EMBO/sd-character-level-ner huggingface dataset:
Lipid-triggered folding of the amphipathic helix of CD-loop peptides and its requirement for vacuole scission.Figure 4a, CD loop folding in hydrophobic buffer. Peptides were synthesized that correspond to the predicted amphipathic helix from S. cerevisiae Atg18 (TRLAREPYVDASRKTMGRMIRYSSQ) or to a sequence with reduced tendency to form amphipathic helices, generated by a swap of two amino acid pairs (indicated by arrows; SLoop). Helical wheel projections of the sequences are shown. b,c Their secondary structure was analyzed by circular dichroism spectroscopy in the presence of increasing concentrations of hexafluoro-2-propanol (HFIP), in the presence of 6 mM small unilamellar vesicles (SUVs, 70% egg PC and 30 % PS, total phospholipid concentration approx. 6 mM), or of or control buffer only (blue). Spectra are shown for b, the wildtype peptide b,c Their secondary structure was analyzed by circular dichroism spectroscopy in the presence of increasing concentrations of hexafluoro-2-propanol (HFIP), in the presence of 6 mM small unilamellar vesicles (SUVs, 70% egg PC and 30 % PS, total phospholipid concentration approx. 6 mM), or of or control buffer only (blue). Spectra are shown for c, the SLoop peptide. d, Vacuole morphology of cells expressing Atg18Sloop-GFP, imaged before and after osmotic shock as in Fig 1a. Scale bar: 5 µm. e, Number of vacuoles per cell in d, assayed as in Figure 1b. n=3. f, Vacuole morphology of cells expressing Atg18DLoop-GFP, imaged as in d. g, Number of vacuoles per cell in e, assayed as in Fig. 1b. n=3.
FIP3 silencing enhances IL2 production.Figure 8A: Jurkat T cells transfected with control or FIP3.1 were activated on anti-CD3-coated plastic plates in the presence of soluble anti-CD28 during 16 h. Concentration of IL2 released in the supernatant was measured by ELISA. Histograms represent the mean ± SD of three experiments. Mann-Whitney test. B: T cells were activated with soluble anti-CD3 and Erk1/2 phosphorylation was analyzed by Western blot. Graph represents the percentage of each band intensity with respect to that of cells transfected with control siRNA and left non-stimulated (t=0). The intensity each band from Western blot was normalized with respect to the intensity of β-actin for each time point. Representative experiment out of three performed.
Figure 2A summary bar graph showing the quantitative real-time PCR data for TMEM16A, TMEM16B, and BEST1 with cDNA samples prepared from the mHb and hippocampus of adult mouse brains (N=3, n=3 each, unpaired Student t-test, data are presented as mean values ± s.e.m.). RNAscope ISH showed an apparent expression of TMEM16A mRNA co-localized with ChAT and Tac1 signals in the mHb compared to the ones in lHb. Enlarged images showed that signals of TMEM16A and Tac1 in the dorsal mHb or ChAT in the ventral mHb reside together at the single cell level. Immunohistochemical staining of 40-µm coronal brain sections with anti-TMEM16A antibody. In the mHb, TMEM16A signals colocalize with the cholinergic marker ChAT (arrows). High magnifications of the indicated areas are shown in the merged panel.
Figure 5Urea production from each group was determined by a commercially available urea assay kit. GC-MS analyses of the major metabolites of Urea cycle in SW-1116-Vector, SW-1116-LOC113230 or SW-1116-shASS1 cells, respectively. Enzymatic activity of ASS1 was examined by di-phosphate release from the argininosuccinate synthesis reaction. whole cell extracts prepared from SW-1116 cells expressing LOC113230, shLOC113230, or vector were subjected for in vitro ASS1 enzymatic assays, and purified enzymatic dead mutant ASS1 K165Q and ASS1 K176Q proteins were used as controls. Arginine production from each group was determined by a commercially available Arginine assay kit. The cells were stimulated with 5 ng/ml TGF-β or DMSO for 24 hrs. Urea production for each group was determined by a commercially available urea assay kit. The cells were stimulated with 5 ng/ml TGF-β or DMSO for 24 hrs.
Figure 3Depletion of Siwi results in segregation of BmQin from BmDcp2-containing P-bodies. Box plot showing that depletion of Siwi (dsSiwi, n=47 cells) reduces colocalization ratio between BmQin and BmDcp2, compared to control (dsRLuc, n=55 cells). Representative data from N = 4 independent experiments are shown. Depletion of Siwi results in segregation of BmSpnE from BmDcp2-containing P-bodies. Box plot showing that depletion of Siwi (dsSiwi, n=55 cells) reduces colocalization ratio between BmSpnE and BmDcp2, compared to control (dsRLuc, n=55 cells). Representative data from N = 6 independent experiments are shown. Depletion of Siwi results in colocalization of BmSpnE and BmVasa. Box plot showing that depletion of Siwi (dsSiwi, n=18 cells) significantly up-regulates colocalization ratio between BmSpnE and BmVasa, compared to control (dsRLuc, n=18 cells). Representative data from N = 3 independent experiments are shown. Depletion of BmVasa results in segregation of Siwi from BmAgo3 foci. Box plot showing that depletion of BmVasa (dsBmVasa, n=12 cells) down-regulates colocalization ratio between Siwi and BmAgo3, compared to control (dsRLuc, n=12 cells). Data points collected from N = 2 independent experiments are shown (statistical test is not performed for N < 3 data). Depletion of BmVasa results in entanglement of Siwi at piP-bodies. Interaction interfaces are indicated by white arrows. Box plot showing that depletion of BmVasa (dsBmVasa, n=45 cells) mildly up-regulates colocalization ratio between Siwi and BmDcp2, compared to control (dsRLuc, n=49 cells). Representative data from N = 5 independent experiments are shown.
Figure 5A. Immunofluorescence images of Sec24C and TRAPPC2 in vehicle-treated and SNS-032-treated (1 µM, 3 h) cells. Graphs show the quantification of Sec24C and TRAPPC2 at ERES (mean intensity) normalized in SNS-032-treated cells relative to vehicle-treated cells (set as 100%). Mean ± s.e.m. of three independent experiments. **p<0.05, ****p<0.0001, Student's unpaired two-tailed t-test. Data information: Scale bars, 10 µm. B. HeLa cells were treated with Dinaciclib (1 µM, 3 h) and permeabilized or not with digitonin A GM130 antibody was added to the buffer of living cells to monitor permeabilization efficiency. Upper panels, non-permeabilized (NP) control cells; middle panels, permeabilized control cells; lower panels, permeabilized Dinaciclib-treated cells. Data information: Scale bars, 10 µm. C. Quantification of Sec24C membrane association after CDK inhibitor treatment. Digitonin-permeabilized cells treated with vehicle (CTRL) or the indicated CDK inhibitor (1 µM, 3 h) were immunostained for Sec24C. The mean intensity of Sec24C in the perinuclear area normalized for the cytosolic Sec24C signal in drug-treated cells is expressed as fold change compared to the control; n= 60-80; mean ± s.e.m. of three independent experiments ****p<0.0001, One-way ANOVA with Dunnett's multiple comparison test. D. HeLa cells transfected with HA-hnRNPK were left untreated (Steady state) or treated with SA (300µM, 30 min) or SA+Dinaciclib (10 µM, 150 min) and then immunostained with anti-HA and anti-G3BP antibodies. Data information Scale bars, 10 µm. E. TRAPPC2 interacts with hnRNPK. (Top) SDS-PAGE and Western blot analysis of endogenous hnRNPK after immunoprecipitation with an anti-TRAPPC2 Ab or IgG, and (bottom) after immunoprecipitation with an anti-HA Ab from cells transfected (+) or not (-) with hnRNPK-HA detected with the indicated antibodies. F. Mock or hnRNPK KD cells treated with SA were immunostained for TRAPPC2 and eIF3. Data information: Scale bars, 10 µm. G. Mock or hnRNPK KD cells treated with SA were immunostained for Sec24C and G3BP. Data information: Scale bars, 10 µm. H. Graphs show quantification of TRAPPC2 and Sec24C redistribution to SGs in mock treated (CTRL) and hnRNPK KD cells. n>100, N=3 mean ± s.d. ***p<0.001.****p<0.0001, Student's unpaired two-tailed t-test.
Figure 1A) Volcano plot of the fold-change (x-axis) plotted against significance (y-axis) of lipids changing upon HF/HS feeding. Lipids are colored according to fold-change (log2, absolute) > 1 (orange), p-value <0.05 (red) or both (green). Pvalues calculated from differential expression using limma B) Heatmap of the fold-change (log2) of each lipid in HF/HS compared to chow diet. Only lipid species detected in all mice are shown. C) Examples of different hepatic lipids within one class that are regulated in different directions in HF/HS fed as compared chow fed mice.
Postnatal inactivation of PR-SET7 in hepatocytes leads to cell death.Figure 2A Macroscopic appearance of livers in 120-day-old (P120) wild-type (WT) and PR-SET7loxp/Alb-Cre (KO) mice. Note, small adenomatous foci in KO livers. B Representative hematoxylin and eosin staining of liver sections from P120 wild-type (WT) and PR-SET7loxp/Alb-Cre (PR-SET7ΔHepA) mice. Arrows show three areas containing morphologically different hepatocytes. Right panels: zoom-in to Area-A-'normal zone', to Area-B-'necrotic zone' and to Area-C-'regenerative zone'. C Immunohistological staining of liver sections from P120 PR-SET7ΔHepA mice and control littermates (WT) with HNF4 antibody. D TUNEL staining of liver sections from P120 PR-SET7ΔHepA mice and control littermates (WT). Note that cells containing enlarged nuclei (white arrows) are TUNEL negative. E Immunohistological staining with γH2AX and albumin (Alb) antibodies.
Figure 4 (A) Graphical view showing SOX9 and SOX10 expression among spinal cord cells analyzed with scRNA-seq  (B) Bar graph shows enrichment of genes bound by SOX10 in rat oligodendrocytes within the differentially expressed gene sets. Yellow bar represents enrichment within NPC/GPC genes, green bar within neuronal genes, blue bar within astrocytic genes and red bar within oligodendrocytic genes. SOX10 ChIP-seq data from [37 7] (C) Venn-diagram shows 2622 SOX9 bound sites in GPCs, which are not co-targeted by SOX3, and their overlap with SOX10 bound sites in mature oligodendrocytes. Bar graph shows that mostly oligodendrocyte-specific genes are sequentially bound by SOX9 in GPCs and by SOX10 in oligodendrocytes. However, SOX9 and SOX10 also bind genes that are selectively expressed in astrocytes. (D) ChIP-seq peak graphics around the oligodendrocyte gene Plekhb1. ChIP-seq peaks are derived from four different experiments; SOX3 ChIP-seq in NPCs, SOX3 ChIP-seq in GPCs, SOX9 ChIP-seq in GPCs and SOX10 ChIP-seq in oligodendrocytes. Both ChIP-seq reads and called peak regions (underlying black lines) are shown for all data sets except for SOX10, for which only called peak regions are shown (black line). Triangle in the SOX10 data set indicate called peak. SOX10 ChIP raw reads could not be transferred from rn5 to mm9 assembly and therefore only SOX10 peak regions are shown in the figure. Scale bar: 5kb (E) Figure shows most strongly enriched DNA binding motifs within 3 specific peak sets; SOX3 and SOX9 bound regions around astrocyte specific genes, SOX9 and SOX10 bound regions around astrocyte specific genes, and SOX9 and SOX10 bound regions around oligodendrocyte specific genes (F) Expression of Nfia, Ehf and Zbtb3 among spinal cord cells analyzed with scRNA-seq. Red color indicates cells with RPKM values above 55 for Nfia, 5 for Zbtb3 and 5 for Ehf. n=3 for SOX9 and 2 for SOX3. P-values (phyper, R) were calculated from the total number of genes for mm10 (23´389)
Figure 4ChIP analysis of NF45 and NF90 binding to the H42.9 loci in CD3+ T cells stimulated with P/I for 6 h. (n=3) qPCR analysis of pre-rRNA in CD3+ T cells treated with P/I for 6 h. (n=3) CD3+ T cells were first silenced with shNC, shNF45 or shNF90, and then treated with P/I to extract total RNA for qPCR analysis of pre-rRNA. (n=3) The analysis of rDNA-luc activity in Jurkat cells with or without NF90 silence upon P/I stimulation or not. (n=3) Confocal images of Jurkat cells to show NF45 (left panel) or NF90 (right panel) (green fluorescence) colocalized with NFATc2 (red fluorescence) in nucleoli. Scale bar, 10 μm. Co-IP analysis of NFATc2 and NFATc1 with NF90 in Jurkat cells treated with P/I. ChIP analysis of NFATc2 binding to the rDNA loci in CD3+ T cells stimulated with P/I for 6 h. (n=3) Jurkat cells co-transfected with rDNA-luc (wild type or mutant plasmid) with NF90 and/or NFATc2 for 24 h, then rDNA-luc activity was detected after P/I sitmulation or not (DMSO). (n=3) ChIP analysis of NFATc2 binding to the H42.9 loci in NF90-slienced CD3+ T cells stimulated with P/I for 6 h. (n=3)
Fig.Figure 2Experimental outline. IL-7 was injected intravenously into HSC-humanized NSG mice before 2x106 t.u. of CD8-LVCD19CAR (CAR), or PBS as control (PBS), were administered. Distinct symbols for each individual animal are used throughout B-F (M16: lilac triangle; M19: blue triangle). Open symbols indicate animals from the vector-injected group devoid of PCR-detectable CAR T cells. CAR T cell levels in the CD8+ and CD8- T cells harvested from blood, spleen and bone marrow of PBS (PBS) or vector-injected (CAR) mice determined by flow cytometry. Detection of CAR T cells by determining vector copy numbers (VCN) in genomic DNA isolated from CD8+ enriched cells harvested from bone marrow. Detection of CAR T cells in bone marrow quantified by flow cytometry. CD19+ B cell levels in blood, spleen and bone marrow determined by flow cytometry. Relative human CD19+ B cell level in blood calculated by normalizing the levels at the day the animals were sacrificed to those before vector administration.
Figure 1A) An illustration of spinal ligament tissues collection. (B) SOFG staining of spinal entheseal tissue and Immunohistochemical analysis of CaSR+ and Runx2+ cells in human spinal tissue. The bottom panels show higher magnification of the boxed area in the top panels. Data information: SP: spinous process; IL: interspinous ligament; SL: supraspinous ligament; UIL: uncalcified interspinous ligament; TZ: transitional zone. Data shown as mean ± SD.. ** P < 0.01 compared between groups. Scale bar: 100μm (C) Quantitative analysis of the number of CaSR+ and Runx2+ cells per area (mm2). n=5, Student's t-test. D) RT-qPCR analysis of expression of CaSR in human spinal tissue. n=3, Student's t-test. E) Western blot analysis of expression of CaSR in human spinal tissue. F) Immunofluorescence analysis of CaSR+ (Green) and Runx2+ (Red) cells in human spinal tissue.H) Immunofluorescence analysis of CaSR+ (Green) and OCN+ (Red) cells in human spinal tissue.
Residues in the Munc18-1 domain 3a essential for MUN-catalyzed transition from the Munc18-1/Syx1 complex to the SNARE complex.Figure 1A Structure overview of the Munc18-1/Syx1 complex (pdb entry: 3C98). Mutating residues are highlighted in red below the domain depiction and shown as yellow sticks in the structure. B Elution volumes (peak positions) of the Munc18-1 mutants in the absence (top) and presence (bottom) of Syx1 using size-exclusion chromatography on Superdex-200 column. C Effects of the domain 3a mutations on MUN-catalyzed transition detected by native-PAGE assay. Components addition was indicated on the top of every lane. D Quantitative analysis of the integrated densities of Munc18-1/Syx1 bands in C. Effects of the QKAA, KKEE and L348R mutations on MUN-catalyzed transition detected by FRET assay. Schematic diagram is shown on the top of the chart. FRET between BODIPY FL-labeled Syb2 (donor) and TMR-labeled SN25 (acceptor) was monitored. F Effects of the QKAA, KKEE and L348R mutations on lipid mixing between liposomes with Munc18-1/Syx1 and liposomes containing Syb2 in the presence of Munc13 (M13), SN25 and Syt1/Ca2+. Illustration of the reactions is shown on the top of the chart.
Figure 6a. Autophagy measured with the pho8∆60 assay in atg18 cells complemented with wt, FGGG, SLoop or DLoop alleles of ATG18. b. Same assay as in a, but in atg18 atg21 cells complemented with wt or SLoop versions of ATG18 and ATG21. Atg21SLoop contains K426F and F429K substitutions. Helical wheel projections for both Atg21 helices are given. The arrows indicate the magnitude of the hydrophobic moment. c. Vacuole fragmentation in atg2 cells, assayed 15 min after a moderate hypertonic shock as in Fig. 1. d. Number of vacuoles per cell in c, assayed as in Figure 1b. n=3.
GCN5 inhibits lysosomal biogenesis.Figure 2A LAMP1 puncta (green) and DAPI (blue) in WT and GCN5 KO HEK293 cells (Scale bars, 10 µm). B Fluorescence-activated cell sorting analysis of WT and GCN5 KO HEK293 cells stained with LysoTracker. Fluorescence intensity of 10,000 cells per sample was measured. C Immunoblot showing lysosomal protein levels in three independent clones of GCN5 KO HEK293 cells. CTSD HC, cathepsin D heavy chain. D LAMP1 puncta in GCN5 KO HEK293 cells overexpressing Myc-tagged GCN5 or GCN5-E575Q (Scale bars, 10 µm). E Quantification of LAMP1 puncta in (A) and (D) (Graph represents data from three independent experiments with ≥ 30 cells per condition; mean ± SEM; ***p < 0.001, Student's t-test). F Hexosaminidase activity in GCN5 KO HEK293 cells (mean ± SEM; n = 3 independent experiments; ***p < 0.001, Student's t-test). G Degradation of EGFR in WT and GCN5 KO HEK293 cells in the presence or absence of CQ. H Representative images of LysoTracker staining (red) and DAPI (blue) in Drosophila larval fat body in which dGcn5 is overexpressed (OE) or silenced (KD). Drosophila (cg-GAL4/+) was used as the control (Graph represents data from three independent experiments with ≥ 30 cells per condition; mean ± SEM; *p < 0.05, **p < 0.01, Student's t-test; Scale bars, 10 µm).
cAMP inhibits Hla-induced autophagy.Figure 1(A) GFP-LC3 CHO cells were preincubated with 1 mM dybutiryl cAMP (+dbcAMP, panels d, e, and f) for 30 min and then were treated for 4 h with 10 µg/ml of α-hemolysin (Hla) or subjected to starvation conditions (Stv) in the continuous presence of dbcAMP. Cells without any treatment were used as control (−dbcAMP, panels a, b, and c). Cells were immediately analyzed by confocal microscopy. A quantification of the percentage of cells presenting LC3 puncta (i.e., stimulated cells) upon incubation with the different conditions is shown in the right panel (n = 100 cells/condition, * p<0.05 paired Student's t-test). These data are representative of three independent experiments. (B) GFP-LC3 CHO cells were incubated with complete medium in the absence (−dbcAMP) or presence of dbcAMP (+dbcAMP) and treated for 4 h with 10 µg/ml of α-hemolysin (Hla) or subjected to starvation conditions (Stv) with (lower panel) or without (upper panel) bafilomycin A1 to block lysosomal degradation. Afterwards, cells were lysed with sample buffer and the samples were subjected to Western blot analysis using a rabbit anti-LC3 and the corresponding HRP-labeled secondary antibody, and subsequently developed with an enhanced chemiluminescence detection kit. These data are representative of three independent experiments. (C) The band intensities of two independent experiments were quantificated with the Adobe Photoshop program, and normalized against tubulin. * p<0.05 (paired Student's t-test).
Figure 4A, B SMD snapshots of NKG2D dissociation with MICA (A) and ULBP3 (B) in the presence of force (directions are indicated by black arrows). C The force vs. extension trajectories for the simulations are shown in (A) and (B). Occurrence of the sudden extension changes are indicated in the shaded area and time points correspond with the snapshots in (A) and (B) are marked. D, E Zoomed-in binding interfaces of NKG2D with MICA (D) or ULBP3 (E), corresponding with the configuration (shown as gray dashed box in (A) and (B), respectively). F Distance vs. time curves for force-induced H-bond formation between indicated residues within NKG2D-MICA binding interfaces. The dashed red lines represent H-bonds whose distances are less than 3.5 Å.
Figure 2A. HEK293T cells were transfected with MYC-FOXP3 and FLAG-USP44 encoding expression constructs using Polyethylenimine. 48hrs post-transfection, cells were harvested, lysed, and anti-FLAG or anti-MYC antibody coated beads were used to immunoprecipitate the given labeled protein along with its binding partner. Co-IP'ed proteins were subjected to SDS PAGE followed by immunoblot analysis. Antibodies recognizing FLAG or MYC tags were used to probe for USP44 and FOXP3, respectively. B. Endogenous co-IP of USP44 and FOXP3 in murine iTregs. iTregs were generated as in Fig. 1 from naïve CD4+ T cells FACS isolated from pooled suspensions of the lymph node and spleen cells of wild type C57BL/6 mice (n=2-3/experiment). iTregs were lysed and key proteins were immunoprecipitated using either anti-USP44 (right panel) or anti-FOXP3 (left panel) antibody. Proteins pulled-down in this experiment were then resolved and analyzed by immunoblot using anti-FOXP3 or anti-USP44 antibodies. C. Endogenous co-IP of USP44 and FOXP3 in murine nTregs. nTregs (CD4+CD25high) isolated by FACS were activated by anti-CD3 and anti-CD28 (1 and 4 ug/ml, respectively) overnight in the presence of IL-2 (100 U/ml). The cells were lysed and proteins were immunoprecipitated using either anti-Foxp3 (left panel) or anti-Usp44 (right panel). Proteins pulled down in this experiment were then resolved and identified with the indicated antibodies. D. Naïve murine CD4+ T cells were isolated by FACS from lymph node and spleen cell suspension of USP44fl/fl CD4Cre+ mice and that of their wild type littermates (USP44fl/fl CD4Cre- mice; n=2-3/group/experiment). iTreg cells were generated from these mice as described for Fig. 1 before incubation on a microscope slide pre-coated with poly-L lysine for 1h. Adhered cells were then fixed by PFA for 0.5 followed by blocking with 1% BSA for 1h, then incubation with the specified antibodies. Representative confocal microscopy images (40X) were visualized for endogenous USP44 (red) and FOXP3 Baxter et al(). DAPI was used to visualize cell nuclei (blue); scale bar 50μm.
Histidine deprivation inhibits nerfin-1 clones via Myc and eIF4E A-D) Representative pictures showing that pros NBs and GMCs are smaller than wildtype (NBs and GMCs are distinguished at telophase, as a doublet of unequal size), quantified in C (n=25,31,21,16).Figure 4A-D) Representative pictures showing that pros NBs and GMCs are smaller than wildtype (NBs and GMCs are distinguished at telophase, as a doublet of unequal size), quantified in C (n=25,31,21,16). pros NBs exhibit similar nucleolus /nuclear ratio (white arrows, nucleolus marked by Fib) compared to wild type and pros;TorDN NBs, quantified in D (n=10,18,15). E-F) Representative pictures showing pros clones (green) generated in myc hypomorphic background did not significantly alter pros nucleolus/nuclear ratio (E-F) pros clones (green) generated in myc hypomorphic background did not significantly alter pros nucleolus/nuclear ratio (E-F), nucleolus labelled with Fib (red) [quantified in G (n=44,43)], clone size [quantified in H (n=41,34)], or the proportion of Dpn+ NBs and Elav+ neurons per clone [quantified in I (n=15,15,15,15)]. J-K) Representative pictures showing that nerfin-1 clones (green) generated in myc hypomorphic background (J-K) nerfin-1 clones (green) generated in myc hypomorphic background (J-K) exhibited significantly reduced nucleolus/nuclear ratio, [nucleolus labelled with Fib (red)] and reduced clone size, quantified in L (n=34,27) and M (n= 106,75), and displayed an increased proportion of differentiated Elav+ neurons, and reduced proportion of Dpn+ NBs per clone, quantified in N (n=20,20,20,20). scale bar =10μm O) Reducing Myc dosage with the hypomorphic Myc allele, mycP0, abolished the increase in nerfin-1 clone size mediated by 2x histidine dietary intake, 2x his (n=41,18,10,57). P) Overexpression of myc restored the growth of nerfin-1 clones inhibited by histidine dietary reduction, 25% his (n=51,36,39,29). Q, R) pros (Q) and nerfin-1 (R) clones are reduced upon Tor inhibition (n=22,63) compared to control (n=51,65), but their growth differentially depend on amino acid transporter Slif ( n=12,32) and downstream growth regulators eIF4E (n= 21,47) and S6K (n=39,37).