EMBO/sd-character-level-ner huggingface dataset:
USP17 is necessary for peripheral lysosome trafficking.Figure 1A HeLa cells were transfected with constructs for a non-targeting shRNA (NT), USP17 shRNA1, or USP17 shRNA2. Cells were stained using an anti-LAMP1 antibody (green) and the nuclei counterstained with DAPI (blue). Lower panels are enlarged images of indicated area in top panels and the cell membrane is marked by dotted line. B HeLa and MDA-MB-231 cells were transfected as in A and the distribution of LAMP1 positive vesicles was plotted as vesicle relative position (see C). C C Vesicle relative position is the ratio between vesicle distance from the nucleus border and the sum of the vesicle's distances from the nucleus and cell border. If the vesicle is located within the nucleus the value is set to 0. If the value is 1 then the vesicle is located at the border of cell. D HeLa cells transfected as in A, in conjunction with a construct coding GFP tagged CD63. The lower panels are enlarged images of indicated area in top panels and the cell membrane is marked by dotted line. E HeLa cells transfected as in A, in conjunction with constructs for either GFP tagged LAMP1 or CD63. The distribution of GFP positive vesicles plotted as vesicle relative position. F F HeLa cells were transfected with empty vector, or expression constructs for USP17, or USP17CS (inactive mutant). 48 hours post-transfection lysates and cell growth media were harvested and immunoblotted for GAPDH, USP17 and CatD, as indicated. G HeLa cells were transfected as in 1F. 48 hours post-transfection lysates were harvested and 5µg of protein used in a CatD/E activity assay. Relative RFU of triplicate samples at 60 minutes, compared to empty vector control are plotted. The results plotted are representative of results obtained in three separate experiments.
Figure 2(A-C) Graph showing the mitotic duration of NPCs and the fate of their progeny in primary cultures generated from E14.5 embryos. Each bar represents one cell; its height represents the amount of time the mother cell spent in mitosis; bar color represents the fate of the progeny. Dashed line is set at 30 mins. WT n = 330 cells, N = 4 embryos; Cep63T/T n = 260 cells, N = 3 embryos; Sas4cKO n = 223 cells, N = 3 embryos. (D-F) Percentage of proliferating, arrested and apoptotic progeny within each group of the indicated mitotic duration, calculated from data shown in A-C; groups with n < 20 cells were excluded from this quantification; #, chi-square test with *, post-hoc analysis, within each genotype, comparisons are made to the 0-30min group. (G) Genome-wide copy number plots of single cells sequenced from dissociated dorsolateral telencephalons of E14.5 embryos. Individual cells are represented in rows with copy number states indicated by colors; arrows on the zoomed in view (right) indicate aneuploid cells. WT n = 38 cells, N = 2 embryos; Cep63T/T n = 40 cells, N = 2 embryos; Sas4cKO n = 42 cells, N = 2 embryos. (H) Percentage of aneuploid cells from the single-cell sequencing data shown in G.
Analysis of TFEB-mediated autophagy and lysosomal pathway in vitroWestern blot analysis of Lamp1 and LC3-I and LC3-II levels in T40PL cells transfected with TFEB-FLAG and harvested at times indicated.Figure 7A Western blot analysis of Lamp1 and LC3‐I and LC3‐II levels in T40PL cells transfected with TFEB‐FLAG and harvested at times indicated. γ‐tubulin was used as a loading control. Ctrl: untransfected. B Fluorescent images of T40PL cells transfected with TFEB and RFP‐GFP‐LC3. Thick white arrows indicate cells with significant LC3 puncta and lower PHF1 compared with nearby cells. Images on the upper and right are higher magnification views of the bracketed area of the LC3‐GFP and LC3‐RFP or Merge panels, respectively. RFP‐only autolysosomes are marked by red arrows; GFP/RFP double‐positive puncta representing an autophagosome is shown in yellow (upper inset). Right insets highlight a cell with colocalization of GFP/RFP‐positive autophagosome with PHF1. Scale bar: 10 μm; in inset: 2.5 μm. C Immunofluorescence images of T40PL cells transfected with TFEB and stained with anti‐LAMP1/MC1 (top) or PHF1 (bottom). Arrow marks cells with nuclear TFEB and its correlation with higher LAMP1 and lower MC1 (top) or PHF1 (bottom) stainings. Arrowhead indicates nearby cells with cytoplasmic TFEB and opposite LAMP1 and MC1/PHF1 patterns. Scale bar: 10 μm. D Western blot analysis of T40PL cells transfected with either pCMV or TFEB, allowed to recover for 24 h, and then treated with 0.5 µg/ml doxycycline and either DMSO or 50-µM Leupeptin for 48 h prior to lysis. Black line denotes cropped lanes from a single immunoblot. E Quantization of the autophagy markers LC3-I, LC3-II (pCMV vs pCMV+Leupeptin P = 0.032, TFEB vs TFEB+Leupeptin P = 0.014), and p62 (pCMV vs pCMV+Leupeptin P = 0.019, TFEB vs TFEB+Leupeptin P = 0.024), and Tau species markers Tau1, PHF1 (pCMV vs pCMV+Leupeptin P = 0.00082, TFEB vs TFEB+Leupeptin P = 0.022), and total Tau normalized for loading to y-tubulin from (D). P-values determined by t-test, n = 4. (*P < 0.05) Each bar represents average ± s.e.m.
Hnf4g drives differentiation of enterocytes.Figure 4 (A) Gene Set Enrichment Analysis (GSEA) of Hnf4g KO organoids compared to WT organoids. Significant changing intestinal cell type gene sets from Haber et al (2017) are shown (FDR < 0.05)  (B) Hematoxylin and (Periodic acid-Shiff) PAS staining of WT (left) and Hnf4g KO (right) intestine. Nuclei are visualized using Hematoxylin and goblet cells stain positive for PAS   (C) Alcian Blue and Nuclear Fast Red staining of WT (left) and Hnf4g KO (right) small intestinal organoids. Cells are visualized using Nuclear Fast Red. Intra- and extracellular mucus that is produced in goblet cells stain positive for Alcian Blue   (D) Strand specific RNA-seq data, Hnf4g ChIP-seq in EN, and promoter specific histone modification H3K4me3 over the Hnf4a locus is shown. Data from different organoid cultures are color coded. RNA-seq reads mapping to the positive strand (sense strand) are plotted in the top window of every sample, while RNA-seq reads that map to the negative strand are mirrored and shown in the bottom window of every sample. The two isoforms of Hnf4a that are expressed are shown in the bottom, together with the antisense transcript Hnf4aos. The "phyloP30way" track from the UCSC genome browser is plotted at the bottom to show sequence conservation   (E) Quantification of the amount of goblet cells per villus for WT and Hnf4g KO intestine. Dots represent the amount of goblet cells counted per villus. (n = 7 to 25 villi from 11 WT mice and 3 to 17 villi from 14 Hnf4g KO mice). P value is from two-tailed Mann-Whitney U test
Figure 6A Comparison of N proteins from seven human coronaviruses. B Principal Component analysis of the seven high-confidence interacting protein (HCIP) lists with bait N protein from different human coronaviruses. C Heat map of the N protein HCIPs identified by SFB-tandem affinity purification. N protein HCIPs were compared among seven human coronaviruses using the preys' spectral counts. Three areas of the heat map are manually selected, enlarged and labeled as (1), (2), and (3), which are enriched by all N proteins of the seven human coronaviruses. Functional characterization for each protein is shown with the red circles below the enlarged images. D The human proteins which binding to N proteins of human coronaviruses were integrated and analyzed by STRING to illustrate the protein-protein interaction among the HCIPs. The minimum required interaction score setting in STRING is 0.9. Different function group or complex were colored differently. The filled circles are proteins, and the lines are the interactions.
a) Autophagy induction in response to 5-FU measured in a GFP::LGG-1 reporter strain fed control RNAi (L4440) or RNAi targeting the indicated genes (scale bars, 10 μm).f3a) Autophagy induction in response to 5-FU measured in a GFP::LGG-1 reporter strain fed control RNAi (L4440) or RNAi targeting the indicated genes (scale bars, 10 μm). (b) Immunofluorescence showing anti-VPS-34 staining in dissected embryos in the absence or presence of 5-FU (scale bar, 5 μm). (c) Western blot analysis of embryonic extracts with and without the addition of 5-FU or the autophagy inhibitor 3-MA, detecting the ~40-kDa GFP::LGG-1 fusion protein and the cleaved ~25-kDa product. (d) F1 survival measured after depletion of BEC-1 (red triangles) and ATG-7 (blue squares) as compared with worms fed control RNAi (black diamonds). The survival curve shows the mean±s.d. for each data point from three independent experiments. (e) Induction of autophagy (% GFP-positive embryos) following depletion of the indicated genes. (f) Immunofluorescence showing anti-VPS-34 staining in N2 and atl-1(tm853) embryos in the absence or presence of 5-FU (scale bar, 5 μm). (g) The fraction (as % of untreated control) of embryos with VPS-34-positive foci following 5-FU treatment. (h) Induction of autophagy (% GFP-positive embryos) in control (GFP::LGG-1) and atm-1(gk186) mutants (atm-1; GFP::LGG-1). (e,g,h) Bar graphs represent mean±s.d. from three independent experiments.
a, Percentage of S.f3a, Percentage of S. Typhimurium coated by the indicated galectins. HeLa cells stably expressing YFP-tagged galectins were treated with the indicated siRNAs. YFP-positive bacteria were counted by microscopy at 1 h p.i. siRNAs are further characterized in Supplementary Fig. 2. b, Analysis of HeLa cells treated with the indicated siRNAs and stained with NDP52 antiserum. NDP52-positive bacteria were counted by microscopy 1 h after infection with S. Typhimurium. siRNAs are further characterized in Supplementary Fig. 2. c, Percentage of bacteria coated by the indicated galectin 8 alleles. HeLa cells stably expressing the indicated galectin 8 alleles fused to YFP were infected with S. Typhimurium. YFP-positive S. Typhimurium were counted by microscopy at 75 min p.i. WT, wild type. d, Binding of galectin 8 to bacteria and HeLa cells. The indicated bacteria and HeLa cells were incubated with His-GST-ubiquitin (Ub), His-GST-galectin 8 or buffer as indicated, followed by murine anti-His antibody and PE-labelled anti-mouse serum. e, Percentage of S. Typhimurium coated by the indicated galectins. Wild-type CHO cells and mutant Lec3.2.8.1 cells stably expressing YFP-tagged galectins were infected with S. Typhimurium. YFP-positive bacteria were counted by microscopy at 1 h p.i. f, Confocal images of HeLa cells expressing the indicated YFP-tagged galectins. Cells were left untreated or were exposed to hypertonic conditions, with or without (w/o) PEG as indicated, followed by hypotonic shock. g, Fold replication of S. Typhimurium in HeLa cells expressing the indicated galectin 3 variants and transfected with the indicated siRNAs. At 2 h and 6 h after infection, cells were lysed and bacteria counted on the basis of their ability to form colonies on agar plates. Galectin 3 proteins are further characterized in Supplementary Fig. 4b. Mean and s.d. of duplicate coverslips>100 bacteria counted per coverslip. Data are representative of at least two repeats. *P< 0.05, Student's t-test. Scale bar, 10 µm.
Figure 2 (A) Schematic representation of the domain composition of the heterodimeric full-length Smc5-Nse2 complex  (B) Bar diagram representation of the relative SUMO conjugation activity of Nse2, full-length Smc5-Nse2, ΔHinge/Smc5-Nse2, ΔHead/Smc5-Nse2 and Arm/Smc5-Nse2 truncation constructs (schematic representation above). Orange bars indicate the presence of ssDNA (virion ϕx174) and red bars absence of ssDNA. Reaction rates were performed at least in three different independent experiments (see Fig EV2). Data values are mean ± s.e.m.; and n=3 technical replicates. Significance was measured by a two-tailed unpaired t-test relative to wild-type. **P<0.01  (C) SYPRO-stained (left) and Western blot (right) time course SUMO conjugation reaction using and ΔHead/Smc5-Nse2 truncation construct in the presence of ssDNA. T7-tagged Smc5 and E1 were immunodetected by an anti-T7 antibody. Reactions were run at 30 °C and stopped at indicated times by adding SDS-loading buffer  (D) SYPRO-stained (left) and Western blot (right) time course SUMO conjugation reaction using and ΔHinge/Smc5-Nse2 truncation construct in the presence of ssDNA. T7-tagged Smc5 and E1 were immunodetected by an anti-T7 antibody. Reactions were run at 30 °C and stopped at indicated times by adding SDS-loading buffer  (E) Western blot of the time-course SUMO conjugation reaction in the presence of ssDNA (virion ϕx174) using either Nse2 (left) or Arm/Smc5-Nse2 complex (right). The reactions were run in the presence or absence of cNse4 external substrate. Reaction was run at 30°C and stopped at indicated minutes by adding SDS-loading buffer (N-S2, cNse4-SUMO2; N-2S2, cNse4-2SUMO2; N-3S2, cNse4-3SUMO2 and pS2, poly-SUMO2)
Figure 3A. Immunoblotting of indicated proteins in melanoma cells treated with siASNS, sicMYC (#1 or #2), or a combination of siASNS and sicMYC (#1 or #2) for 72 hr. B. RT-qPCR analysis of indicated transcripts in melanoma cells 36 hr after treatment with siASNS, sicMYC, or both. C. Immunoblotting of indicated proteins in melanoma cells 72 hr after treatment with siASNS, sicMYC, PLX-4032, or a combination of siASNS and sicMYC, or siASNS and PLX-4032. D. Immunoblotting of indicated proteins in A375 cells 72 hr after treatment with siASNS, HA-GSK3-β (S9A), or both. E. RT-qPCR analysis of indicated transcripts in melanoma cells after 36 hr treatment with siASNS, HA-GSK3-β (S9A), or both. F. A375 cells were treated as indicated for 72 hr followed by immunoblotting of indicated proteins. Proliferation of melanoma cells over indicated times after treatment with siASNS, sicMYC, or both (G), Proliferation of melanoma cells over indicated times after treatment with siASNS, HA-GSK3-β (S9A), or both (H).
SNAREs are locked in an assembled form in LSD endolysosomal membranes.Figure 6(A) SDS‐resistant complexes containing Vti1b were detected by immunoblotting analysis of nonboiled samples corresponding to total, detergent insoluble (DRM) and detergent soluble (Sol.) endo‐lysosomal membrane fractions derived from MSD and WT MEFs. The SDS‐resistant complexes were also visualized after loading WT MEFs with cholesterol and after treating MSD MEFs with MβCD. Immunoblots revealed the presence of low molecular weight complexes (*, 50-60 kDa) and high molecular weight complexes (**, >80 kDa). The percentage of Vti1b in SDS‐resistant complexes in total endolysosomal membranes (bottom‐left chart) and the amount of Vti1b‐containing SDS‐resistant complexes in DRM and soluble fractions (bottom‐right chart) were calculated by the densitometric quantification of the correspondent immunoblots (ImageJ). Values represent the mean±s.e.m. values of three independent measurements. *P0.05, Student's t‐test: WT versus MSD, WT versus WT+cholesterol and MSD versus MSD+MβCD (bottom‐left chart); WT versus MSD, WT versus WT+cholesterol and MSD versus MSD+MβCD for each fraction (bottom‐right chart). (B) Syntaxin 7 and VAMP7 were co‐immunoprecipitated with Vti1b using anti‐Vti1b antibodies in WT (untreated or cholesterol treated) and in MSD (not treated or MβCD treated) MEFs. The amount of Vti1b precipitated in each cell line is also shown. (C) SDS‐resistant complexes are decorated by anti‐syntaxin 7 antibodies in total endolysosomal membrane fraction from WT and MSD MEFs. (D) Membrane‐associated α‐SNAP and its release in the cytosol were evaluated by western blot analysis on total cell lysates (total), intracellular membranes recovered after centrifugation from a post‐nuclear supernatant fraction (membrane associated) and cell lysates devoid of membranes (cytosolic released) derived from MSD (untreated or MβCD treated) and WT (untreated or cholesterol treated) MEFs.
Figure 4A, Diagram illustrating the Golgi recruitment mechanisms of PI4KIIα. B, Confocal images of wild-type and PI4KIIα-depleted (sgPI4K2A) HEK293-CAS9 cells expressing P4M-YFP, treated with or without U18. Bottom panels show enlarged views of PtdIns4P in a Golgi region. C, Quantitative analyses of P4M-YFP mean fluorescence intensity distributed in Golgi normalized to the cytoplasm under different conditions. Control: n=19; U18: n=13, sgPI4K2A: n=19; sgPI4K2A+U18: n=17; sgDHHC3: n=11; sgDHHC3+U18: n=11. * represents a significant difference (P<0.05) between control and other non-U18 treated groups; # represents a significant difference (P<0.05) between U18-treated and other U18-treated sgRNA groups. D. Confocal images of control (top) and NPC1I1061T (bottom) patient fibroblasts immunolabeled for DHHC3 and Giantin. Insets show enlarged views of DHHC3 and Giatin regions. E. Analysis of anti-DHHC3 mean fluorescence intensity in a Golgi region relative to cytoplasm. Control: n=19; PC1I1061T: n=17. F. Confocal images of control (1st column) and NPC1I1061T fibroblasts (2nd column) expressing mCherry-P4M or control fibroblasts expressing P4M-mCherry and GFP-DHHC3 (3rd column). G. Quantification of Golgi mCherry-P4M. Control: n=11; NPC1I1061T: n=14; control+DHHC3: n=12. H. Control or GFP-DHHC3 expressing tsA201 cells fixed and stained for PI4KIIα or PI4KIIIβ. Bottom panels show enlarged views of DHHC3, PI4KIIα, or PI4KIIIβ at Golgi regions. I. Quantification of PI4KIIα (left; control: n=42, DHHC3: n=24), or PI4KIIIβ (right; control: n=39, DHHC3: n=26) intensity relative to cytoplasm. J. Top: HEK293-CAS9 cells expressing P4M and either vehicle control, sgDHHC3, U18, and U18 with sgDHHC3. Bottom: expanded views of P4M in the black dashed rectangles.. K. Control (left) and NPC1I1061T (right) patient fibroblasts expressing P4M-YFP (top row) or P4M-YFP and DHHC3C157S (DN-DHHC3; bottom row). Inset panels show enlarged views of P4M-YFP distribution at Golgi. L. Quantification of P4M-YFP signal in the Golgi region relative to cytoplasm. NPC1I1061T: n=20; NPC1I1061T+sgDHHC3: n=14. M. Confocal images of control (top row), NPC1I1061T (middle row), and NPC1I1061T treated with sgDHHC3 (bottom row) patient cells expressing P4M, LAMP1 and CAS9. Insets are zoomed regions from LAMP1 positive areas. N. Quantification of P4M changes at endomembranes. Control: n=149, U18: n=251, sgPI4KIIa +U18: n=131, sgDHHC3+U18: n= 271. Each n represents a single enodolysosome.
QC divisions correlate negatively with the number of CSC layers.Figure 4The combined results of the SCN staining 3 are shown as 2D plots to visualise the correlation of the CSC layer and QC division phenotypes. Number of CSC layers are shown on the y axis and the QC division phenotype is shown on the x axis. The darker the colour, the more roots show the respective phenotype (see colour gradient top right indicating the frequencies in percent). Col wild-type roots show one layer of CSCs and no EdU stained cells (no QC division) after 24 h EdU staining. Number of analysed roots (n) (biological replicates) is indicated for each genotype and results from 2-5 technical replicates per genotype.
Figure 1(A) Waterfall plot showing the percentage of tumor volume change in olaparib-treated tumors compared to the tumor volume on day 1. +20% and -30% are marked by doted lines to indicate the range of PR, SD and PD. The box underneath summarizes different characteristics of each model and the clinical context at the moment of PDX implantation. TNBC, Triple Negative Breast Cancer; ER+BC, Estrogen Receptor positive Breast Cancer; P, primary; M, metastasis. Error bars indicate SEM from independent tumors (n ≥ 3). (B) Graph showing the percentage of tumor volume change during olaparib treatment in PDXs from cohort-1. Olaparib-sensitive models are represented with discontinuous lines. Acquisition of PARPi-resistance in PDX302, STG201 and PDX093 after prolonged exposure to olaparib is shown.
Pharmacological action profiling of the entire compound library and confirmed hits.Figure 2.Pharmacological action profiling of the entire compound library and confirmed hits.The 54 final hits were selected and sorted into 43 pharmacological action categories. Gray and black bars indicate the distribution of all screened compounds and confirmed hits with a therapeutic index (TI) >6, respectively. The vertical axis displays counts of each compound on a log scale with +1 added to the count to prevent negative values.
Figure 4(A, B) C57BL/6 mice were left untreated (NT) or were intraperitoneally treated with indicated amounts of AG for 3 d in the absence or presence of the MMP inhibitor marimastat (10 mg/kg) given intraperitoneally prior to AG stimulation. Lung sections stained with H&E (A) and quantification of lung lesion burden from H&E stained sections (B). Data information: Data in (A) are representative of n=3 independent experiments. Data in (B) are means + SD of indicated numbers of mice from one of n=3 independent experiments and each symbol represents data from 1 mouse. One-way ANOVA followed by Dunnett's post hoc test (B) was used for statistical analysis. ns, not significant; ****, p<0.0001. Scale bar, 200 μm.
Figure 2Representative western blot showing level of IKKε in IKBKE (IKKε)-silenced (A) T47D breast cancer cell lines. Representative western blot showing level of IKKε in IKBKE (IKKε)-silenced (B) MDA-MB-468 breast cancer cell lines. (C-D) Heatmap and hierarchical clustering of metabolite concentrations in (C) IKBKE (IKKε)-silenced T47D cells and (D) IKBKE (IKKε)-silenced MDA-MB-468 cells (n=5 technical replicates). metabolite levels were normalised to the internal standard HEPES. (E) Glycine production, representative of serine production, from glutamine (glycine m+1, 15N-glutamine labelling) and glucose (glycine m+2, 13C6-glucose labelling), and contribution of pyruvate and glucose-derived carbon to TCA cycle metabolites (citrate m+2, malate m+2, 13C6-glucose labelling) in IKBKE (IKKε)-silenced T47D cells. (n=5 technical replicates). metabolite levels were normalised to the internal standard HEPES. (F) Serine production from glutamine (serine m+1, 15N-glutamine labelling), and serine and glycine production from glucose (glycine m+2, serine m+3, 13C6-glucose labelling) as well as contribution of pyruvate and glucose-derived carbon to TCA cycle metabolites (malate m+2, 13C6-glucose labelling) in IKBKE (IKKε)-silenced MDA-MB-468 cells.
Figure 3Intracellular NAD levels in brain tissues from 6-month- old G1 and G3 Tert-/- female mice. immunoblots of the expression of NAD consuming related proteins and their activities in brain tissues from 6-month- old G1 and G3 Tert-/- female mice. The CD38 antibody was validated using the CD38-/- mouse brain tissues. C Quantification of immunoblots from the indicated proteins in B. Data information: All values are presented as mean ± SD of G1 Tert-/- mouse brain tissues versus G3 Tert-/- mouse brain tissues. n=4 in each group. P values on the basis of Student's t test. D NADase activity in G1 and G3 Tert-/- mouse brain lysate was determined at various time points during the reaction. NADase activity was not detectable in the extracts treated with the CD38 inhibitor, 78c, at a concentration of 50nM.
Figure 4A) Representative MEA trace (left) showing the effect of 100 nM AST and increasing concentrations of LUF7346 in the presence of AST on FP contour, measured in LQT1R594Q-CMs. Average data (right) of the effect of AST and LUF7346 in the presence of AST on QT interval duration compared to baseline in CMs derived from all the hiPSC lines used in this study. * = p<0.05 vs respective baseline. ° = p<0.05 vs AST. Colour in the heatmap defines the magnitude of QT prolongation (red) and QT shortening (blue), respectively, after treatment with AST and with increasing concentrations of LUF7346 in the presence of AST. N: 5-10. B) Representative AP from WT-, LQT1R594Q- and JLNSR594Q-CMs, paced at 1 Hz, under baseline conditions (black), after application of 100 nM AST (red), and after addition of increasing concentrations of LUF7346 in the presence of AST (colour-code is shown). C) Effect of AST and LUF7346 in the presence of AST on APD90, APD70, and APD50 in WT- (grey), LQT1R594Q- (red) and JLNSR594Q-CMs (black) paced at 1 Hz. * = p<0.05 vs respective baseline; ° = p<0.05 vs AST. N: 11-16. D) Effect of AST and LUF7346 in the presence of AST on APA and Ediast in WT- (grey), LQT1R594Q- (red) and JLNSR594Q-CMs (black) paced at 1 Hz. N: 11-16.