EMBO/sd-character-level-ner huggingface dataset:
Figure 2(A) Principal component analysis (PCA) of control (CTNSWT), CRISPR-generated cystinotic (CTNS-/-), and patient-derived cystinotic (CTNSPatient) cells based on the metabolites measured. Each dot represents one sample, and the dots of the same colour are biological replicates (n = 3). (B) Heatmap analysis of top 25 metabolites distinctively expressed in control and CTNS-/- cells. Metabolites significantly decreased (P < 0.05) were displayed in green, while metabolites significantly increased (P < 0.05) were displayed in red (n = 3). (C) Global test pathway enrichment analysis of the intracellular metabolic interactions distinctively affected in CTNS-/- cells compared to healthy control cells (n = 3). Larger circles further from the y-axis and orange-red colour show higher impact of pathway. (D) List of metabolites that were shared and significantly altered in both the CTNS-/- and CTNSPatient cells compared to control cells (n = 6). (E) Plasma levels of αKG (µM) in healthy individuals (n=4) and cystinotic patients (n=6). All cystinotic patients were on cysteamine treatment at the time of blood sampling. For this experiment, non-parametric Mann-Whitney t-test was used to demonstrate the significance. (F) Principal component analysis (PCA) of the measured proteins in CTNSWT, CTNS-/-, and CTNSPatient (n = 3). (G) Volcano plot illustrates differentially abundant proteins (n = 3). The -log10 (Benjamini-Hochberg corrected P-value) is plotted against the log2 (fold change: CTNSWT/CTNS-/-). (H) Gene ontology (GO) analysis illustrates classes of proteins differing between CTNSWT and CTNS-/- cells (n = 3). (I) List of proteins that were significantly upregulated and downregulated in CTNS-/- cells compared to control cells (n = 3). AKGDH; Alpha-ketoglutarate dehydrogenase, LIPA; Lysosomal acid lipase, ACP2; Lysosomal acid phosphatase, CTSS; Cathepsin S, CTSC; Dipeptidyl peptidase, IGF2R; Cation-independent mannose-6-phosphate receptor, SORT1; Sortilin, CAT; Catalase, CTSA; Lysosomal protective protein, and SOD; Superoxide dismutase.
| IgD BCR expression requires Pten A, B | Splenocytes from mice of the indicated genotypes, (A) Ptenf/f, Ptenf/f x mb1-cre and Ptenf/f x mb1-cre 3-83ki mice on the respective backgrounds, (B) Ptenf/f x MD4tg and Ptenf/f x mb1-cre x MD4tg mice in presence and absence of antigen (ML5tg), were analyzed by flow cytometry.Figure 3Splenocytes from mice of the indicated genotypes, (A) Ptenf/f, Ptenf/f x mb1-cre and Ptenf/f x mb1-cre 3-83ki mice on the respective backgrounds were analyzed by flow cytometry. B cells were pre-gated by B220 and CD19 expression and the IgM/IgD surface expression (dot plots, left) was determined. Histograms (right) show a comparison of intracellular IgD expression (Ig-δHC ic) between the splenic populations of B cells (orange, identified by B220 and CD19 expression) and non-B cells (gray). Numbers in the histograms indicate the percentages of the positive populations. Data are representative of 11 - 35 individual mice per genotype. (B) Ptenf/f x MD4tg and Ptenf/f x mb1-cre x MD4tg mice in presence and absence of antigen (ML5tg), were analyzed by flow cytometry. B cells were pre-gated by B220 and CD19 expression and the IgM/IgD surface expression (dot plots, left) was determined. Histograms (right) show a comparison of intracellular IgD expression (Ig-δHC ic) between the splenic populations of B cells (orange, identified by B220 and CD19 expression) and non-B cells (gray). Numbers in the histograms indicate the percentages of the positive populations. Data are representative of 11 - 35 individual mice per genotype. C | Quantification of surface IgD mean fluorescence intensity (MFI) in Ptenf/f x MD4-trangenic mice in presence (mb1-cre +/+) and absence (mb1-cre +/ki) of Pten. Single dots represent the average MFI of 4 independent measurements per mouse (n), mean ±SD. Statistical significance was calculated by using an unpaired two-tailed t-test. D | Serum anti-HEL titers. Sera from MD4tg mice of the indicated genotypes were adjusted to an IgM concentration of 500 μg/ml and applied in triplicates to HEL-coated plates in dilution steps of 1 : 3, mean ±SEM. Statistical significance was calculated by using the Kruskal-Wallis test (see also Table S4). Statements of significance in the figure refer to the comparison between Ptenf/f x MD4tg (filled red squares) and Ptenf/f x MD4tg x MD5tg sera (filled orange triangles), ** p ≤ 0.01; *** p ≤ 0.001. E | Immunohistochemistry of sections from spleens of Ptenf/f, Ptenf/f x mb1-cre, Ptenf/f x mb1-cre x 3-83ki and Ptenf/f x mb1-cre x MD4tg mice for CD169 (green), CD5 (red) and IgM (cyan) at 10x magnification. Pictures in the second row show enlarged areas indicated by the white squares, respectively. Double-arrows indicate the distance between T cell (red) and marginal zone (green). Shown pictures are representative of 2 - 3 mice per genotype
Figure 1A. SDS-PAGE analysis of purified T4SS3-10+D4 complex. Left panel: Coomassie-stained SDS-PAGE gel of the T4SS3-10+D4 complex. Right panel: Sypro Ruby-stained SDS-PAGE gel of the T4SS3-10m and T4SS3-10+D4 complexes. Molecular weights of MW markers are shown on the left. Identification of bands is shown in the middle. "*" indicates TrwK/VirB4 degradation products. "**" indicates minor contaminants OmpA and OmpC. B. Stoichiometry measurement. Fluorescence scan of an SDS-PAGE gel analysis of the T4SS3-10+D4 complex where cysteine residues were reacted with AlexaFluor 633 C5 maleimide. Signals corresponding to the proteins coupled to AlexaFluor 633 were detected at a wavelength of 633 nm. Fluorescent Trw protein bands are labelled and the derived stoichiometry for TrwB/VirD4 is indicated. C. SDS-PAGE analysis of complexes purified from cell expressing deletion mutants of the pBADM11_trwN/virB1-trwE/virB10Strep_trwD/virB11_HistrwB/virD4 constructs termed pBADM11_ΔtrwX/ΔvirBY (see main text). The same molecular weights marker has been used on this gel as in the gel presented in Figure 1A. * indicates degradation products of TrwK/VirB4.
Figure 5(A) Representative images of root explants from WT, atg2-2 and atg5-1 in CIM (6 days) or CIM + SIM (6 + 21 days). Scale bar: 2 cm (B) Quantification (%) of explants presenting shoots after 21 days after incubation on SIM. Results were obtained from 3 independent experiments with at least 20 calli per condition. Bars marked with an asterisk (*) are statistically significant (P<0.05) according to the T -test.
Figure 2qRT-PCR analysis showing the relative expression of CBF1 in 4-d-old wild-type (Col) seedlings grown at 22°C in darkness (D) or continuous W, R, FR, and B light. Error bars represent SD of three technical replicates. Different letters represent significant differences by one-way ANOVA with Duncan's post hoc test (P < 0.05). GUS staining of 4-d-old homozygous CBF1p:GUS transgenic seedlings grown at 22°C in D or continuous W, R, FR, and B light. Scale bar = 1 mm. qRT-PCR data showing that CBF1 expression is induced by different light regimes. Wild-type (Col) seedlings were first grown at 22°C in D for 4 d, and then transferred to continuous W, FR, R, or B light for the indicated times ranging from 1 h to 24 h. Error bars represent SD of three technical replicates. Different letters represent significant differences by one-way ANOVA with Duncan's post hoc test (P < 0.05). Immunoblots showing CBF1 protein levels in nuclear fractions from 4-d-old wild-type (Col) seedlings grown at 22°C in D or continuous W, R, FR, and B light. Anti-H3 was used as a sample loading control. Numbers below the immunoblots indicate the relative band intensities of CBF1 normalized to those of loading control, respectively. The ratio of the first clear band was set to 100 for each blot. Immunoblots showing CBF1-myc protein levels in 4-d-old CBF1-myc seedlings grown at 22°C in D or continuous W, R, FR, and B light. anti-HSP was used as a sample loading control. Numbers below the immunoblots indicate the relative band intensities of CBF1-myc normalized to those of loading control, respectively. The ratio of the first clear band was set to 100 for each blot. F, G Immunoblots showing the levels of CBF1-myc proteins after exposure to white light. CBF1-myc seedlings were first grown at 22°C for 4 d in D, and then transferred to continuous W light for the indicated time periods within 1 h (F) or ranging from 1 h to 24 h (G). anti-HSP was used as a sample loading control. Numbers below the immunoblots indicate the relative band intensities of CBF1 or CBF1-myc normalized to those of loading control, respectively. The ratio of the first clear band was set to 100 for each blot. Immunoblots showing the levels of CBF1-myc proteins after exposure to FR (H), R (I) or B (J) light. CBF1-myc seedlings were first grown at 22°C for 4 d in D, and then transferred to continuous FR, R or B light for the indicated time periods ranging from 1 h to 24 h. anti-HSP was used as a sample loading control. Numbers below the immunoblots indicate the relative band intensities of CBF1 or CBF1-myc normalized to those of loading control, respectively. The ratio of the first clear band was set to 100 for each blot.
BACE1 inhibition in AppNL-G-F mice by AAV-VHH-B9 delivery significantly reduces neuroinflammation.Figure 4(A) (left) Representative images of anti-GFAP immunostaining in Prefrontal Cortex and Amygdala from AppNL-G-F mice receiving either AAV-VHH-B9 or AAV-GFP; (right) quantification of the GFAP+ cell number in the indicated brain regions across experimental cohorts. (B) (left) Representative images of anti-IBA1 immunostaining in dentate gyrus and amygdala from AppNL-G-F mice receiving either AAV-VHH-B9 or AAV-GFP; (right) quantification of the IBA1+ cell number in the indicated brain regions across experimental cohorts.
Figure 6Lethally irradiated WT mice were reconstituted with BM from Lyzs-Cre (Cx BMLyzs-Cre) or p38γ/δLyzs-KOmice (Cx BMp38γ/δLyzs-KO). Two months after the transplant, mice were fed the HFD for 10 weeks. (A) Freshly prepared CD45.2 whole BM mononuclear cells (2 × 107) were transplanted into lethally irradiated B6.SJL (CD45.1) mice, and engraftment by CD45.2 cells (%) was analyzed by antibody staining and FACS of peripheral blood and liverCD45+ cells. Charts show CD45.1 and CD45.2 expression in blood cells (left) and liver cells (right) isolated from transplanted mice (n = 3). Representative FACS dot plots of CD45.1 and CD45.2 expression are shown beneath the charts. Lethally irradiated WT mice were reconstituted with BM from Lyzs-Cre (Cx BM Lyzs-Cre) or p38γ/δLyzs-KO mice (Cx BM p38γ/δLyzs-KO). Two months after the transplant, mice were fed the HFD for 10 weeks. (B) Representative H&amp;amp;E and oil red-stained liver sections. Scale bar: 50 μm. Lethally irradiated WT mice were reconstituted with BM from Lyzs-Cre (Cx BMLyzs-Cre) or p38γ/δLyzs-KOmice (Cx BMp38γ/δLyzs-KO). Two months after the transplant, mice were fed the HFD for 10 weeks. (C) Livertriglyceride content. Lethally irradiated WT mice were reconstituted with BM from Lyzs-Cre (Cx BM Lyzs-Cre) or p38γ/δLyzs-KO mice (Cx BM p38γ/δLyzs-KO). Two months after the transplant, mice were fed the HFD for 10 weeks. (D) Plasma transaminase ALT activity. Lethally irradiated WT mice were reconstituted with BM from Lyzs-Cre (Cx BMLyzs-Cre) or p38γ/δLyzs-KOmice (Cx BMp38γ/δLyzs-KO). Two months after the transplant, mice were fed the HFD for 10 weeks. (E) Fasted glucose, detected at the end of the diet period in overnight-fasted mice. Data are means ± SEM. (n=5-10) *P<0.05; **P<0.01; ***P<0.001 (2-way ANOVA coupled to Bonferroni's post tests).
Ref(2)P is a component of protein aggregates in flies defective in autophagy and D.Figure 4.(A and B) Protein aggregates in atg8aKG07569 mutants exhibit sequestosome morphology and are positively labeled for Ref(2) P (A) and ubiquitin (B). Protein aggregates are surrounded by small electron-dense vesicles (arrows). (C) Western blot analysis of cell lysates of wild type and atg8aKG07569 young adult heads demonstrating a significant increase of Ref(2) P protein levels in atg8aKG07569 flies. (D, left) Ref(2) P protein migration profile in ref(2) P loss-of-function alleles. Ref(2) P mutant protein for the PB1 domain has an apparent molecular mass of 85 kD (ref(2) Pod2/ref(2)Pod2) and the Ref(2) P mutant protein for the UBA domain has an apparent molecular mass of 69 kD (ref(2) Pod3/ref(2)Pod3). (D, right) Western blot analysis of insoluble protein fractions of adult heads of double mutants for atg8a and ref(2) P probed for ubiquitin. Genotypes: (1) atg8aKG07569/Y; ref(2) Pod2/Cyo, (2) atg8aKG07569/Y; ref(2) Pod2/ref(2) Pod2, (3) atg8aKG07569/Y; ref(2) Pod3/CyO, and (4) atg8aKG07569/Y; ref(2) Pod3/ref(2) Pod3. The insoluble ubiquitinated protein profile of the double mutants is diminished compared with the one of the respective control heterozygous for ref(2) P. (E and F) Neuronal cells mutant for atg1 (green) accumulate Ref(2) P- (E) and ubiquitin-positive (F) structures (arrows), whereas the normal surrounding cells do not exhibit positive staining. Genotypes: (E and F) yw/UAS-CD8-GFP, hs-flp; tubGal4/+; atg1Δ3D, FRT80/tubGal80, FRT80 (G) Confocal micrographs of the optic lobe (medulla region) of young flies expressing a mutated form of human tau protein exhibiting Ref(2) P- and tau-positive aggregates (arrows). Genotypes: (G) elav/+;UAS-E14/+.
Figure 2A Amino acid sequences of PR8 PB1-F2, 1918 PB1-F2, and the cloned PB1-F2 mutants. B-D A549 cells were transfected with the indicated plasmids encoding Flag-tagged PB1-F2 derived from the PR8 or 1918 strain and chimeric PB1-F2 mutants. At 18 h after transfection, cells were treated with or without MG132 for 6 h. The expression of PB1-F2 and control genes was detected by semi-quantitative RT-PCR (upper panel) or Western blotting (lower panel). Results shown are representative of three independent experiments. E Representative immunofluorescence images of PB1-F2 expression in A549 cells. Cells transfected with the Flag-tagged PB1-F2 plasmid were stained with anti-Flag (red) antibody and analyzed by immunofluorescence assay. Magnification, ×400; scale bar, 50 μm.
Tollip and Parkin are in complex in a CUE domain-dependent manner, but independent of autophagy and Tom1.Figure 3(A) HeLa cells expressing mycBioID-Tollip and HA-Parkin were either left untreated or treated with 5 μM antimycin A/10 μM oligomycin B (AO) or AO/100 μM bafilomycin A (BfnA1) for 6 hours in the presence of biotin. Cells were lysed and streptavidin pulldowns performed overnight to isolate biotinylated proteins. Proteins in whole cell extracts and pulldowns from each condition were then separated by SDS-PAGE and membranes probed with specific antibodies. Immunoblotting for Tom1 and mycBioID-Tollip were used as positive controls. Cells cultured in media lacking biotin were used as a negative control to assess background levels. Biotinylation of Parkin suggested Tollip specifically interacted with Parkin under these conditions. (B) HeLa cells expressing mycBioID alone and HA-Parkin were used to confirm the specificity of the Tollip-Parkin interaction. (C) Hela cells co-expressing GFP-Tollip and HA-Parkin were either left untreated or treated with AO for 2 hours prior to lysis. Cell lysates were subjected to either a normal rabbit IgG IP or HA IP and western blot analysis was performed using antibodies against Parkin and GFP. (D) HeLa cells expressing mycBioID-Tollip containing the CUE domain mutation (CUEmut) and HA-Parkin were subjected to biotinylation and streptavidin pulldowns, followed by western blot analysis. (E) HeLa wild-type (WT), Tom1 KO and ATG5 KO cells expressing mycBioID-Tollip and HA-Parkin underwent the same conditions as those above and immunoblotted for the indicated proteins.
Figure 4(A) A summary of significantly different plasma inflammatory protein markers compared to time baseline in metabolic cofactor supplementation and control studies. Red and blue colors denote the increased and decreased plasma levels compared to baseline, respectively. * Denotes significance (one-way ANOVA; FDR <0 .05). (B) Proteins significantly different between supplementation and control studies. Red and blue colors denote the increased and decreased plasma level in supplementation study compared to control study, respectively. * Denotes significance (one-way ANOVA; FDR <0 .05). (C-F) Changes in the plasma level of key proteins compared to time baseline based on untargeted metabolomics measurement in supplementation and control studies. The grey shaded area represents the 95% confidence level interval. For boxplots limits, the middle line represents the median. The upper and lower box limits represent the 25% quantiles. The upper and lower error bars correspond to 75% quantiles. The p-values are derived from one-way ANOVA (FDR< 0.05).
Figure 6A Comparison of P-body RNA and total RNA levels with and without AKT and PIM inhibitor treatment by anota2Seq. B Specific RNA subtypes found in P-body RNAs purified from DMSO treated GFP-EDC3 containing PC3-LN4 cells.
Spreading of K18-ATTO 594 fibrils in CAD cells.Figure 1(A) Transfer of K18-ATTO 594 fibrils from donor cells to H2B-GFP-expressing acceptor cells. Representative confocal images of each population are in the upper panels, below are pictures after 24 hours of co-culture of the two populations (left), and of acceptor cells treated with conditioned medium from donor cells for 24 hours (SN, right). In white is the cell membrane labelling with WGA (wheat germ agglutinin) coupled to Alexa 647. The arrows point to acceptor cells containing fibrils, scale bars are 10 μm. (B) Quantification by flow cytometry of the percentage of K18-ATTO 594-positive acceptor cells after co-culturing donor and acceptor cells (total), or culturing acceptor cells with donor-conditioned medium for 24 hours (secretion). The total transfer is arbitrarily set at 100%, and cell-cell contact transfer is calculated by subtracting secretion transfer from total transfer. Data represent the means (+SD) of 4 independent experiments, with statistical analysis by two-tailed unpaired t-test (****P = 4.64E-08). (C) Quantification by flow cytometry of the relative percentage of K18-ATTO 594-positive acceptor cells upon treatment with CK666 during the co-culture. Left, data represent the means (132%) +SD, normalized to non-treated co-culture arbitrarily set at 100%, of 3 independent experiments, with statistical analysis by two-tailed unpaired t-test (*P = 0.015). The hatched area of each bar represents the part due to secretion (respectively 4.8 and 4.3% in the absence and presence of CK666). Right is the same analysis when the cells were cultured in sparse conditions, not allowing cell-to-cell contacts (mean= 47.7%, 3 independent experiments, *P = 0.020). (D) Representative confocal images (40x) of CAD cells treated with 1 µM K18-ATTO 594 fibrils, 24h after fibrils addition, fixed and stained with WGA-Alexa 488 (green) and DAPI (blue in the merge panels). Bottom panels are a bottom slice corresponding to the substrate-attached surface of cells (z3), upper panels correspond to slice 10 of the same picture, (z10), not attached to the substrate). On the right and below z10 pictures, are the orthogonal views (xz and yz) of the same region covering 27 slices over 11 μm in total. The arrows point to red fibrils into a WGA-positive TNT. Scale bars are 10 μm.
BC network dilation is accompanied by increased apical myosin levels during regeneration A, C Fluorescence stainings for F-actin (a) or pMLC (c) and the apical marker CD13 in the PV area on liver tissue sections from untreated mice or animals at indicated time points post PH.Figure 2Fluorescence stainings for F-actin and the apical marker CD13 in the PV area on liver tissue sections from untreated mice or animals at indicated time points post PH. Arrows indicate BC. Scale bar, 5 µm Quantification of apical F-actin intensity from images as representatively shown in (a, as well as Appendix Fig. S2A,B, at indicated time points post PH (solid line) or sham OP (dashed line). Data is normalized to untreated animals (timepoint 0). Mean ± s.e.m, n=3-5 (b) mice per timepoint. Fluorescence stainings for pMLC and the apical marker CD13 in the PV area on liver tissue sections from untreated mice or animals at indicated time points post PH. Arrows indicate BC. Scale bar, 5 µm Quantification of pMLC (d) intensity from images as representatively shown in (a,c) as well as Appendix Fig. S2A,B, at indicated time points post PH (solid line) or sham OP (dashed line). Data is normalized to untreated animals (timepoint 0). Mean ± s.e.m, n=2-5 (d) mice per timepoint.
a, HeLa cells transfected with control siRNA (siCTL) or siCaspase 8 were stimulated with 2.f2a, HeLa cells transfected with control siRNA (siCTL) or siCaspase 8 were stimulated with 2.0 μM staurosporine or 20 ng ml−1 TNF + 10 μg ml−1 CHX. b, HeLa cells transfected with control siRNA or siCaspase 9 were stimulated as in a. c, HeLa cells transfected with control, caspase-3- or caspase-7-specific siRNAs were stimulated with 20 ng ml−1 TNF + 10 &amp;amp;mgr;g ml−1 CHX. d, Wild-type or caspase-3-knockout macrophages were stimulated with 2.0 µM staurosporine or 20 ng ml−1 TNF + 10 µg ml−1 CHX. Data in a-c represent 2 independent experiments; data in d represent 4 independent experiments.
Fig.Figure 3(A-D) 3D views of representative 232 x 232 μm2 (A, B) and 155 x 155 μm2 (C, D) fields of view from confocal imaging of an uninfected control (A), and infected chips without macrophages at days 2 (B) and 3 (C, D) post-infection, respectively. Actin and nuclear labelling are shown in azure and indigo, respectively. (B) At 2 dpi, adjacent areas of endothelial cell clusters identified by a stronger nuclear stain and brighter actin staining (yellow arrows) and cells with increased permeability and loss of confluence (white arrows) are indicated. (C) At 3 dpi, a significant loss of barrier integrity is observed in the endothelial layer, whereas the AT layer directly above this field of view (D) is relatively intact. A small area of missing cells in the AT layer is indicated with a white arrow in (D). (E, F) Plots of density of nuclei (number of nuclei per 1E4 μm2) on the AT (E) and endothelial (F) faces of infected LoCs both with and without macrophages for characterization of the cellular layer integrity. The bars represent the mean value, the solid line represents the median and the error bars represent the standard deviation from at least 4 fields of view each from two independent LoCs for each timepoint. Data from uninfected controls corresponding to the 3 dpi timepoint are labelled '3C'. Data information: P-values are calculated using a one-way Kruskal-Wallis ANOVA test, * represents p≤0.05, ** represents p≤0.01, ** represents p≤0.001. (G-J) The fields of view in (C, D) are shown also in (G, H) respectively, SARS-CoV-2 spike (S) proteins labelled via immunofluorescence are false-colored in amber. Zooms in (I) and (J) show clear evidence of intracellular spike protein within both endothelial and ATs, respectively. (K) A 3D view of the field of views in (C, D) and (G, H) respectively highlights the relative damage to the endothelial layer versus the epithelium, as well as the basolateral transmission of S protein via junctions in the pores of the membrane (identified with yellow arrows). The white arrows define the XYZ axes.