMP/ATP ratios between PCF trypanosomes grown in the absence (−Tet) or presence (+Tet) of tetracycline to induce RNAi, and with or without glucose (Gl), expressed as fold increase (means±s.f4(a) Comparison of AMP/ATP ratios between PCF trypanosomes grown in the absence (−Tet) or presence (+Tet) of tetracycline to induce RNAi, and with or without glucose (Gl), expressed as fold increase (means±s.d., n=3; *P0.05; **P0.01, Student's t-test). (b) Comparison of AMP/ATP ratios between (−Tet) and (+Tet) PCF trypanosomes grown in a glucose-depleted medium (SDM-80) containing 5.2 mM L-proline for 2 days after 2-day growth in SDM-79±tetracycline, expressed as fold increase (means±s.d., n=3, **P0.01, Student's t-test). (c) Fluorescence microscopic images of TbATG8.2 in PCF trypanosomes cultured in SDM-79 in the absence (WT) or presence (RNAi) of tetracycline to induce RNAi; scale bars, 10 μm. (d) Quantification of autophagosome formation in RNAi PCF trypanosomes grown in SDM-79 or SDM-80 medium in the presence (+Tet) or absence (−Tet) of tetracycline. Over 500 cells from three experiments with 20 random fields/experiment were analysed (means±s.d., n=3, **P0.001, Student's t-test). (e) Western blot analysis of ATG8.2 or tubulin in PCFtrypanosomes cultured in SDM-79 or SDM-80 in the absence (−Tet) or presence (+ Tet) of tetracycline to induce RNAi. Cell lysates (30 μg) were fractionated in 12% urea-SDS-PAGE gel. Immunoblots were labelled with anti-ATG8.2 or anti-tubulin antibody. (f) Quantification of ATG8.2-II/(ATG8.2-II+ATG8.2-I) presented as mean ratios±s.d. (n=3, **P0.01, Student's t-test). (g) Groups of five mice were infected with WT T. brucei (black line) or trypanosomes transfected with the construct for RNAi of TbMCU (red line). Doxycycline (green line; 200 μg ml−1) was given in the drinking water throughout a 30-day period. (h) Parasitemia levels in surviving mice were monitored at 2-day intervals after infection. The results are means±s.d. (n=2).
METPlatform is compatible with the evaluation of the metastasis-associated microenvironment.Figure 2A Schema of the experimental design. B Representative images of organotypic cultures with established metastases with various glial components of the microenvironment labelled. Scale bar: 75 µm. Each individual condition was evaluated in several organotypic cultures (3-6 slices). C Quantification of the bioluminescence signal emitted by established H2030-BrM brain metastases in each organotypic culture at day 3 normalized by their initial value at day 0 (before the addition of DMSO or any compound). The final value in the graph is normalized to the organotypic cultures treated with DMSO. Values are shown in box-and-whisker plots where the line in the box corresponds to the mean. The boxes go from the upper to the lower quartiles and the whiskers go from the minimum to the maximum value (n=5-6 organotypic cultures, 1 independent experiment). P value was calculated using two-tailed t-test. D Quantification of LDH levels in the conditioned media of organotypic slices cultured during 3 days relative to a lysate of the same preparation. Values are shown as mean + s.e.m. (n=3 organotypic cultures per experimental condition, 1 independent experiment).
Figure 71) The Bem1-Cdc24 complex is recruited to the plasma membrane via multivalent interactions with anionic lipids. The BC motifs in Bem1 provide the strongest affinity for anionic lipids at this step. 2) Upon their recruitment to anionic lipids, the Bem1 BC motifs may influence the local membrane environment, contributing to local Cdc42 activation by Cdc24 and Cdc42 nanoclustering.
Figure 1A Screen design: generation of mammary cancer cell lines LB-mHR1 (HR1) from PIK3CAH1047R mutant transgenic animals and transfection with the piggyBac (PB) system (HR1 Ctrl and HR1.PB cells) (Ivics et al, 2009). PB transposon (ATP1-Puro) and transposase (pFS250.PBase) plasmid design. PB-3'/5', piggyBac inverted terminal repeats; CβASA, Carp β-actin splice acceptor; En2SA, Engrailed-2 exon-2 splice acceptor; SD, Foxf2 exon-1 splice donor; pA, bidirectional SV40 polyadenylation signal; PGK, mouse phosphoglycerate kinase 1 promoter; Puro, puromycin resistance; CAG, cytomegalovirus enhancer and chicken beta-actin promoter; Tet-ON, tetracycline-responsive element-tight promoter; PBase, PB transposase; UbC, Ubiquitin C promoter; rTTA3, reverse tetracycline transactivator 3; IRES, internal ribosomal entry site; Neo, Neomycin/G418 selection marker. The cell pools were treated with doxycycline (1 μg/ml) for 96 h in vitro. B In vivo screen design: generation of lung metastatic mammary cancer cell lines (LM) from HR1.PB cell lines after orthotopic injections of HR1.PB or HR1.Ctrl. C Bar graph showing the percentage of unique insertions in the top 7 genes normalized to the total number of insertions in a given sample. Genes are annotated with a gene symbol when available otherwise with UCSC ID transcript names. Data are from all the sequenced samples (tumour samples: 16 primary tumours; metastatic samples: 3 lung micro-metastases, 6 lung macro-metastases and 9 LM cell lines). Means ± s.d., two-tailed Mann-Whitney U-test, n.s. = not significant. D Bar graphs showing the percentage of unique insertions in Nfib and Foxp1 in tumours, LM1, LM8, LM9, and other LM cell lines normalized to the total number of insertions in a given sample. Dots represent individual samples (Tumours n = 16, LM1/8/9 n = 9, Other LM n = 9), means ± s.d., two-tailed Mann-Whitney U-test, n.s. = not significant. E Bar graphs representing mean Nfib mRNA expression normalized to HR1 cells. n = 2 biological replicates and n = 2 technical replicates, means ± s.d., two-tailed Student's t-test, FC= fold change.G Immunoblot analysis of LM and HR1 cell lines showing NFIB and FOXP1 protein levels. ERK2 served as a loading control.
miR-155 promotes mycobacterial phagosome maturation.Figure 3RAW 264.7 cells were transiently transfected with control or miR-155 mimic for 24 h and then infected with Texas Red-labeled BCG for 1 h. Lysosomes were immunolabeled with CD63 antibody followed by Alexa Fluor 488-conjugated goat anti-rabbit IgG antibody (A) The colocalization of BCG with lysosome was detected by confocal microscopy. The percentage of co-localization of BCG with CD63-positive (B) RAW 264.7 cells were transiently transfected with control or miR-155 mimic for 24 h and then infected with Texas Red-labeled BCG for 1 h. Lysosomes were immunolabeled with CD63 antibody followed by Alexa Fluor 488-conjugated goat anti-rabbit IgG antibody (A), or labeled with a fluorogenic substrate for proteases, DQ-Green (C). The colocalization of BCG with lysosome was detected by confocal microscopy. The percentage of co-localization of BCG with CD63-positive (B) or DQ-Green labeled (D) lysosomes was quantified, respectively. Cells treated with rapamycin were used as a positive control. Arrows indicate the co-localization of BCG with lysosomes; scale bar = 5 µm. Quantification of data are shown as the mean ± SEM of three independent experiments (n = 100 phagosomes). **, p<0.01.
Figure 8HEK293 cells were transfected with M3R and either control vector (CTRL), FUS, FUSR521C or FUSR518K and then treated with vehicle (DMSO) or 1 M AR-A014418 for 16 hours as indicated. Release of ER Ca2+ was induced by treatment of cells with OxoM. Representative Rhod2 fluorescence traces showing mitochondrial Ca2+ are shown along with bar chart displaying normalized peak values.
STAMP2 expression is associated with development of castration resistance of PCa.Figure 4A STAMP2 expression was determined by IHC of a neoadjuvant hormone therapy (NHT) TMA containing samples from hormone naïve (untreated) (n = 28), NHT 1-6 months (n = 31), NHT 7-12 months (n = 41), and patients with PSA recurrence (n = 21) as indicated. Representative images are presented.B Quantitative presentation of the data from (A). Unt, untreated; Recur, PSA recurrence. The Kruskal-Wallis test was used for significance analysis. *P = 0.0499. Error bars indicate SEM. C Boxplots of STAMP2 expression levels in normal prostate tissue, primary prostate cancer, and metastatic CRPC were analyzed in two independent GEO datasets (GSE6919 and GSE35988). The values of individual samples are presented as dots. Thick horizontal lines represent the median, with the box representing the upper and lower quartile. The whiskers represent the 5th and 95th percentiles. The statistical significance, indicated in the tables at the bottom, was determined by Student's t-test. D CWR22 xenografts were grown in nude mice, and tumors were collected at different times after castration. mRNA was extracted from the tumors and used for qPCR analysis of STAMP2 expression. The results are presented as boxplots. Thick horizontal lines represent the median, with the box representing the upper and lower quartile. The whiskers represent the 5th and 95th percentiles, and the outlier is presented as an open circle. The statistical significance was determined by one-way ANOVA with a post hoc test. n = 3 in group week 0, 1, 2, and 4; n = 5 in refractory group. *P = 0.011; **P = 0.049. Error bars indicate SEM.
NBS1 in trans with MR and pCtIP cleaves DNA similarly as MRN-pCtIP.Figure 1A representative nuclease assay with MR, MBP-NBS1-his (denoted MBP-NBS1), MRN and pCtIP on 5'-end labeled 70-bp dsDNA with all ends blocked with streptavidin. Samples were separated on 15 % denaturing polyacrylamide gel. Quantitation of nuclease assays such as in (A). Averages shown; n ≥ 3; error bars, SEM.
Figure 4mTORC2 in adipose tissue is not required for cold-induced mitochondrial uncoupling and β-oxidation. (A) mRNA levels of the indicated genes in BAT of AdRiKO and control mice housed at 22°C or at 4°C for 8h (n=6/group). (B) Immunoblot analysis of BAT from AdRiKO and control mice housed at 22°C or at 4°C for 8h for the indicated proteins (n=6/group, each lane represents a mix of 3 mice). (C) mRNA levels of the indicated genes in BAT of AdRiKO and control mice housed at 22°C or at 4°C for 8h (n=6/group, each lane represents a mix of 3 mice). (D) Immunoblot analysis of BAT from AdRiKO and control mice housed at 22°C or at 4°C for 8h for the indicated proteins (n=6/group, each lane represents a mix of 3 mice). (E) Mitochondrial DNA content of BAT from AdRiKO and control mice housed at 22°C or at 4°C for 8h (n=6/group). (F) Representative electronmicrographs of BAT from AdRiKO and control mice housed at 22°C or at 4°C for 4h (n=3/group). (G) Oxygen consumption rate (OCR) of BAT explants from AdRiKO and control mice housed at 22°C or at 4°C for 4h (n=7/group). H) Maximal respiration (VO2 max) of AdRiKO and control mice housed at 22°C or at 4°C for 8h (n=9 (control 22°C), n=7 (AdRiKO 22°C), n=8 (control 4°C), n=8 (AdRiKO 4°C). (I) Respiration (VO2) of AdRiKO and control mice upon cold exposure (n=8/group). Data represent mean ± SEM. Statistically significant differences between AdRiKO and control mice were determined with unpaired Student's t-test and are indicated with asterisks (*=p<0.05; **=p<0.01, ***=p<0.001). Statistically significant differences between temperatures were determined with unpaired Student's t-test and are indicated with a number sign (#=p<0.05; ##=p<0.01; ###=p<0.001). The exact p-value for each significant difference can be found in Appendix Table S2.
HMGCL and βOHB promote pancreatic tumor aggressivenessA, B Quantification of volume with representative images (A) and weight (B) of sg-CTRL (n=15 for A and n=9 for B), sg HMGCL #2 (n=19 for A and n=8 for B), #3 (n=7 for A and B) pancreatic tumors.Figure 4A, B Quantification of volume with representative images (A) and weight (B) of sg-CTRL (n=15 for A and n=9 for B), sg HMGCL #2 (n=19 for A and n=8 for B), #3 (n=7 for A and B) pancreatic tumors. Data are expressed as mean of tumor volume or weight ± SEM. Significance was defined by Mann-Whitney test. *: p<0.05, **p<0.01, ***p<0.001. C Extracellular matrix quantification following trichrome staining in sg-CTRL and sg-HMGCL #2 pancreatic tumors sections (n=8 and 9 mice/group respectively, left panel). Data are expressed as mean of percentage of total tissue area ± SEM. Significance was defined by Mann-Whitney test. **: p<0.01. Representative images of trichrome staining in sg-CTRL and sg-HMGCL pancreatic tumors. Scale bar: 100 µm (right panel). D, E Quantification of volume (D) and weight (E) of sg-CTRL pancreatic tumors treated with 0.9% NaCl (i.p.) (n=4), and pancreatic tumors from two different clones of sg-HMGCL treated with 0.9% NaCl (i.p.) or βOHB (100mg/kg/bi-weekly, i.p.) (n=10/group). Data are expressed as mean of tumor volume or weight ± SEM. Significance was defined by Mann-Whitney test. ns: not significant, **p<0.01. F Histological characterization and representative picture of liver from mice orthotopically xenografted with sg-CTRL or sg-HMGCL PANC-1 cells and treated with 0.9% NaCl (i.p.) or βOHB (100mg/kg/bi-weekly, i.p.). Number of mice displaying healthy or metastatic liver in each experimental group is reported. Metastatic area (orange star) is separated from liver (green circle) by dotted lines. Scale bar: 100µm (inset images scale bar: 20µm). G Schematic showing isotopomer transition from [U-13C]βOHB to label TCA-cycle intermediates, glutamate, and proline. Grey filled circles indicate 13C carbon derived from labeled βOHB. Empty circles illustrate unlabeled 12C-species. H [U-13C]βOHB tracing into TCA intermediate: citrate in sg-CTRL and sg-HMGCL #2 and #3 PANC-1 cells cultured in indicated glucose concentrations. Data are expressed as mean ± SEM. (n=2 independent experiments). Significance was defined by one-way ANOVA followed by a Bonferroni's multiple comparisons test, only significances between sg-HMGCL #2, #3 PANC-1 cells and sg-CTRL PANC-1 cells under the same culture condition are mentioned. ***p<0.001.
| The REST/NRSF binding motif was enriched in DNA hypo-methylation regions.Figure 3A. Volcano plots showing GO terms enriched for genes near hypo-methylated regions relative to other RefSeq genes. Data information: Data are presented in the volcano plot format. Statistical significance was determined using Fisher's exact test. B. Volcano plots showing MGI phenotypes enriched for genes near hypo-methylated regions relative to other RefSeq genes. Data information: Data are presented in the volcano plot format. Statistical significance was determined using Fisher's exact test. C. Enrichment of the REST/NRSF binding motif in hypo-methylated DMRs (19/96) in aged mice. D. Gene lists near the 37 hypo-methylated genomic regions with REST/NRSF binding confirmed using the ChIP-Atlas database. Blue highlights indicate DMRs in the intron of overlap genes. Magenta highlights indicate DMRs in intergenic regions between upstream and downstream genes. Gray highlights indicate DMRs with the REST/NRSF binding motif. The black circle indicates ASD-related genes (SFARI genes), and the black triangle indicates schizophrenia-related genes (SZ genes).
Mec1 phosphorylation on Serine 1991 helps cells cope with replication stress (A) The domain structure of Mec1, which is typical for PI3K-related protein kinases, including the N-terminal HEAT repeat domain, FAT domain, kinase domain and the C-terminal FAT domain.Figure 2(A) The domain structure of Mec1, which is typical for PI3K-related protein kinases, including the N-terminal HEAT repeat domain, FAT domain, kinase domain and the C-terminal FAT domain. The Mec1 consensus SQ/TQ sites identified in silico are marked in black, while in vivo validated phosphorylation sites induced by genotoxic agents are in red. (B) A 10-fold dilution series of cells from exponential SC cultures of the indicated strains were spotted on SC +/- the indicated dose of genotoxic agents. (C) Asynchronous exponentially growing cells were treated with 0.2M HU (+HU), then HU was removed from the medium (-HU). SDS-PAGE of total protein extracts taken at the indicated times were used to detect the kinetic of Rad53 phosphorylation upshift (Rad53-P=**). Red and green asterisk indicate the time where mec1-S1991 phosphomutants show altered Rad53 recovery. A 5-fold dilution series of cells from exponential SC cultures of the indicated strains were spotted on SC +/- the indicated dose of HU. A 5-fold dilution series of cells from exponential SC cultures of the indicated strains were spotted on SC +/- the indicated dose of HU.