EMBO/sd-character-level-ner huggingface dataset:
Rap1 ectopic expression reverts the telomeric defects of cay1∆ cellsTelomere length analysis of ApaI‐digested DNA from cells harvested at early (left) and late generations (right) after expression of YFP‐tagged proteins.Figure 7Telomere length analysis of ApaI‐digested DNA from cells harvested at early (left) and late generations (right) after expression of YFP‐tagged proteins. Northern blot analysis of ARIA, ARRET, and 18S rRNA (loading control) at early generations after expression of YFP‐tagged proteins. Numbers at the bottom are ratios between ARRET and 18S rRNA signals, expressed as fold increase over wt. ChIP analysis of subtelomeric H3K9ac, H3K9me3, and total H3 in the indicated strains at early generations. Immunoprecipitated DNA is normalized to input DNA and expressed as fold increase over wt after subtraction of values obtained for negative control immunoprecipitations performed with beads only. Bars and error bars are averages and s.d. from 3 independent experiments. Statistical significance was assayed using the unpaired, two‐tailed Student's t‐test. *P 0.05, **P 0.01, ***P 0.001 relative to wt. Serial dilutions of early generation cay1+ and cay1∆ cells expressing Cay1‐YFP and Rap1‐YFP were spotted on complete medium and grown at 30°C and 20°C. Quantification of cell length in strains grown for 24 h in complete medium at 30°C or 20°C. Each dot represents a single cell and median cell length (m) is indicated for each sample. Data are from 3 independent experiments and at least 100 cells were scored per sample in each experiment. Quantification of chromosome bridges of strains grown for 24 h in complete medium at 30°C or 20°C. Bars and error bars are averages and s.d. from three independent experiments and at least 200 cells were scored per sample in each experiment.
Figure 1A Representative images of alkaline phosphatase (ALP), Alizarin Red S (ARS) and Van Gieson's staining of human MSCs  B, C Quantitative analyses of the ALP activity and calcium mineralization. Results are shown as mean ± SEM; n=6; **: p<0.01 and ***: p<0.001 by t test D, E Quantitative RT-PCR and western blot analyses of the expression of osteogenic markers. Cells were cultured in osteogenic medium for 5 days. Results are shown as mean ± SEM; n=3; *: p<0.05, **: p<0.01 and ***: p<0.001 by t test  F H&E staining of the MSCs-mediated ectopic bone formation. Scale bar, 50 μm G Quantitative analysis of bone volume versus total tissue volume (BV/TV). Results are shown as mean ± SEM; n=6. *: p<0.05 by t test
Aggregates from short GA25-50 repeat lengths are mobile, while those from longer repeats are more stationary Primary rat cortical and motor neurons underwent live-cell imaging after 48 hours of eGFP-GAn expression to determine aggregate mobility within neurites.Figure 2Primary rat cortical neurons underwent live-cell imaging after 48 hours of eGFP-GAn expression to determine aggregate mobility within neurites. A) Example of a cortical neuron expressing eGFP-GA100 (green) with brightfield overlay, 60x magnification, scale bar indicates 10μm. Inset in upper right shows enlargement of boxed area, to better visualize aggregates within neuronal processes. Inset scale bar indicates 5μm. Primary rat motor neurons underwent live-cell imaging after 48 hours of eGFP-GAn expression to determine aggregate mobility within neurites. B) Example of a motor neuron expressing eGFP-GA25 (green) with brightfield overlay, 60x magnification, scale bar indicates 10μm. Inset in upper right shows enlargement of boxed area, to better visualize aggregates within neuronal processes. Inset scale bar indicates 5μm. C) Quantification of eGFP-GAn aggregate velocity within cortical neurons. D) Quantification of eGFP-GAn aggregate velocity within motor neuron neurites.
Figure 4(A) UgpB overexpression confers bile salt resistance, and a ugpB deletion mutant is slightly CHO sensitive. (B) CHO-induced MDH protein aggregation analyzed by monitoring light scattering. (C) CHO-induced MDH protein aggregation analyzed by a solubility assay using centrifugation (cfg) at 16,000 g for 20 min. The supernatant (Sup) represents the soluble fraction and the pellet the insoluble fraction; 12 µM MDH was used as a model aggregation-prone substrate (see Methods). Addition of increasing quantities of UgpB increasingly solubilizes MDH. The molar ratios of MDH:UgpB that were used (left to right) are as follows: 1:0, 1:0.1, 1:0.2, 1:0.5, and 1:1. 5-fold more of the pellet fraction was loaded relative to the supernatant fraction. (D) The supernatant/pellet assay was performed using a 1:1 MDH:chaperone ratio for Spy, SurA, and OsmY. The pellet fraction was concentrated 5-fold. (E) 12 µM of MDH alone and a 1:1 ratio of either MDH:UgpB or MDH:MBP were incubated at 37°C for 60 min in the presence of CHO and then fractionated via centrifugation for use in a solubility assay as described above (C) . The pellet fraction was concentrated 5-fold. In some samples (+G3P), 120 µM of G3P was added prior to the addition of any other protein. Addition of G3P inactivates UgpB's ability to inhibit protein aggregation. (F) 800 μg of E. coli total lysate and 60 µM of UgpB/MBP were used for the supernatant/pellet assay. 600 µM of G3P was added before adding proteins. The pellet fraction was concentrated 10-fold. As before, addition of UgpB inhibited aggregation, but only in the absence of G3P. (G) The anti-aggregation activity of tryptophan single mutant (W169S) and double mutant (W169S/W172S) was examined by CHO-induced MDH aggregation assay. Light scattering values were monitored. (I) The anti-aggregation activity of the UgpB variants (E66A and R374V) was examined by CHO-induced MDH aggregation assay. CHO-induced MDH protein aggregation analyzed by monitoring light scattering. (J) Solubility of MDH is also monitored by supernatant/pellet assay. 12 µM of MDH alone and a 1:1 ratio of MDH:UgpB proteins, 120 µM of G3P were used.
Figure 4A Immunostaining of representative images of KV embryos at 8 somite stage in which mRNA encoding for mCherry or mCherry-Rab35 WT was injected. Staining of acetylated tubulin was used as cilia marker. Inset shows a higher magnification image of the cilia region, displaying colocalization of mCherry-Rab35 and acetylated tubulin. Scale bar, 10 µm. B,C Immunoblot analysis with anti-Rab35 and anti-GAPDH antibodies of 40 µg of total lysates of zebrafish embryos injected with Rab35 morpholino (MO) at 8 somite stage, compared with non-injected embryos. The plot in (C) shows Rab35 protein level in non-injected and Rab35 morphants, normalized to GAPDH protein level. Data are mean ± S.E.M. of 3 independent experiments (n > 50 embryos per condition). D Organ situs was determined by observing the heart position at 30 hours post-fertilisation (hpf), and the liver position at 53 hpf, by in situ hybridisation with a probe for foxa3, in controls (Mismatch MO and non-injected [WT]) and Rab35 MO. Situs solitus means left heart and liver; situs inversus means right heart and liver; and heterotaxia means any other possible combination of the heart and liver position. E Whole-mount in situ hybridization with the dand5 probe at 8 somite stage (13 hpf) in wild type non-injected embryos and mismatch morphants, both presenting right-sided distribution of dand5, and Rab35 morphants presenting bilateral localization of the dand5 probe. Scale bar, 50 µm. Graph shows quantification of embryos (%) with bilateral or right-sided dand5 localisation from both conditions. Statistical significance according to Fisher Exact test (** P = 0.0002) F-I Immunostaining of representative images of KV embryos at 8 somite stage in which: (F) Mismatch or Rab35 MO was injected, as well as a co-injection of Rab35 MO with mRNA encoding mCherry-Rab35 WT [Rescue]; (H) mRNA encoding mCherry or mCherry-tagged Rab35 WT, Rab35-Q67L or Rab35-S22N were injected. Acetylated tubulin is shown in green as cilia marker. Quantification of KV cilia length in (F) and (H) is shown respectively in (G) and (I) as box-and-whisker plots of 3 independent experiments. N ≥ 8 embryos and n > 300 cilia per experimental condition. Horizontal lines show 25, 50 and 75th percentiles; whiskers extend to minimum and maximum values. Statistical significance according to Kruskal-Wallis followed by Dunn's post-hoc test (** P < 0.01, *** P < 0.001, ns. not significant; Mismatch vs Rab35 MO, P < 0.001; Cherry vs Rab35-SN, P = 0.0063; ). Scale bars, 10 µm.
Figure 5Effect of TFL457 on MEF2-promoter activity. Cultures transfected with pMEF2 (two minimal wild type MEF2 elements) or pMEF2mut (mutant) were preincubated with CPPs (25 μM, 30 min) and treated with NMDA (100 µM, 2 h). Means ± SEM of luciferase activities obtained in excitotoxicity, relative to values found in cells treated with same peptide and no NMDA, are presented. Effect on CRE-promoter activity. pCRE contains two minimal CREs. Peptide preincubation was as above with or without KG-501 (10 µM). Mean ± SEM luciferase activities are given relative to values in cells treated with the same peptide but no NMDA or KG-501. Effect on NMDAR-subunit promoters. Cells transfected with pGluN1 or pGluN2A were treated and analyzed as in panel A (pGluN1, *p = 0.016; pGluN2A, *p = 0.029; n = 14). Effects on BDNF promoter III or TrkB promoter. Cells transfected with pIIIBDNF (n = 9), pTrkB (n = 7) or pCRE (n = 5), as a control, were processed as above but using 20 µM NMDA for 4 h. Data are presented and analyzed as in panel A (pCRE, **p = 0.0098; pIIIBDNF, *p = 0.029; pTrkB, *p = 0.033). Effects of TFL457 on mRNA levels of CREB/MEF2-regulated genes. Total RNA was extracted from cultures preincubated with CPPs (25 μM, 30 min) and treated or not with NMDA (100 µM, 4 h). Levels of mRNA were normalized to NSE (genes expressed in neurons, left panel) or GAPDH (neuronal and glial expression, right panel). Means ± SEM of levels obtained in excitotoxicity relative to values found in cells treated with same peptide and no NMDA are presented. Differences found in excitotoxicity were analyzed by ANOVA test followed by post-hoc Tukey HSD test (GluN1, ***p = 0.00096; GluN2A, p = 0.98; TrkB-FL, ***p = 0.00037; BDNF, *p = 0.045; n = 8).
Promastigote differentiation and infectivity.Figure 7(A) Proportion of metacyclic promastigotes in stationary phase cultures, assessed using the PNA assay. Values shown are the means ± SD from three independent experiments. *, differed significantly (p<0.05). (B) Western blot analysis of extracts of 107promastigotes at stationary phase of growth probed with α-HASPB. α-Cysteine Synthase was used as a loading control [56]. (C) Infectivity and survival of promastigotes in peritoneal macrophages in vitro, infected at a ratio 5∶1, with the infection rates being assessed after 1 and 5 days. *, differed significantly (p<0.01). (D) Lesion progression in BALB/c mice inoculated with 5×105 stationary phase promastigotes. Values shown are the means ± SD from 5 mice. *, infection level of Δatg5 differed significantly from WT (p<0.01) and Δatg5::ATG5 (p<0.05). (E) Morphologies of cells isolated from mouse lesions and analysed by SEM. Scale bar: 2 µm.
Figure 2(A) RNA-seq of MCF7 and MDA-MB-231 transfected with miR-515-5p revealed the down-regulation of five transcripts, NRAS, FZD4, CDC42BPA, PIK3C2B and MARK4. (B-D) The effect of miR-515-5p overexpression on NRAS, FZD4, CDC42BPA, PIK3C2B and MARK4 mRNA levels in MCF7 (B), MDA-MB-231(C), A549 and H1299 (D) was determined by qPCR 48 h following transfection of the indicated cell line with non-targeting control miR (NC) or miR-515-5p. Data is displayed as the normalised mean ± SEM. P values were calculated by t-test between miRNA conditions and their respective NC conditions (*, P<0.05; **, P<0.01; ***, P<0.001). (E-F) miR-515-5p directly interacts with NRAS, MARK4 and PI3KC2B's 3'UTR. Relative luciferase activity levels were measured 24 h after co-transfection of MCF-7 (E) and MDA-MB-231 (F) with 3'UTR-luciferase reporter constructs and either with miR-515-5p or miR NC. Data shown are normalised mean of three independent experiments ± SEM. (B-F) P values were calculated by t-test between miRNA conditions and their respective NC conditions (*, P<0.05; **, P<0.01; ***, P<0.001).
Disease-associated Parkin mutations are defective in mitophagy.Figure 1.(A) MEFs were transfected with GFP-tagged wild-type (WT) or mutant Parkin expression plasmid followed by an 18-h treatment of CCCP. Cells are immunostained with a Tom20 antibody to visualize mitochondria (red). GFP-parkin-transfected cells are marked by dotted lines. Arrows indicate parkin-positive mitochondria or mitochondrial aggregates. Bar, 10 µm. (B) The average percentages of mitochondria-free cells from three independent experiments from A are presented with standard deviation as error bar. **, P < 0.01 (C) MEFs were transfected and treated with CCCP as described in A, followed by an immunoblotting analysis with antibodies for cytochrome c, actin, and parkin. Note that levels of parkin R275W and R42P mutant were lower, as previously reported (Wang et al., 2005).
Figure 3A. Schematic of the methodology adopted for the identification of protein interacting partners of veal2 using a RAP-MS based approach. B. RAP- MS followed by western blotting validated Prkcbb and Ephb3 as interacting protein partners with veal2. C. RNA immunoprecipitation of veal2 was performed by pull-down of Prkcbb and Epbhb3, followed by qRT-PCR. IgG pull-down was performed as control. Gel image shows the amplification of veal2 in different RNA immunoprecipitation samples, along with 10% input control, as marked by arrowheads. D. qRT-PCR based quantification of veal2 transcript across Prkcbb, Ephb3 and IgG immunoprecipitations. actb was used as normalization control. Data from three different biological replicates represented as mean fold change values ± standard deviation. E. Relative kinase activity of human PRKCB2 under standard conditions and in the presence of various concentrations of the WT veal2 RNA, veal2-Δ8 RNA, veal2-AS RNA. 52nM of the PRKCB2 protein was used per reaction. Data from three different experiments plotted as mean fold change values ± standard deviation. F-K. Enzastaurin treatment rescues hemorrhage phenotype in veal2gib005Δ8/+ zebrafish embryos indicated by rescue of the vascular integrity defects in veal2gib005Δ8/+. Arrowheads show the presence of hemorrhage due to the vascular integrity defects. Experiment was repeated in biological replicates and total no of embryos scored are mentioned on figure. (F-I) Magnification-5X, Scale bar-100μm, (J-K) Magnification-20X, Scale bars-50μm. L. Relative number of animals that displayed the hemorrhage phenotype across the progeny of the outcross of veal2gib005Δ8/+ zebrafish. Number of phenotypic animals upon treatment with Enzastaurin was normalized with the number of phenotypic animals when treated with DMSO. Data from three different experiments plotted as mean fold change values ± standard deviation.
a) Ganglioside composition of human fibroblasts, iPSCs, differentiated iPSCs and enriched iPSC-derived neurons (after FACS) from control (line C1-1), GBA-PD (line PD1-1) and GD (line GD1-1) subjects.f4(a) Ganglioside composition of human fibroblasts, iPSCs, differentiated iPSCs and enriched iPSC-derived neurons (after FACS) from control (line C1-1), GBA-PD (line PD1-1) and GD (line GD1-1) subjects. Cell lipids were metabolically labelled with [1-3H]sphingosine and visualized by digital autoradiography. (b) Activities of glycohydrolases (GCase, GBA2, β-galactosidase, β-hexosaminidase) in control iPSCs and iPSC-derived enriched neurons at DIV65. Enzyme activities are expressed as pmoles mg−1 h−1. Data are represented as mean+s.d.; experiments were independently repeated three times in triplicate. *P0.01; Student's t-test. c) GBA1, GBA2, β-galactosidase and HEXA and HEXB gene expression levels in control iPSCs and iPSC-derived enriched neurons at DIV65. Data are represented as mean+s.d.; experiments were independently repeated three times in triplicate. *P0.05; **P0.01, Student's t-test. (d) Representative western blots showing GCase, GBA2, β-galactosidase and HEXA and HEXB protein levels in control iPSCs and corresponding iPSC-derived neurons (DIV65).
Figure 8TGFβ1-injured epithelium activates TAK1 and actomyosin remodelling, which subsequently induces nuclear translocation of TAK1 and actin. Nuclear TAK1 mediates the phosphorylation of EZH2 on T311 and facilitates the release of EZH2 from PRC2. The liberation of EZH2 is accompanied by an EZ switch to EZH1-PRC2, which is required to maintain H3K27me3 at TGFβ1 non-target genes. Simultaneously, EZH2 establishes the fibrotic transcriptional complex with POL2 and nuclear actin to promote the metaplastic differentiation of AECs and triggers the fibrotic crosstalk with MCs. Perturbing this fibrotic complex blocks the fibrotic cascade, reinforces tissue repair and restores homeostasis.
Figure 7The effect of miR-1236 KO on AGCT metastasis was assessed, using an in vivo xenograft mice model. (A) Representative images of tumor nodules (white arrows) formed in the intestines of nude mice xenografted with control or miR-1236-/- KGN cells are shown (left and right; scale bar = 5 mm). Hematoxylin and eosin staining confirmed the pathological characteristics of the metastasized GCT nodules (middle, black arrows; scale bar = 50 µm). The black dashed circles are metastasized nodules of xenografted KGN cells found in mouse intestines. (B) The number of tumor nodules formed in the intestines was counted in control (n = 8) and miR-1236-/- (n = 8) mice. (C) Allele-specific real-time RT-PCR analysis of the WT and 402C>G variant FOXL2 mRNAs was performed using RNA extracted from tumor nodules from control or miR-1236-/- mice. The p values were analysed by unpaired, two-tailed Student's t-test (**p < 0.01, ***p < 0.001). D-F Box-and-whisker plots showing the relative expression of miR-1236 (D), variant FOXL2 mRNA (E), and WT FOXL2 mRNA (F), respectively, in 32 patients with non-metastasized AGCTs and 14 patients with metastasized AGCTs. X- axis indicates patient subgroups depending on whether they exhibit metastasized AGCTs (meta) or not (non-meta). The relative miR-1236 levels were measured using a TaqMan® microRNA RT-qPCR assay, with expression normalized to RNU6B. The levels of 402C>G or WT FOXL2 mRNA were determined by real-time RT-PCR, and the data were normalized to paired-gDNA levels. The relative levels of miR-1236 and FOXL2 mRNAs were quantified by setting the levels of AGCT #1 to 1. Real-time RT-PCR was performed in triplicate for each specimen. The box plot represents the minimum value, first quartile, median, third quartile and maximum value of a data set. The whiskers extend to the most extreme data points not considered outliers, and the outliers are represented as dots. Comparisons between groups were performed using Student's t-tests, and p values are presented. G, H The estimated regression line superimposed on the scatter plot of miR-1236 levels with 402C>G FOXL2 mRNA (G) or WT FOXL2 mRNA (H) in AGCT samples (n = 46) is shown, along with correlation coefficient (r) and p values. I The proposed model for FOXL2 haploinsufficiency induced by the 402C>G mutation during AGCT development.
PINK1 accumulates in the mitochondrial and postmitochondrial supernatant fractions upon knockdown of Lon.Figure 2PINK1 accumulates in the mitochondrial and postmitochondrial supernatant fractions upon knockdown of Lon.(A) Mitochondrial (Mito) and postmitochondrial supernatant (Sup) protein fractions isolated from the heads of flies of the indicated genotypes were analyzed by western blot with antibodies to Myc, Complex V β (Comp V β), NADH dehydrogenase (ubiquinone) Fe-S protein 3 (NDUFS3), voltage-dependent anion channel (VDAC) and Actin. elav-GAL4 was used to drive expression of the RNAi constructs used in these experiments. (B) Densitometry of the MPP-PINK1 bands in the mitochondrial fractions from the indicated genotypes was performed using Fiji software. PINK1 band intensities were normalized to VDAC intensity, and this ratio was in turn normalized to the MPP-PINK1/VDAC ratio in Control-R animals. (C) Quantification of the Rho-PINK1 band intensities in mitochondrial fractions from the indicated genotypes was performed as described in B. (D) Quantification of the AFG-PINK1 band intensities in mitochondrial fractions from the indicated genotypes was performed as described in B. (E) Quantification of the MPP-PINK1 band intensities in the supernatant fractions from the indicated genotypes was performed as described in B except that Actin rather than VDAC was used as the loading control. (F) Quantification of the Rho-PINK1 band intensities in supernatant fractions from the indicated genotypes was performed as described in E. (G) Quantification of the AFG-PINK1 band intensities in supernatant fractions from the indicated genotypes was performed as described in E. Results shown are representative of at least three independent subcellular fractionation experiments per genotype. Error bars represent s.e.m. *p<0.05 by Student t test.
Genetic inhibition of PI3KC2β reduces vascular leakage, edema, cerebral infarction and improves neurological outcomes in ischemia/reperfusion stroke models.Figure 1WT, heterozygous (C2βWT/D1212A) and C2βD1212A/D1212A mice were subjected to tMCAO (1h ischemia followed by 24h reperfusion). (A) Representative photographs of coronal brain sections and graph quantification of Evans blue (EB) dye leakage (dashed orange line) and edema volume. Data represent mean ± SEM (EB leakage: n=5-16 mice per group; Edema volume: n=14-30 mice per group; *p<0.05, **p<0.01 Mann-Whitney test). (B) Representative photographs of coronal brain section stained with TTC (dashed white line) and graph quantification of infarct volume measurements. Data represent mean ± SEM (n=14-27 mice per group; *p<0.05; ***p<0.001, unpaired t-test). (C) Mortality between day 0 and day 1 after tMCAO (n=30-39 mice per group; *p<0.05, Fisher's test). WT, heterozygous (C2βWT/D1212A) and C2βD1212A/D1212A mice were subjected to tMCAO (1h ischemia followed by 24h reperfusion). (D) Neurological scores evaluated 24 hours after reperfusion by Bederson test (left panel) and Grip test (right panel) based on a five point system. Data represent mean ± SEM (n=14-27 mice per group; *p<0.05, unpaired t-test). E WT and C2βD1212A/D1212A mice were subjected to thromboembolic stroke model. Representatives T2-weighted MRI of coronal section taken 24 hours after the onset of in situ clot formation by α-thrombin were shown. Graph quantification of infarct volume measurements. Data represent mean ± SEM (n=23-25 mice per group; **p<0.01, Unpaired t-test).
Figure 7A. In vitro pull-down assay of hTrmt13 with USF1. USF1 with an N-terminal GST tag (GST-USF1) co-purified by hTrmt13 with a C-terminal His tag (hTrmt13-His) using Ni-NTA resin. GST-USF1 and hTrmt13-His were expressed separately in E. coli BL21. B. The gel filtration chromatography of hTrmt13-His or USF1-GST or their mixture on Superdex 200 Increase. C. The binding shift assays of hTrmt13, USF1, and their mixture for synthesized DNA from the TGFB1 promoter. In the reaction system, 10 ng/μL DNA was used, and 1 μM hTrmt13 or 1 μM USF1 or a mixture (0.5 μM hTrmt13 + 0.5 μM USF1) was applied. D. The binding shift assays of hTrmt13 for HctRNAGly(GCC) with the presence of various concentrations of USF1. In the reaction system, 0.3 μM HctRNAGly(GCC) and 0.5 μM hTrmt13 were used, and a varied amount of USF1 (0, 0.5, 1, 2, 3, 4 μM) were used. E. The binding shift assays of hTrmt13 (0.5 μM), or USF1 (0.5 μM) or the hTrmt13-USF1 complex (0.5 μM) for both tRNA (0.1 μM) and DNA (15 ng/μL) in the same reaction. F. A proposed model based on the above results showing that standalone hTrmt13 binds either to tRNA or synthesized DNA, while the complex of hTrmt13 and TFs (e.g., USF1) excludes the binding with tRNA but not DNA.