EMBO/sd-character-level-ner huggingface dataset:
Bcl-xL regulates the interaction between FUNDC1 and HSC70.Figure 3Co-IP of HSC70 with FUNDC1 in HeLa cells treated with MG132 as indicated. Immunoblot analysis of the HeLa cells transfected with or without HA-Bcl-xL and treated with DMSO or MG132 for 6 hours. Ni2+-NTA precipitation of HeLa cells transfected with indicated vectors. S13A-Myc-His, Myc/His6 double-tagged FUNDC1-S13A mutant. D, E. Fractionation analysis of HeLa cells transfected with indicated vectors and treated with DMSO or MG132. Quantification is shown in (E).
Figure 3(A) mtDNA NGS performed in 84 hiPSC clones distributed in non-mitochondrial disease group (n=43 hiPSCs derived from 16 individuals) and MELAS group (n=41 hiPSCs derived from 3 patients). See also Table S1 and Table S2. (B) Distribution of GPMs with mutation load lower or greater than 10%, in control and MELAS groups. The majority of GPMs are present at levels lower than 10% in either group. (C) Location of GPMs greater than 10%, in mtDNA. (D) PolyPhen 2 Damaging Score corresponding to those GPMs residing in protein coding genes. Potentially damaging mutations (score close to 1.0) are present in both groups.
Figure 5(A) Correlation analysis of EZH2 and p53 mRNA expression measured by RT-qPCR in primary and metastatic prostate cancer specimens of a cohort of patients from University of Washington. (B) IHC analysis of EZH2 and p53 protein expression in human prostate cancer tissues of a cohort of patients from Mayo Clinic. Case 1 and 2 show low and high expression in primary tissues, respectively; case 3 shows high expression in lymph node metastasis. Scale bar, 50 µm. Scale bars in insets, 10 µm. (C) Correlation analysis of EZH2 and p53 protein expression in primary and metastatic prostate cancer specimens of the Mayo Clinic cohort. (D) IHC analysis of EZH2 and p53 protein expression in commercially purchased TMA with patient specimens of brain, colorectal, pancreatic cancer and sarcoma. Scale bars in low magnification images, 200 µm; Scale bars in high magnification images, 50 µm. Arrows, indicated the zoom out-box. (E) IHC data as shown in (D) were quantified and used for correlation analysis of EZH2 and p53 protein expression in patient specimens of brain, colorectal, pancreatic cancer and sarcoma. Pearson correlation r and P values in each cancer type are indicated.
Cytotoxic activity of NK cells in response to the MICA-129Met and MICA-129Val isoforms.Figure 3The degranulation (CD107a expression) of purified IL-2-stimulated (100 U/ml for 4 days) NK cells in response to immobilized MICA-129Met-Fc and MICA-129Val-Fc, and as negative control, OVA-Fc fusion proteins was determined by flow cytometry after 1 h (n = 3). Displayed are means and SD of the MFI (left panel) and the percentage of CD107a+ cells (right panel). Differences between MICA-129Met-Fc- and MICA-129Val-Fc-induced NK-cell degranulation were analyzed by two-way ANCOVA adjusted for MICA protein concentration, and the respective P-values are indicated. The degranulation of IL-2-stimulated LAK cells (100 U/ml for 4 days) exposed to L-MICA-129Met (n = 27) or L-MICA-129Val clones (n = 27) for 1 h was determined by flow cytometry. CD107a cell surface expression was analyzed after gating on CD56+ NK cells. In parallel, the MICA expression on target cells was determined. Displayed are the linear regressions of the MFI of CD107a on NK cells (upper panels) or the proportion of CD107a+ NK cells (lower panels) and the MICA expression intensity on target cells (MFI) for the L-MICA-129Met (left panels) and L-MICA-129Val clones (right panels). The coefficients of determination (R2), the regression coefficients (reg. coeff.), and the P-values for Pearson correlation are indicated. A representative of 21 experiments is shown demonstrating the specific cytotoxic activity of LAK cells against an L-MICA-129Met and an L-MICA-129Val clone. L-con cells served as a negative and K562 cells as a positive control. The means of specific lysis of triplicates plus SD at different E:T ratios (200:1 to 3:1) were measured in an 51chromium-release assay. The MICA expression intensity and the binding of a recombinant NKG2D-Fc fusion protein to the target cells were determined in parallel by flow cytometry, and the MFIs are indicated.
Figure 7(A) Scatterplot depicting the expression measurements with ecdysone induction versus measurements without ecdysone for all tested promoters separated by promoter architecture (Fig EV3). Each color represents one architecture (color-code indicated in the insert). Three types of line are used to indicate the expression fold change with no increase (y = x; solid line), 2-fold increase (y = x + 1; dotted line) and 4-fold increase (y = x + 2; dashed line). Red vertical dashed line: log2 basal expressions = 2. Log2 expressions > 2 on the right of the red dotted line. (B) Expression fold changes (ecdysone inducibility) versus measurements without ecdysone and grouped by native core promoter sequences. The colors refer to different core promoter architectures. Three types of line are used to indicate the expression fold change with no increase (y = x; solid line), 2-fold increase (y = x + 1; dotted line) and 4-fold increase (y = x + 2; dashed line). Red vertical dashed line: log2 basal expressions = 2. (C) Heatmap depicting the ecdysone inducibility fold changes caused by individual knockout of motifs in different core promoters. Disrupted INR (highlighted with the black dotted line rectangle) had a slightly negative effect on changing the core promoter responsiveness to ecdysone. (~ 2.3-fold reduction on average, Wilcoxon rank-sum test p = 2.1×10-5). Constitutive and developmental promoters highlighted in red and green, respectively.
TBK1 overexpression affects subcellular localization and reduces aggregation and cytotoxicity of HTT: (A) Representative western blot of soluble HTT (ab-MAB5492), HTT pS13 (ab-CHDI-90001039-1) and HTT pT3 (ab-CHDI-90001528-1) upon coexpression of HTTex1 16Q eGFP or 72Q eGFP or its phosphorylation-incompetent mutants with the indicated kinases for 48 hour in HEK293T cells.Figure 3(A) Representative western blot of soluble HTT (ab-MAB5492), HTT pS13 (ab-CHDI-90001039-1) and HTT pT3 (ab-CHDI-90001528-1) upon coexpression of HTTex1 16Q eGFP or 72Q eGFP or its phosphorylation-incompetent mutants with the indicated kinases for 48 hour in HEK293T cells. (B) The graph indicates the fold change in HTT in samples like illustrated in A, relative to empty vector controls normalized to GAPDH. (C) Representative immunoblot of the HTT (ab-MAB5492) after filter retardation assay from the insoluble cellular fraction upon expression of HTTex1 16Q eGFP or 72Q eGFP or its phosphorylation-incompetent mutants with the indicated kinases for 48 hour in HEK293T cells. (D) Quantification of the fold change in HTT aggregates compared to EV expressing HTTex1 16Q from the experiments like in C. (E) Representative immunofluorescence images of HTT aggregates (eGFP) and TBK1 (ab-anti-MYC) upon coexpression of HTTex1 72Q eGFP or its phosphorylation-incompetent mutants with the indicated kinases for 48 hour in HEK293T cells (scale bar 20μm). (F) Quantification of the percentage of co-transfected cells presenting aggregates upon expression of the indicated kinase, from the experiments like in E. (G) Quantification of the percentage of cells presenting nuclear aggregates from the experiments like in E. (H) Representative western blots of HTT (ab-MAB5492), HTT pS13 (ab-CHDI-90001039-1) in soluble fraction and aggregates from HEK293T cells transfected first with HTTex1 72Q eGFP and then after 48 hour (to enable the formation of aggregates before the kinase expression); with TBK1; cells were lysed at the indicated time after kinase transfection. (I) Quantification of the percentage of cells presenting aggregates and kinase expression from the experiments in H.
IGF-I Neutral Chromatography and Proposed Mechanism of Action of PAPP-A2 Deficiency.Figure 4• A Concentrations of endogenous insulin-like growth factor (IGF)-I in the different fractions obtained by neutral chromatography on a Superdex 200 column. Serum samples from the four siblings of family 1 were analyzed and compared to a non-related prepubertal control, as all siblings were prepubertal. The amount of IGF-I detected in the fractions corresponding to the ternary complex (TC) was markedly greater in the homozygous affected subjects (II·1 and II·3) compared to the unaffected siblings (II.2 and II.4) and the prepubertal control. No IGF-I was detected in the fractions corresponding to the binary complex (BC) or free IGF-I in any of the subjects. This is most likely due to the fact that under physiological conditions 80-85% of IGF-I circulates in the TC and after the separation procedure, the amount of IGF-I in the BC or free was below the level of detection of our IGF-I assay. These results clearly demonstrate that in the affected patients the amount of IGF-I circulating in the TC is markedly elevated. •B Cartoon of hypothesized mechanism underlying growth failure due to lack of PAPP-A2. There is an increase in the formation of ternary complexes, due to decreased proteolysis subsequent to the lack of PAPP-A2 activity. This results in reduced concentrations of free IGF-I (fIGF-I) in serum and most likely at specific target tissues. This also decreases the negative feedback effect of IGF-I on growth hormone (GH) production, contributing to the increased circulating concentrations of GH. Increased serum GH concentrations cause increased IGF-I, IGFBP-3 and ALS concentrations. This increase in IGFBP-3 and ALS would then contribute to further ternary complex formation and an increase in total IGF-I and IGF-II concentrations.
Figure 1 (A) The egfl7 gene (about 13.8 kb) comprises 10 exons with 7 of them encoding for the EGFL7 cDNA (lower left). Intron 7 harbors the parasitic miRNAs (miR) 126 and 126* (lower right) (B) Methylation arrays of primary glioblastoma (GBM) specimens revealed that CpG island 1 (egfl7 promoter) was mostly unmethylated with the exception of some samples in the RTKII subgroup (*). CpG island 2 (miR-126 promoter) was found methylated in most cases except the G34R subgroup (**). G34R and K27M - GBMs with mutations in a gene for the H3.3 histone variant; RTKI and II - GBMs with a mutation in the receptor tyrosine kinase type I (proneural subtype) and II (classical subtype); MES - GBMs with a deleterious mutation in the NF1 gene (mesenchymal subtype); IDH1 - GBMs with a mutation in isocitrate dehydrogenase 1 (C) Quantification of human EGFL7 by qRT-PCR in primary GBM biopsies and corresponding PDX xenografts upon implantation of GBM-derived organotypic spheroids (n = 3; Mann-Whitney U-test; mean ± SEM); B - GBM biopsy; G - generation (D) Alignment of EGFL7 expression along with stromal (VWF, VEGFR1, VEGFR2, ACTA2, CD248, ITGAX, AIF1, CD68, CD4, and PTPRC) or tumor markers (EGFR, PDGFRA, VEGFA, VIM, and NES) in primary GBM biopsies or GBM-derived organotypic spheroids (PDX xenografts) (E) Immunohisto­chemical (IHC) analyses revealed EGFL7 expression in blood vessels (arrow) and neurons (arrowheads) of healthy gray matter. Expression in malignant brain tumor specimens was restricted to blood vessels, in particular with a large diameter (arrows). Scale bar represents 50 µm (F) Quantitative scoring of EGFL7 staining in glioma specimens yielded about 25-40% EGFL7-positive blood vessels (n = 13 astrocytoma WHO° grade II, n = 25 astrocytoma WHO° grade III, n = 24 GBMs WHO° grade IV)
Figure 2Survival curve of smc5 cells treated with different concentrations of cisplatin (CDDP). Data represents means ± SD of 3 experiments. Survival curve of smc5 cells complemented or not with chicken SMC5 cDNA and treated with different concentrations of cisplatin. The experiment is carried out at 39.5ºC. Data represents means ± SD of at least 3 experiments. Survival curve of smc5 ku70 cells treated with different concentrations of cisplatin. Data represents means ± SD of the 3 experiments. Survival curve of smc5 fancc cells treated with different concentrations of cisplatin. Data represents means ± SD of 3 experiments. WB analysis of CHK1-P levels at steady state or after treatment with 1 μM cisplatin for 3 hours. CHK1 total protein is used as loading control. Quantitative analysis of CHK1-P levels at steady state versus total CHK1 is shown in the bottom panel. Data represents means ± SD of 4 experiments. WB analysis of FANCD2 ubiquitination levels at steady state after treatment with 1 μM cisplatin for 3 hours. Analysis of the ratio between FANCD2 ubiquitinated/non-ubiquitinated forms is shown on the bottom panel. Data represents means ± SD of 3 experiments.
Figure 3 (A) Brains were dissected from w1118, alix1, alix-l/CyO; alix1 and mud4 larvae and stained with anti-Bazooka (red), anti-Cnn (white) and Hoechst (blue). Representative confocal images are shown. Scale bars, 5 μm. The graph summarizes the average variation of the relative spindle angles in w1118 (n=74), alix1 (n= 96), alix-l/CyO; alix1 (n= 78) and mud4 (n=32) NBs (four separate experiments) (±S.E). Compared to w1118 or alix-l/CyO; alix1 NBs both alix1 and mud4 NBs showed greater variation in MS orientation (***p<0.001, ANOVA)  (B) Schematic illustration of the calculation of the relative spindle angle between the polarity axis (blue line, perpendicular to the line aligning the apical Bazooka crescent (red dotted line) and the MS axis (blue dotted line) going through the two centrosomes. The basal crescent is here indicated in green (C) Brain lysates of w1118, alix1 and alix-l/CyO; alix1 larvae were immunoblotted for ALIX, α-tubulin and GAPDH (loading control) (D) Time-lapse imaging of Asl-YFP, α-tubulin-GFP in control (left panel) and alix3 (right panel) mutant SOPs of the pupal notum. SOPs were identified by the apical meshwork of MTs (not shown). Time is indicated in min:sec. Mitotic cells are indicated with arrows. Scale bar represents 5 μm (E) Quantification of the rotation angle (±S.E) in control (n=26) and alix3 (n=45) mutant SOPs (at least three separate experiments). **p<0.01 (Student's t-test) compared to control (F) Schematic view of the calculation of the rotation angle. The angle α1 represents the angle made between the midline (horizontal line) and the axis of the two centrosomes immediately prior to nuclear envelope breakdown (when the two centrosomes compress the nucleus). The angle α2 represents the angle between the midline and the axis of spindle at anaphase onset. The rotation angle is equal to the difference α1-α2 (G) Western blotting analysis of lysates from control Asl-YFP, α-tubulin-GFP and Asl-YFP, α-tubulin-GFP; alix3 pupae showed absence of ALIX in the alix3 homozygous mutant and unchanged expression of α-tubulin and GAPDH (loading control)
A working model illustrating regulation of the malate–aspartate shuttle by GOT2 acetylationShown is a working model depicting how acetylation of GOT2 at 3K regulates the malate–aspartate shuttle and promotes pancreatic tumor proliferation.Figure 6A working model illustrating regulation of the malate-aspartate shuttle by GOT2 acetylationShown is a working model depicting how acetylation of GOT2 at 3K regulates the malate-aspartate shuttle and promotes pancreatictumorproliferation. In pancreatictumorcells, reduced SIRT3 expression leads to an increase of GOT2 acetylation. GOT2 acetylation at 3K enhances the protein interaction between GOT2 and MDH2, thereby stimulating the net transfer of cytosolicNADH into mitochondria to support ATP production. Meanwhile, GOT2 3K acetylation promotes cellular NADPH production to suppress ROS that are produced during rapid cellproliferation. Glc, glucose; Gln, glutamine; Asp, aspartate; and Mal, malate.
Figure 1(A) Quantification of CXCR4+ cells by flow cytometry and Sox17+ cells by immunostaining at day 7 following differentiation in the presence or absence of 3 µM CH. Average and standard deviation (SD) of eight independent experiments. (B) Gene expression profiling by RT-PCR at day 7 differentiation. Average and SD of three independent experiments. (C) Immunostaining for Ecadherin (left) and FoxA2 (right) at day 7 of differentiation in presence of 3 µM CH. (D) Quantification of CXCR4+ by flow cytometry over a seven-day differentiation time course in the presence of 3 µM CH. Average and SD of three independent experiments. (E) Immunostaining for Sox17 at day 4 to day 7 of differentiation in the continuous presence or absence of 3 µM CH. Scale bars = 200 µM
Tyrosine Kinase inhibition restores parkin-Beclin-1 interactionGraph represents brain levels of TKIs over a 24 hr period after IP injection with Imatinib, Nilotinib, Bosutinib and DMSO (N = 7).Figure 1Graph represents brain levels of TKIs over a 24 hr period after IP injection with Imatinib, Nilotinib, Bosutinib and DMSO (N = 7). [Correction added after publication on 4 July 2013: The y axis of the graph shown in Fig. 1A has been corrected from "concentration (mM)" to "concentration (nM)"] WB in Tg-APP total brain lysates on 4-12% NuPAGE SDS gel (Invitrogen) show total Abl (top blot) T412 Abl (2nd blot), soluble parkin level (3rd blot), and LC3 (4th blot) relative to MAP-2 (N = 9). Quantitative ELISA showing soluble (STEN extract) and insoluble (4 M urea extract) mouseparkin in Tg-APP (N = 9). Parkin−/−brains were used as a specificity control. (D) Blots represent immunoprecipitatedBeclin-1 in mice probed with parkin antibody (D) Blots represent immunoprecipitatedBeclin-1 in mice probed with parkin antibody, and (E) control IgG in parallel with immunoprecipitates. (F) immunoprecipitatedBeclin-1 probed with parkin and the reverse experiment in post-mortem human AD cortex analyzed on 4-12% SDS-NuPAGE gel. In situ proximity ligation assay (PLA) shows endogenous parkin-Beclin-1 complexes in (G) WT C57BL/6mice (N = 5) and (H) parkin−/− as control.PLA in Tg-APPmice IP injected once daily for 3 weeks with (I) DMSO (J) 5 mg/kg Bosutinib and (K) 10 mg/kg Nilotinib (N = 5). PLA in human post-mortem brains in the (L) cortex of a normal subject and (M) cortex of an AD patient; the hippocampus of (N) a normal subject and (O) an AD patient; the caudate of (P) a normal subject and (Q) an AD patient. Graph represents human Aβ1-42ELISA in rat B35 neuroblastoma cells transfected with human cDNA Aβ1-42 (or LacZ) or Beclin-1 shRNA for 24 hr, and then treated with 1 μM Bosutinib for an additional 24 hr (N = 12). *Significantly different to control or as indicated, Mean ± SEM, ANOVA with NeumannKeuls multiple comparison.
Figure 9A. Primary mouse bone marrow-derived macrophage cells (BMDM) from adult male WT-mLRRK2 BAC transgenic mice were treated with DMSO, 1 μM Rab10 anti-sense oligonucleotide for 4 days, 100 nM MLi2 for 12 hours, as indicated, before stimulation with 100 nM CCL5 for the indicated time (hours). Representative immunoblots from n=3 biologically independent experiments are shown. B. Relative pSer473-Akt levels were normalized to total Akt and calculated as fold of naïve cells (without CCL5 stimulation). Data shown are group means ± SEM from n=3 biologically independent replicates, with significance assessed by one-way ANOVA with Tukey's post hoc test with treated groups compared to matched control groups, with ** representing P< 0.01. C. BMDM cells, as described in panel (A), were additionally treated with DMSO (control), EIPA, or Akti-1/2(Akt1/2 inhibitor) as indicated, for 1 hour after CCL5 stimulation. Representative immunoblots from n=3 biologically independent experiments are shown. D. Relative pSer473-Akt levels were normalized to total Akt and calculated as fold of naïve cells (without CCL5 stimulation). Each dot represents one experiment, with error bars showing ± SEM. Significance was assessed by one-way ANOVA with Tukey's post hoc test, with *** representing P< 0.0005, ****P< 0.0001. E. BMDM cells, as described in panels (A and C), were seeded in Transwell (Sigma) chemotaxis assays using CCL5 as a chemoattractant in bottom chambers. After two hours, the cells failing to migrate through the matrices were removed with a cotton swab. Migrated cells were counted with DAPI images, with representative images shown at low magnification. Representative images from n=3 biologically independent experiments are shown. F. Relative chemotaxis activity was calculated as fold of naïve cells that migrated without any added CCL5. Each dot represents the mean of images collected from one experiment. Data are group means ± SEM, with significance assessed by multiple t test., with* representing P<0.05.
Co-stimulation of CD8+ T cells by the MICA-129Met and MICA-129Val isoforms.Figure 6MACS-separated CD8+ T cells were cultured in triplicate on an immobilized anti-CD3 mAb (0.005 μg/ml [upper panel] or 0.01 μg/ml [lower panel]) in combination with recombinant MICA-129Met-Fc, MICA-129Val-Fc, and OVA-Fc proteins at various concentrations (1.0, 0.5, 0.1, 0.0 μg/ml). After 72 h, 25% of the supernatant was harvested and IL-2 concentrations were measured by ELISA. The harvested medium was replaced by the same volume containing 1 μCi 3H-labeled thymidine. After 12 h, the plates were completely harvested and the DNA-bound radioactivity was determined. The means and SD of the stimulation index (SI) are displayed (n = 4). Significant differences between MICA-129Met/Val-Fc and OVA-Fc proteins were found when the antigen-specific signal (anti-CD3) was limited (*P < 0.05, t-test; upper left panel: 1.0 μg/ml: MICA-129Met-Fc versus OVA-Fc P = 0.0372 and MICA-129Val-Fc versus OVA-Fc P = 0.0366; upper right panel: 1.0 μg/ml: MICA-129Met-Fc versus OVA-Fc P = 0.0499 and MICA-129Val-Fc versus OVA-Fc P = 0.0192; 0.5 μg/ml: MICA-129Met-Fc versus OVA-Fc P = 0.0164 and MICA-129Val-Fc versus OVA-Fc P = 0.0357; lower left panel: 0.5 μg/ml: MICA-129Met-Fc versus OVA-Fc P = 0.0287 and MICA-129Val-Fc versus OVA-Fc P = 0.0232; lower right panel: 1.0 μg/ml: MICA-129Met-Fc versus OVA-Fc P = 0.0171 and MICA-129Val-Fc versus OVA-Fc P = 0.0484). Purified CFSE-stained CD8+T cells were stimulated by immobilized anti-CD3 (0.005 μg/ml) in combination with recombinant MICA-129Met-Fc, MICA-129Val-Fc, OVA-Fc proteins, or co-stimulatory mAb (anti-CD28, anti-NKG2D) or an isotype control (mIgG1). The proliferation of CD3+CD8+T cells was assessed at 60 h by flow cytometry. Results of a representative out of 6 experiments are displayed. Untreated CFSE-stained CD8+T cells are included for comparison. The percentage of proliferating cells and MFI for CFSE are indicated.The MFI of CFSE in unstimulated CD8+T cells (control) was set to 100% in individual experiments (n = 6), and the relative decrease due to proliferation was calculated. Means + SD are shown. Significant differences (*P = 0.0277, Wilcoxon test) between MICA-129Met-Fc versus MICA-129Val-Fc and OVA-Fc proteins were found at slightly higher anti-CD3 concentrations (0.1 and 0.05 μg/ml) than at later time points (see A).Anti-CD28 and anti-NKG2D mAb were used in parallel as a positive control, mean + SD are shown, and significant differences (*P = 0.0277, Wilcoxon test) to the isotype control (mIgG1) are indicated (n = 6).
Figure 4(a) Control, UK5099-treated and DCA-treated HE cells were visualized together by UMAP and divided into 7 clusters. (b) Heatmap showing scRNAseq data of endothelial or hematopoietic genes expressed in the 7 clusters. (c) Clusters 6 (559 cells) and 7 (280 cells) were assessed independently and dot plots show expression levels of the indicated genes detected by scRNAseq and based on percent expressed (size of the dots) and average level of expression (color intensity). (d) Dot plots show expression levels of the indicated hematopoietic transcription factors in clusters 6 and 7 for HE ctrl, HE + UK5099 and HE + DCA conditions, detected by scRNAseq and based on percent expressed (size of the dots) and average level of expression (color intensity).