EMBO/sd-character-level-ner huggingface dataset:
Figure 3The Mac2 and Paip1M interaction was impaired by removing the N-terminal 16 residues as revealed by the gel-filtration assay. Isothermal Titration Calorimetry (ITC) assay of Mac2 binding Paip1M. The raw calorimetric curve is displayed on top while the fitted binding isotherm curve is at the bottom. The measured values are: N = 0.94 ± 0.01 (binding stoichiometry); Kd = 15.5 ± 1.5 μM (dissociation constant); ΔH = -6778 ± 149 cal/mol (standard molar enthalpy change for binding); ΔS = -0.716 cal/mol/K (standard molar entropy change). Δ16-Mac2 lost all binding affinity to Paip1M in the ITC experiment. ITC result of Mac1−2 interacting with Paip1M. The experimental results are: N = 1.14 ± 0.02; Kd = 18.6 ± 2.5 μM; ΔH = -3730 ± 100 cal/mol; ΔS = 9.14 cal/mol/K. Δ16-Mac2 cannot bind Paip1M, Mac1−2 binds to Paip1M in the split-YFP assay. Scale bars represent 50 µM.
Endothelial specific deletion of Foxp2 leads to defective collecting vessel and valve morphogenesis.Figure 3Genetic constructs and breeding strategies used to generate pan-endothelial Foxp2flox/flox;Tie2-Cre (grey) or Foxp2flox/-;Tie2-Cre (blue) deletion. Whole-mount immunofluorescence of P7 mesenteries, showing abnormally short integrin-α9+ lymphatic valve leaflets (B) and selection of non-recombined FOXP2+ LECs (C) in Foxp2flox/flox;Tie2-Cre mice. Whole-mount immunofluorescence of P7 mesenteries, showing efficient FOXP2 depletion in Foxp2flox/-;Tie2-Cre vessels. Dotted line indicates vessel width. Quantification of mesenteric collecting lymphatic vessel width in P7 Foxp2flox/-;Tie2-Cre and littermate controls (n = vessels [mice], as indicated). Lymphatic valves in P7 mesenteric vessels of control and Foxp2flox/-;Tie2-Cre mice. A spectrum of defects in the mutants ranging from shortened integrin-α9+FN-EIIIA+ valve leaflets (middle panels) to a complete loss of leaflets (right panels) are shown. Quantification of the proportion of valves with integrin-α9+ leaflets (G), and leaflet length (H) in P7 Foxp2flox/-;Tie2-Cre and littermate controls (n = valves [mice], as indicated). Leaflet length = 0 corresponds to valves with no leaflets. Images of P7 intestine (on the left) and mesentery (on the right) showing submucosal reflux (arrows) and leakage (arrowheads) in the Foxp2flox/-;Tie2-Cre mice. Chyle phenotypes in P7 Foxp2flox/-;Tie2-Cre and littermate controls (n = number of mice as indicated).
gene methylation as a prognostic biomarker in breast cancer patients A.Figure 6A. Schematic representation of the RASSF1 gene showing the domains in RASSF1A and C. Bars shaded dark and light green denote the CpG islands associated with RASSF1A and RASSF1C respectively. B. Kaplan-Meier plots of overall survival of 496 stage I and II breast cancer patients with different levels of promoter/CpG island methylation of RASSF1A, previously reported to be associated with outcome in breast cancer patients. P values were obtained from a log-rank test. C. Left: heatmap depicting methylation patterns across the RASSF1 gene. In light green, RASSF1-2γ CpG island is depicted; in dark green, RASSF1-1α CpG island is depicted. Right: Kaplan‑Meyer survival curves depicting interaction of RASSF1A and RASSF1C methylation to predict the outcome of early stage breast cancer patients. P values were calculated from a log-rank test. Color legend: yellow, low RASSF1A and low RASSF1C; green, low RASSF1A and high RASSF1C; red, high RASSF1A and low RASSF1C; grey, high RASSF1A and high RASSF1C. D. Model representing how RASSF1C expression in RASSF1A‑silenced breast cancer cells (MDA-MB-231) activates a signaling cascade involving SRC kinase, RhoGDI and culminates in RhoA activation and generation of amoeboid contractility and cancer stemness features. RASSF1C-driven amoeboid cancer stem cells contribute to alteration of the tumor microenvironment by rendering recipient cells (MCF7, T47D) more invasive and able to upregulate stem cell markers, due to EV-mediated communication.
Figure 2Scatter plot elucidating fold change (FC) in gene expression between samples treated with JNKi compared to vehicle (Y-axis) and expression of constitutively active JNK (MKK7-JNK) versus vector control (X-axis) from biological triplicates. Representative examples of genes that are specifically induced by JNK in metastatic breast cancer cells are highlighted in red. Biological functions regulated by the JNK pathway in MDA231-LM2 cells determined by gene ontology using DAVID analysis. False discovery rate (FDR) < 0.1. BP, biological process; CC, cellular compartment; BH, Benjamini-Hochberg. GSEA graphs showing (C) enrichment of a mammary stem cell signature (Pece et al, 2010) in MDA231-LM2 cancer cells expressing MKK7-JNK NES, normalized enrichment score. GSEA graphs showing under-representation of the same mammary stem cell signature in cancer cells treated with JNKi. NES, normalized enrichment score. GSEA showing significant enrichment of four distinct signatures of mammary stem cells within data sets from MDA231-LM2 cells either expressing MKK7-JNK or treated with JNKi. Mammary stem cell signatures are indicated as follows: a, up in mammary stem cells b, up in fetal mammary stem cells ; c, up in adult mammary stem cells and d, up in mammary repopulating units Heat map of the JNK signature composed of 68 genes that are induced by expression of MKK7-JNK and repressed by JNKi, Proportion of JNK signature-positive tumors in breast cancer patients from the METABRIC data set classified according to PAM50 intrinsic biological subtypes JNK-S, JNK signature as in panel F. Subtype patient numbers: Luminal, n = 1213; HER2, n = 240; Basal-like, n = 331.
Oligopaint IF-FISH reveals differential old versus new H3 inheritance at key genes for maintaining stem cell fate or promoting differentiation in WT female GSCs.Figure 4(A-B) Old (red) versus new (green) histone H3 distribution patterns and percentage of signal strength with a single genomic locus labeled with fluorescent probes (cyan) for dad (A) and bam (B) in WT telophase female GSCs, marked by anti-H3S10p (gray). (C) Quantification of the probe association ratios with old versus new H3 for each candidate gene in WT female GSCs. Data gathered over 8 separate experiments. D
Urinary triclosan levels are elevated in TC mothers following 6 months of exposure.Figure 1Fig 1: Urinary triclosan levels are elevated in TC mothers following 6 months of exposure. Summaries are provided as the interquartile range with median of triclosan molecules in a picogram/microliter (pg/L). Urinary triclosan measurements are available for 38 mothers (17 TC, 21 nTC) and 33 infants (15 TC, 18 nTC). The p-value for the mothers in Figure 1 (represented by ***) is p=5.07e-5 (Mann-Whitney U test).
High-resolution transcriptome-wide maps of binding sites for different Sen1 variants.Figure 6A-C) Comparison of the distribution of Sen1 and RNAPII (Rpb1) at mRNAs and CUTs. Values on the y axis correspond to the median coverage. Note that the scale of Sen1 and Rpb1 datasets have been modified to be able to visualize both in the same plot. D-F) IGV screenshots of two mRNAs and one CUT exemplifying the trend observed in the metagene plots. G) Metagene analysis of the distribution of the different Sen1 variants at mRNAs using either the TSS or the PAS as reference point. Values correspond to the median of Sen1 data normalized by the RNAPII signal obtained in the corresponding background. H-I) Screenshots of two well-characterized protein-coding genes that are regulated by NNS-dependent premature termination. Values correspond to unnormalized Sen1 occupancy. J) Profile of Sen1 binding at CUTs calculated as for mRNAs. K-L) Examples of CUTs exhibiting differences in Sen1 occupancy upon deletion of the NIM or the Nter. Plots correspond to unnormalized data. M-O) Analysis of the distribution of the different Sen1 versions around snoRNAs performed as for CUTs. P) Comparison of the total levels of the different Sen1 variants at mRNAs, CUTs and the termination region of snoRNAs (i.e. a window of 200 nt downstream of the 3' end of the mature snoRNA, snoRNA-ter). Boxes in box-plots include all the values between the 25th and the 75th percentiles and the horizontal line indicates the median. The upper error bars indicate the distance between the largest value smaller than or equal to the upper limit of the box + 1.5 *IQR. The lower error bars indicate the distance between the lowest value greater than or equal to the lower limit of the box - 1.5 *IQR. Datasets for the different Sen1 versions were compared using a t-test (bilateral distributions, unpaired data). p-values≥ 0.05 are indicated as ns (not significant), one asterisk is used when 0.05>p-value≥0.01, two asterisks when 0.01>p-value≥0.001 and three asterisks for p-value <0.001.
Energetic model of the combined effects of aggregation gatekeepers and molecular chaperones.Figure 9N=Native fold, U=unfolded state, A=aggregated state, T=transition state. A) Effects of GK placement on the energetics of the native, unfolded and aggregated states. GKs generally come with a cost to the native fold stability, with negative GKs being more destabilizing on average owing to their shorter sidechains and resulting increased burial costs (a). On the other hand, acidic residues more effectively destabilize the aggregated state than basic residues (b). Consequently, structural constraints in the native state may lead to APRs that are inadequately protected by their basic GKs. Hsp70 specifically recognizes these APRs through their inadequate GKs, and effectively compensates for the difference in destabilization of the aggregated state between basic and acidic residues (c). B) Energetic landscape of the model in (A). Acidic GKs are indicated in red, basic GKs in blue and the non-gatekeeped energy landscape in grey. GKs thermodynamically destabilize and kinetically slow down the folding reaction, resulting in higher energies for N and TN⇋U. Acidic residues have a similar effect on the aggregated state, decreasing aggregate formation kinetics and thermodynamic stability. Basic residues on the other hand, only marginally affect aggregate stability and mostly work through deceleration of the aggregation process through an increase in TU⇋A energy. Through specifically binding positive residues, Hsp70 slows aggregate formation kinetics for positively gatekeeped APRs. Hence, Hsp70 increases GK potential of basic residues, preventing aggregation and allowing for a stable native fold.
IGF1R inhibition alters the expression level of BICD1 A) Western blot of PMN treated with 1 µM PPP or 50 ng/ml IGF1 for 60 min.Figure 5Western blot of PMN treated with 1 µM PPP or 50 ng/ml IGF1 for 60 min. Cells were lysed and the extracts assessed for expression levels of dynein adaptor proteins, all proteins were assessed from the same blot. Hook3 expression didn't change after treatment with PPP or IGF1 compared to controls (DMSO, 100%; PPP, 102.16 ± 4.3%, P=0.67, Student's T-test, N=3 independent experiments; water, 100%; IGF1, 93.48 ± 4.2%, P=0.26, N=3 independent experiments, data shown are mean ± SEM). However, BICD1 expression was significantly increased in PMN treated with PPP compared to control (DMSO, 100%; PPP, 137.1 ± 7.3%, **P=0.002, Student's T-test, N=7 independent experiments). In contrast, IGF1R stimulation by IGF1 had no effect on BICD1 expression (water, 100%; IGF1, 88.1 ± 14.0%, P=0.49, Student's T-test, N=3 independent experiments, data shown are mean ± SEM). BICD2 expression didn't change after treatment with PPP or IGF1 compared to controls (DMSO, 100%; PPP, 104.44 ± 6.5%, P=0.51, N=3 independent experiments: water, 100%; IGF1, 106 ± 7.5%, P=0.51, Student's T-test, N=3 independent experiments, data shown are mean ± SEM).
mediated gene regulation and Treg function in vitro.Figure 5A. Jurkat T cells were transfected with the indicated combinations and relative doses of expression constructs encoding MYC-FOXP3, FLAG-USP44 along with an IL-2 promoter-driven firefly luciferase reporter. After 8 hours stimulation with PMA/Inomycin, cells were lysed and luciferase activity, which was observed and normalized to renilla luciferase activity. B. Tregs (CD4+/CD25High) were recovered from the lymph nodes and spleens of wild type C57BL/6 (WT) and USP44-/- mice by FACS purification. Cells were activated with anti-CD3/CD28 agonist antibodies overnight, and RNA was isolated, and cDNA was generated from these samples for the measurement of several signature Treg-associated transcripts by qRT-PCR. The relative expression level of these by WT and KO-derived Tregs is shown. C, D. Analysis of the in vitro suppressive function of Tregs isolated from WT mice or age- and sex-matched USP44-/- mice (n=3/group/experiment). Tregs were mixed with CFSE-labeled responder naïve T cells from CD45.1+ C57BL/6 mice (n=3/experiment) for three days. CFSE dilution in CD45.1+ T cells was observed by flow cytometry and the percent suppression of responder cell proliferation by the co-cultured Tregs was determined.
Figure 3A, B The enriched gene sets from upregulated (A) and downregulated (B) Age-DEGs analysed by WormCat. Categories 1 and 2 are differentiated by capitalisation and bold fonts. Category 2 are shown below the corresponding Category 1. The numbers of tissues enriched with indicated WormCat Category 1 are shown in the cyan blocks.
Figure 1A. Modified Atkins diet (mAD) study protocol. PEO patients followed mAD for 4-11 days; the healthy control subjects for four weeks. Sampling times of controls were matched with their corresponding PEO patients. B. Beta-hydroxybutyrate (ketone body) concentrations, plasma. C. Creatine kinase (CK) concentrations, plasma. D. Myoglobin concentrations, plasma. Dashed line: upper limit of control range. The individual patients are shown separately, and controls as mean with standard deviation (n=10). Arrow: mAD endpoint in PEO patients; lines under graphs: duration of mAD. E-G. PEO patient (P1) muscle before diet:E. Ultrathin section of abnormal muscle fiber (arrowhead) with subsarcolemmal accumulations of mitochondria. Scale bar 50 µmF. Electron micrograph of subsarcolemmal accumulation of abnormal mitochondria (same cell as in E).G. Enlargement of the area marked in F; mitochondria with paracrystalline "parking lot" inclusions (arrow) and concentric cristae (asterisk). Scale bar 1µmH-J. PEO patient (P1) muscle after mAD (H-J):H. Ultrathin section, lytic muscle fiber with centrally located organellar debris (arrow). Scale bar 50 µmI. Electron micrograph of a degrading muscle fiber with an invaded macrophage (arrow) (same cell as in H).J. Enlargement of the fiber in I: paracrystalline inclusions within mitochondria (asterisk). Scale bar 1µmK. Quantification of necrotic or apoptotic cells in PEO muscle from ultrathin sections. n.a., not available.
Figure 4SPP1 and TNC expression in the indicated breast cancer cells treated with increasing concentrations of JNKi. SPP1 and TNC expression in MDA231-LM2 cells transduced with pLVX-puro lentivirus without a transgene (control) or expressing constitutively-active JNK (MKK7-JNK) or mutated inactive JNK (MKK7-JNK(mut)). SPP1 and TNC expression in pleural effusion (1 and 2) and ascites (3 and 4) samples from four different patients treated with JNKi (4 μM, 48h) in culture. Values are mean ± SD from triplicates. The results from all four patient samples were included to calculate significance of JNKi on gene expression. SPP1, P = 0.0079; TNC, P = 0.0002. Analysis of SPP1 and TNC mRNA levels in SUM159-LM1 cells grown as monolayer or spheres and treated with vehicle or JNKi. For panels A-D, values represent the mean of qPCR triplicates ± SD. ChIP analysis of c-Jun binding to sequences in SPP1 or TNC promoters. (E) Diagrams of SPP1 and TNC promoters showing positions of primer pairs, flanking c-Jun/AP-1 consensus sites, that were used for qPCR following ChIP with an antibody against c-Jun. ChIP analysis of c-Jun binding to sequences in SPP1 or TNC promoters. Analysis of SPP1 and TNC promoter fragments pulled down by c-Jun and IgG antibodies. Values represent the mean of qPCR triplicates ± SD. Correlation analysis of SPP1 and TNC with JUN expression in dissected metastatic nodules from breast cancer patients (GSE14020). N = 65 metastasis samples. Lung metastasis in Spp1+/- mice implanted with MDA231-LM2 cells expressing control shRNA (control), and Spp1-/- mice implanted with MDA231-LM2 cells independently expressing one of two different SPP1 shRNAs (SPP1-deficient). Ex vivo lung luminescence was analyzed, panel H. Boxes show the median value with upper and lower quartiles. Whiskers represent minimum and maximum values. Control n = 16 mice, SPP1 deficient (1) n = 14 mice and SPP1 deficient (2) n = 15 mice. P values were determined by a two-tailed Mann-Whitney test. (I) Representative histological images of metastatic foci (top) and ex vivo lung bioluminescence (bottom). In histological samples, immunohistochemistry was used to determine metastatic nodules based on expression of human vimentin (arrows). Scale bar, 100 µm. SUM159-LM1 cells expressing either control shRNA (control), or one of two different TNC shRNAs (shTNC 1 and 2), were injected intravenously into NSG mice. (J) Metastatic colonization was quantified by lung bioluminescence. Control and shTNC (1) n = 8 mice per group, shTNC (2) n = 7 mice. P value was determined by a two-tailed Mann-Whitney test. (K) Representative images of metastatic foci (top) and ex vivo lung bioluminescence (bottom). Arrows denote vimentin-expressing cancer cells in metastases. Kaplan-Meier analysis of breast cancer patients (combined GSE2603 and GSE5327 datasets) showing a link between high SPP1 and TNC expression and poor lung metastasis-free survival. Patient samples, SPP1 and TNC low n = 70; SPP1 and TNC high n = 70. HR, Hazard ratio.
Figure 3A MAGE-A3/6 mRNA levels do not change upon nutrient deprivation. RNA was isolated from HeLa cells treated with EBSS for the indicated times. RT-QPCR analysis was performed to detect both MAGE-A3/6 with a common primer set. Data was normalized to 18S rRNA levels. Quantitation represents average of n=3 with standard deviation shown. B, Proteasome inhibition rescues MAGE-A3/6 protein levels upon nutrient deprivation. HeLa cells were treated for the indicated times in EBSS alone or EBSS containing lysosome inhibitor bafilomycin A1 (BafA1, 50 nM) or proteasome inhibitor MG132 (10 µM). C Proteasome inhibition rescues MAGE-A3/6 protein levels upon nutrient deprivation. HeLa cells were treated for the indicated times in EBSS alone or EBSS containing lysosome inhibitor bafilomycin A1 (BafA1, 50 nM) or proteasome inhibitor MG132 (10 µM). Quantitation represents average of n=3 with standard deviation shown. Data information: Asterisks indicate *** p<0.001. ns indicates p>0.05 using 2-way ANOVA. D Nutrient deprivation increases ubiquitination of endogenous MAGE-A3/6. HeLa cells were treated with MG132 (10 µM) in complete media or EBSS for 6 hrs. Cell extracts were incubated with control agarose or agarose-TUBE2 to isolate ubiquitinated proteins. Input and pulldown samples were probed for the indicated proteins. E MAGE-A3/6 protein instability upon nutrient deprivation is not rescued by knockdown of its associated E3 ubiquitin ligase, TRIM28. HeLa cells were treated with control or TRIM28 siRNAs for 6 days before incubation in EBSS for the indicated times. F, MAGE-A3/6 degradation upon nutrient starvation is dependent on a Cullin E3 ubiquitin ligase. HeLa cells were treated with EBSS alone or EBSS containing the NAE1 inhibitor MLN4924 (1 µM) that blocks the activity of Cullin E3 ubiquitin ligases for the indicated times. G MAGE-A3/6 degradation upon nutrient starvation is dependent on a Cullin E3 ubiquitin ligase. HeLa cells were treated with EBSS alone or EBSS containing the NAE1 inhibitor MLN4924 (1 µM) that blocks the activity of Cullin E3 ubiquitin ligases for the indicated times. Quantitation represents average of n=3 with standard deviation shown. Data information: Asterisks indicate *** p<0.001. ns indicates p>0.05 using 2-way ANOVA. H Degradation of MAGE-A3/6 upon nutrient deprivation is dependent on a Cul4 E3 ubiquitin ligase complex. HeLa cells were transfected with vector control or the indicated dominant negative (D/N) Cullin constructs for 48 hrs before harvesting cells after 0 or 4 hrs EBSS treatment. I Degradation of MAGE-A3/6 upon nutrient deprivation is dependent on Cul4A/B. HeLa cells were transfected with control siRNAs or siRNAs targeting both Cul4A and Cul4B. 72 hrs after treatment, cells were incubated for the indicated times with EBSS before harvesting and immunoblotting. Note, time 0 samples between two siRNA groups were normalized to more readily discern differences in MAGE-A3/6 degradation upon nutrient deprivation.
a–g) Four-month-old C57BL/6 male mice kept on HFD for 2 months were subjected to daily administration of phosphate-buffered saline (Con, n=4) or verapamil (Ver, 25 mg per kg body weight, i.f7(a-g) Four-month-old C57BL/6 male mice kept on HFD for 2 months were subjected to daily administration of phosphate-buffered saline (Con, n=4) or verapamil (Ver, 25 mg per kg body weight, i.p., n=4) for 10 days. Systolic blood pressure (a), LV wall mass (b), diastolic LV volume (c), systolic LV volume (d), ejection fraction (e, % EF), stroke volume (f, SV) and cardiac output (g, CO) were analysed by tail-cuff method (a) or echocardiography (b-g). All data are shown as mean±s.e.m. *P0.05 (Student's t-test).
Calcineurin affects the phospho-regulation of XErp1.Figure 2Schematic representation of the XErp1 variants and their characteristics used in this study. CSF extract was supplemented with Myc-XErp1 DSG- DSA- ZBR- (S33N S38N S284N S288N C583A) IVT and as indicated treated with DMSO or CsA. Both reactions were treated with calcium and samples were taken at the indicated time points. Samples were immunoblotted for the Myc-tag, cyclin B2 and α-tubulin. A pseudocoloured representation of the Myc immunoblot is shown in Fig EV2A. Arrow marks the meiotic phosphorylation state of Myc-XErp1. Asterisk indicates unspecific bands. CSF extract was supplemented with Myc-XErp1 DSG- DSA- ZBR- (S33N S38N S284N S288N C583A) IVT that was either wild-type or mutated to alanine at the p90RSK-target sites S335, T336 and S342 (Rsk3A). Both reactions were divided and treated with DMSO or CsA. Calcium was added and samples were taken at the indicated time points. Samples were immunoblotted for the Myc-tag and cyclin B2. The cyclin B2 membrane was stripped and reprobed for α-tubulin. Asterisk indicates unspecific bands. CSF extract was treated with Myc-XErp1 CaMKII- ZBR- (T195A C583A) IVT. An empty IVT reaction not expressing XErp1 was used as control. The extracts were treated with DMSO or CsA as indicated. Calcium was added, samples were taken at the indicated time points and immunoblotted for cyclin B2, pSer335 XErp1 and α-tubulin. The pSer335 XErp1 membrane was stripped and reprobed for the Myc-tag. Asterisks indicate unspecific bands. Myc-XErp1 CaMKII- ZBR- (T195A C583A) IVT was in vitro phosphorylated by recombinant PKA and isolated by α-Myc immunoprecipitation. The immunoprecipitate was supplemented with calcium and treated with recombinant hs_CaN and calmodulin as indicated. Samples were taken at the indicated time points and immunoblotted for the catalytic calcineurin subunit CnA and pSer335 XErp1. The pSer335 XErp1 membrane was stripped and reprobed for the Myc-tag. Asterisk indicates the IgG heavy chain.