EMBO/sd-character-level-ner huggingface dataset:
Figure 5A Interactions between LAMTOR1 and TRPML1 in mouse hippocampus. Binding of LAMTOR1 to TRPML1 in vivo was disrupted by systemic administration of the TAT-2031 peptide. Wes protein analysis with anti-LAMTOR1 and -TRPML1 antibodies of immunoprecipitation performed with anti-LAMTOR1 antibodies or negative control anti-HA antibodies using whole hippocampal homogenates from naïve, TAT or TAT-2031-treated mice. B Quantification of the relative abundance of TPRML1 pulled down by LAMTOR1 in naïve, TAT or TAT-2031-treated mice. N = 3 mice for each group. Data information: Data with error bars are represented as means ± SEM. Statistical significance was assessed by one-way ANOVA with Dunnett's post-test **P < 0.01, ***P < 0.001. C Representative images from proximity ligation assay (PLA) performed on brain slices from naïve, TAT or TAT-2031-treated mice. Evidence of proximity between LAMTOR1 and TRPML1 is indicated by the appearance of red puncta. Nuclei are counterstained with DAPI (blue). Scale bar, 10 μm. D Quantification of the number of PLA signals in CA1 from naïve, TAT or TAT-2031-treated mice. N = 6 mice for each group. Data information: Data with error bars are represented as means ± SEM. Statistical significance was assessed by one-way ANOVA with Dunnett's post-test **P < 0.01, ***P < 0.001. E TAT-2031 treatment increased lysosomal mobility. Neurons were infected with shLAMTOR1 or shSc AAV; they were treated with TAT or TAT-2031 (10 µM) and imaged with LysoTracker to visualize lysosomal trafficking in dendrites. N = 17-22 neurons from 3 independent experiments. Data information: Data with error bars are represented as means ± SEM. Statistical significance was assessed by two-way ANOVA with Tukey's post-test **P < 0.01, ***P < 0.001.
Figure 3Truncation analysis revealed the regions of Nbr1 involved in its interactions with Ams1, Ape4, Lap2, and Ape2. Schematic at the top left shows the domain organization of Nbr1. Pil1 co-tethering assay data are shown in panel B The ZZ1 fragment of Nbr1 interacted with Ams1 and Ape4 in the Pil1 co-tethering assay. In this assay, a bait protein is fused to Pil1, which forms filamentary structures, and a prey protein that can interact with the bait exhibits co-localization on the same structures. Scale bar, 3 μm. The ZZ1-L1 fragment of Nbr1 co-immunoprecipitated with Ams1.   The ZZ1-L1 fragment of Nbr1 co-immunoprecipitated with Ape4.   Ape4 competes with Ams1 for Nbr1 binding. GST-tagged ZZ1-L1 fragment of Nbr1 was bound to glutathione beads and incubated with FLAG2-His6 (FFH)-tagged Ams1 proteins and different amount of FFH-tagged Ape4. GST-tagged Nbr1 was purified from E. coli. FFH-tagged cargo proteins were purified from S. pombe. Left, SDS-PAGE showing the purify and amounts of the proteins used as input; Right, immunoblotting analysis of the proteins retained on the glutathione beads after washing. Overexpressing of Ape4 inhibited vacuolar targeting of Ams1. FFH-tagged Ape4 was expressed under the strong Pnmt1 promoter. Scale bar, 3 μm. The ZZ1 region but not the ZZ2-ZZ3 region in Nbr1 is necessary for vacuolar targeting of Ams1. Scale bar, 3 μm.   The ZZ1 region but not the ZZ2-ZZ3 region in Nbr1 is necessary for vacuolar targeting of Ape4. Scale bar, 3 μm.   The ZZ2-ZZ3 region but not the ZZ1 region in Nbr1 is necessary for vacuolar targeting of Ape2. Scale bar, 3 μm.   The ZZ2-ZZ3 region but not the ZZ1 region in Nbr1 is necessary for vacuolar targeting of Lap2. Scale bar, 3 μm.
Figure 5A Enrichment analysis of KEGG pathways (FDR <0.05) of up- or down-regulated genes common for all three conditions (+/-1.5-FC and p<0.01). Relevant pathways are labelled in bold. B Heatmaps of genes involved in the relevant pathways Colour code: red represents upregulation, white, unchanged, and blue downregulation of expression. C qPCR analysis of up- and down-regulated genes Pooled RNA was prepared from sorted B220+GFP+ spleen B cells of MaxKO-cd19 (n=4), MycKO-cd19 (n=4), or DKO-cd19 (n=4) and control HET-cd19 (n=4) mice activated Genes were randomly selected and qPCR was performed using specific primers for cd72, med13l, card11, ssh2, cdc7, mrpl12, rpl39, and cysc. max and c-myc were included as controls and actin was used for normalization.
Figure 4(A) Normalized Hi-C contact maps for untreated (left) and chloramphenicol-treated cells (right). (B) Hi-C contact maps for intra-chromosomal arm interactions (dashed box in panel A) rotated 45o clockwise with directional preference plots below. Images were resized in each dimension after rotation, but otherwise unprocessed relative to panel A. Left- and rightward preferences are shown as green and red bars, respectively. Vertical dashed black lines indicate the positions of the largest ribosomal protein cluster and the two rRNA loci in the Caulobacterchromosome.
Figure 1Heatmap of the differentially expressed cholesterol-related genes between normal brain tissues (n = 28) and glioblastomas (n = 217) from the Rembrandt dataset. Gene expression values are z-transformed and colored red for high expression and blue for low expression, as indicated in the scale bar. Volcano plot showing the fold-change (log2) in cholesterol-related gene levels based on GBM vs normal brain tissue. Data were obtained from the Rembrandt dataset. Representative images of IHC staining for CYP46A1 protein in normal brain and different pathological grades of gliomas (n = 64). Scale bar = 30 μm. Quantification of CYP46A1 IHC staining in normal brain (n = 6) and different pathological grades of gliomas (n = 58). Representative images of CYP46A1 IHC staining in GBM and adjacent brain tissues from one specific case. Scale bar = 30 μm. CYP46A1 expression levels in tumours from the TCGA dataset using 2016 WHO classification. Data are shown as the mean ± the standard error of the mean (SEM; n = 667). ***P < 0.0001. Statistical significance was determined by one‐way ANOVA. CYP46A1 expression levels in different molecular subtypes from the Rembrandt GBM dataset. Shown are means and SEM (n = 245). ***P < 0.0001. Statistical significance was determined by one‐way ANOVA. Kaplan-Meier analysis for patient OS and PFS based on high vs low expression of CYP46A1 in LGG and GBM. Data were obtained from the CGGA dataset. P-values were obtained from the log-rank test.
Figure 5(A) Transactivation assay in HEK293 cells with an integrated 4xSLP-HRE2 E1B 12 TATA Luc reporter and transiently transfected with human WT or W752R AR followed by stimulation with increasing DHT concentrations. (B) Specific ligand binding assay in COS cells with overload of cold ligand and increasing concentrations of radioactive labeled DHT. Values are normalized to protein expression. (C) Western blot on cytoplasmic, nuclear and whole cell extracts derived from Hela cells transfected with human WT or W752R AR followed by stimulation with increasing concentrations of DHT. Both panels represent the same blot. The blot was cut in two parts and antibodies against HSP90 and Lamin A/C were used to confirm cellular fractionation of cytoplasmic and nuclear proteins. For AR visualization (upper panel), longer exposure time was used. # = residual AR expression (lower panel). (D) EMSA using nuclear extracts of COS-7 cells transfected with AR WT or AR W752R and incubated with radiolabeled ARE, more specifically TAT-GRE (Denayer et al., 2010). Shift (S) occured through binding of the AR dimer on the ARE. Supershift (SS) occurred after addition of an AR-antibody. S = shift and U = unbound ARE. Western blot on AR, depicted on the right, shows similar expression levels for WT and mutated receptors.
Figure 5A. Verification of ChIP-seq results by ChIP-qPCR analysis of the indicated genes. Data information: statistical analysis was performed using t-tests, and the error bars indicate mean ± SD for three independent experiments. B. The mRNA level of the indicated genes was measured by RT-qPCR. Data information: statistical analysis was performed using t-tests, and the error bars indicate mean ± SD for three independent experiments. C. Luciferase-based reporter system to monitor the transcriptional level changes of indicated genes under overexpression of hTrmt13; MDA-MB-231 cells were co-transfected with TGFB1-Luc (left), PKN2-Luc (middle), or MEF2A-Luc (right) and expression construct for wild-type hTrmt13 (WT) or catalytic inactive mutant (E463A) compared with control (Vector). Relative luciferase activity was calculated as firefly luciferase activity divided by renilla luciferase activity and shown relative to the control. Data information: statistical analysis was performed using t-tests, and the error bars indicate mean ± SD for three independent experiments.
a) Confocal microscopy of differentiated GFP-LC3+ THP-1 cells left untransfected (Control) or transfected for 2 h with poly(dA:dT) (1.figf3(a) Confocal microscopy of differentiated GFP-LC3+ THP-1 cells left untransfected (Control) or transfected for 2 h with poly(dA:dT) (1.5 μg/ml), then immunostained for endogenous AIM2 (b) Confocal microscopy of differentiated THP-1 cells transfected for 3 h with poly(dA:dT), then immunostained for LAMP-1 and AIM2. (c) Confocal microscopy of differentiated THP-1 cells left untreated or treated for 2 h with LPS (500 ng/ml) and ATP (3 mM), then immunostained for endogenous LC3 and NLRP3. Scale bars, 10 μm. Data are representative of at least three experiments.
Figure 1Bone marrow cells were polarized in the presence of 40 ng/ml GM-CSF and treated with 0.2 mM of the indicated compounds for 7 days. Gr1-CD11b+ cells were sorted and lysed for microarray. The hierarchical clustering was based on the different expression genes between BSA (Con) and oleate group. Signature genes involved in lipid metabolism, dendritic cell maturation, macrophage maturation, tumor associated macrophages (TAMs) phenotype, MHCII complex and innate immune response are listed and validated via flow cytometry or catalytic activity assay. Data are expressed as mean±SD from two to four independent experiments. Unpaired Student's two-tailed t tests were performed to compare the expression level of indicated proteins in control and oleate groups. *p<0.05; **p≤0.01
Figure 5A ERp57flox/flox mice were crossed with Nestin Cre transgenic mice to generate nervous system specific ERp57 deficient animals. The levels of ERp57 protein in the spinal cord were monitored by Western blot. ERp57WT (n=4), ERp57Nes+/- (n=5) and ERp57Nes-/- (n=4) mice. HSP90 levels were determined as a loading control. Right panel: Quantification of ERp57 levels was performed relative to Hsp90 levels. B Body weight measurements were performed for indicated time points in ERp57WT (n=50), ERp57Nes+/- (n=32) and ERp57Nes-/- (n=19) mice. C Rotarod performance was performed ERp57WT (n=20), ERp57Nes+/- (n=15) and ERp57Nes-/- (n=8) mice. D Hanging test performance was assessed in ERp57WT (n=41), ERp57Nes+/- (n=32) and ERp57Nes-/- (n=12) mice. E Kaplan-Meier survival curve for ERp57Nes-/- mice (N = 19) that prematurely died or had to be sacrificed because of health reasons between the ages 22 and 73 days. Mean survival of this subgroup of animals was 57 days. ERp57WT (n=50) and ERp57Nes+/- (n=32) mice are shown as a reference. F Histological analysis of NeuN and GFAP staining was performed in spinal cord tissue from ERp57WT and ERp57Nes-/- mice in three animals per group using indirect immunofluorescence. The nucleus was stained with Hoechst. Representative images from one mouse per group are shown. Scale bar: 50 μm. G Stereological analysis of the spinal cord from ERp57WT (n = 4), ERp57Nes+/- (n = 4) and ERp57Nes-/- (n = 4) mice. Alternate series of sections from the spinal cord of the mice were either stained for Nissl (top row images) or processed for immunohistochemistry for the cholinergic cell marker Choline Acetyl Transferase (ChAT, bottom row images). The nucleoli of the motoneurons, as stained in the Nissl series, were counted inside the motoneurons pools previously defined using the adjacent ChAT series (contours not shown). Cell densities of the three genotypes are shown on the right plots. No significant differences were found between the genotypes. Scale bar represents 200 μm and 50 μm on large and inset images, respectively. H Representative images of the spinal cord myelination of ERp57WT and ERp57Nes-/- mice.
Figure 4HEp-2 cells were incubated with 20 µg/ml of whole cell extract of Y. pseudotuberculosis expressing full-length CNFY, N- or C-terminal deletion variants fused to GFP (green) for 90 or 180 min. Cells were fixed and processed for fluorescence microscopy. The red fluorescent signal represents late endosomes (CellLight Late Endosomes-RFP (Rab7a)). Nuclei were stained with DAPI (blue). A merged image of the different channels is shown, and smaller images are magnified views of boxed areas. White scale bar is 10 µm.
Figure 5A. Representative images showing morphological changes in spheroids from 3D cultures of HRPTEpi cells following Stx2a (10 ng/mL) treatment for 72 h in the presence or absence OSMI-1 (10 µM, final). B. Quantification of estimated spheroid diameter in (A) [n = 11 independent spheroids (biological replicates)]. The effects of Stx2a in vehicle controls were compared with those of spheroids maintained in the absence of Stx2a, and OSMI-1 treatment was compared with that of the vehicle (DMSO) controls in the presence of Stx2a. Data information: Error bars for bar graphs are presented as mean ± S.E.M. Statistical analysis was performed using two-tailed Student's t-test. *P < 0.05; **P < 0.01; and ***P < 0.001. C. ELISAs were used to analyze the inhibitory effect of OSMI-1 on IL-8 and CCL-2 production from 3D cultures of HRPTEpi cells exposed to Stx2a (10 ng/mL) for 72 h (n = 3 biological replicates). The effects of OSMI-1 were compared with those of the vehicle (DMSO) controls. Data information: Error bars for bar graphs are presented as mean ± S.E.M. Statistical analysis was performed using two-tailed Student's t-test. *P < 0.05; **P < 0.01; and ***P < 0.001. D. Representative images showing morphological changes in spheroids from 3D cultures of human mini-kidneys following Stx2a (10 ng/mL) treatment for 72 h in the presence or absence OSMI-1 (10 µM, final). E. Quantification of estimated spheroid diameter in (D) [n = 11 independent spheroids (biological replicates)]. The effects of Stx2a in vehicle controls were compared with those of spheroids maintained in the absence of Stx2a, and OSMI-1 treatment was compared with that of the vehicle (DMSO) controls in the presence of Stx2a. Data information: Error bars for bar graphs are presented as mean ± S.E.M. Statistical analysis was performed using two-tailed Student's t-test. *P < 0.05; **P < 0.01; and ***P < 0.001. F. ELISAs were used to analyze the inhibitory effect of OSMI-1 on IL-8 and CCL-2 production from 3D cultures of human mini-kidneys exposed to Stx2a (10 ng/mL) for 72 h (n = 3 biological replicates). The effects of OSMI-1 were compared with those of the vehicle (DMSO) controls. Data information: Error bars for bar graphs are presented as mean ± S.E.M. Statistical analysis was performed using two-tailed Student's t-test. *P < 0.05; **P < 0.01; and ***P < 0.001. G, H. Human apoptosis antibody array analysis of multiple proteins using pooled lysate from 3D-human mini-kidney spheroids following Stx2a (10 ng/mL) treatment for 72 h in the presence or absence OSMI-1 (10 µM, final). (G) Antibody spots representing signal differences are indicated in red boxes. (H) The graph shows the average of relative spot intensities for each protein compared to those measured in the control in the absence of Stx2a exposure (n =2 biological replicates). Dashed line represents the reference point of the fold change. Raw values of fluorescence intensities are provided in the Source Data. Data information: Error bars for bar graphs are presented as mean ± S.E.M. Statistical analysis was performed using two-tailed Student's t-test. *P < 0.05; **P < 0.01; and ***P < 0.001.
Figure 7Bumetanide interferes with the interaction of CRTH2 with LARP6 in MRC-5 human lung fibroblasts MRC-5. MRC-5 cells were co-transfected with myc-LARP6 and flag-CRTH2 expressing plasmids for 24 h, and then treated with Bumetanide (0.1, 2 μM) for an additional 24 h. Whole cell lysates from MRC-5 cells were immunoprecipitated and immunoblotted with the indicated antibodies. Effect of Bumetanide on LARP6 binding to Col1a1, Col1a2, and Col3a1 mRNA in cultured MRC-5 cells. MRC-5 cells were treated with TGF-β1 and Bumetanide (2 μM) for 24 h. The cells were ultraviolet-cross-linked and RNA-bound proteins were immunoprecipitated with LARP6 antibody followed by RNA extraction for qRT-PCR. *P < 0.05 vs Vehicle, #P < 0.05 vs Control (two-way ANOVA); n = 6 for all groups. , Effect of Bumetanide on Col1a1 mRNA expression in MRC-5 cells. GAPDH was used as housekeeping gene. *P < 0.05 vs vehicle, #P < 0.05 vs Control (two-way ANOVA); n = 6 for all groups. , Effect of Bumetanide on Col1a2 and Col3a1 mRNA expression in MRC-5 cells. GAPDH was used as housekeeping gene. *P < 0.05 vs vehicle, #P < 0.05 vs Control (two-way ANOVA); n = 6 for all groups. Effect of Bumetanide on Collagen (Col) I and III protein expression in MRC-5 cells.
SCV-associated ubiquitinated aggregates are autophagy substrates.Figure 4(A) Quantification of the percentage of infected cells with SCV-associated ubiquitinated aggregates. HeLa cells were infected with ΔsseL mutant bacteria for 12 h. At 8 h post-infection, cells were subjected to the indicated treatments for 4 h before fixation and immunolabelling. A minimum of 50 cells were counted per condition in each experiment and values are the mean ± SEM of at least 3 independent experiments. p values are relative to the mean value of mock-treated cells. * p<0.05; ** p<0.01; *** p<0.001. (B) Single confocal sections of HeLa cells infected with GFP-expressing ΔsseL mutant bacteria (blue), treated and processed as in (A) and analysed by fluorescence microscopy for ubiquitin (Ub, red) and LC3 (green) (scale bars, 5 µm). The far right panels show merged images of LC3, ubiquitin and Salmonella.
a–d) USP33 inhibits assembly of SEC5–beclin-1 complexes.figf5(a-d) USP33 inhibits assembly of SEC5-beclin-1 complexes. The indicated proteins were immunoprecipitated from cells expressing GFP shRNA or USP33 shRNA with anti-Flag (M2), anti-HA or anti-SEC5 antibody and analysed for co-precipitation by immunoblotting using specific antibodies as indicated. (e) USP33 potentiates the interaction between EXO84 and beclin 1. Endogenous beclin 1 was immunoprecipitated from HEK293T cells, expressing either empty vector (V), HA-USP33 wild type (WT) or the catalytically inactive mutant C194S-H683Q, using anti-beclin 1 antibody. Immunoprecipitates were analysed for co-precipitation by immunoblotting using anti-EXO84 or anti-beclin 1 antibodies. (f,g) RALB but not RALA undergoes deubiquitylation under nutrient deprivation. 6xHis-tagged ubiquitin and the indicated RAL mutants were overexpressed in HEK293T cells. At 48 h after transfection cells were deprived of nutrients and incubated in Hank's buffered salt solution (HBSS) medium for 90 min. Ubiquitylated RALproteins were purified by Co2+ metal affinity chromatography and analysed by immunoblotting using anti-RALA or anti-RALB antibodies. (h) Nutrient starvation results in the accumulation of USP33. HEK TE cells were incubated in HBSS medium for the indicated periods of time. Immunoblotting was performed using anti-USP33 antibody. (i) USP33 co-localizes with RALB-positive vesicles in the absence of nutrients. At 48 h after co-transfection with RALB-G23V-EGFP and USP33-mCherry, HeLa cells were incubated in HBSS medium for 90 min. Cells were imaged for EGFP and mCherry fluorescence. Scale bars, 10 μm. The outlined areas are shown at higher magnification in the bottom panels. Arrows indicate USP33 co-localization to RALB-positive vesicles. (j) Beclin 1 co-localizes with USP33. HeLa cells were transfected with beclin-1-HA and USP33-mCherry. At 48 h after transfection, cells were incubated in HBSS medium for 90 minutes and treated as in i. Scale bars, 10 μm. The outlined areas are shown at higher magnification in the bottom panels. Uncropped images of blots are shown in Supplementary Fig. S7.
Figure 6A Silver stain (left) and CTCF immunoblot (right) of 3xFLAG-CTCF purified from transfected HEK293T cells. B EMSA with purified CTCF and PCR generated CD45 exon 5 probes with a wildtype or mutated CTCF binding site. Cold competition was performed with unlabeled wildtype probe. C EMSA with purified CTCF and PCR generated 72 base pair probes symmetrically flanking the exon 5 CTCF binding site. Substitution of modified dCTP allowed for variable CTCF binding site methylation. Cold competition was performed with unmethylated wildtype probe. D EMSA with purified CTCF and PCR generated 72 base pair exon 5 probes generated in the presence of cytosine or 5caC. Cold competition was performed with unmethylated or 5caC-containing wildtype probe, as indicated. Supershift was accomplished with anti-CTCF antibody. E Quantification of EMSA from D. The volume of free probe and CTCF complexed probe was determined with ImageQuant software (Molecular Diagnostics). Percent shift represents the complex volume divided by the sum of complex plus free probe. F MedIP with antibody specific to 5caC followed by qPCR with the indicated CD45 primers in naïve and activated CD4+ T cells. Values represent means +/- SD from three independent IP experiments. p= two-tailed Student's t test comparing the indicated values.
Figure 2Macroscopic assessment of the gastrointestinal tract of Mex3a KO and WT mice. These images are representative of the phenotype observed in mutant animals euthanized at different postnatal days. Boxed areas depict the distal small intestinal section (ileum) used for subsequent immunohistochemical analyses. H&E staining of a Mex3a KO and WT littermate at P19. Inserts depict high magnification of the boxed areas. Average crypt depth (C) of WT animals (n = 3, > 40 crypts counted per animal) and Mex3a mutants (n = 6, > 20 crypts counted per animal). Also shown is the average villi height (D) of WT animals (n = 3, > 25 villi counted per animal) and Mex3a mutants (n = 6, > 15 villi counted per animal). Data is represented as mean ± standard error between P16 and P18. *P = 0.03, ***P = 0.0006, Student`s t test. Immunohistochemistry staining for (E) the tuft cell marker DCLK1 (black arrowheads indicate positive cells), (F) the Paneth cell marker LYZ1 Immunohistochemistry staining for the proliferation marker KI67. mRNA ISH staining for the stem cell markers (H) Lgr5, (I) Olfm4, (J) Lrig1, and (K) Hopx in ileal sections of Mex3a mutants and littermate controls at P19. Inserts depict high magnification of the boxed areas. qPCR analysis of the expression level of the indicated stem cell markers in freshly isolated crypt fractions from Mex3a KO and WT animals (n = 3 of each genotype). Data is represented as mean fold-change plus standard error in Mex3a KO mice relatively to WT animals (dashed line). *P = 0.0208, **P < 0.01, ***P = 0.0007, Student`s t test.
Figure 4A Enzyme-linked immunosorbent assay (ELISA) for Activin A expression in the supernatant of C57#1 and C57#3 DLP mouse prostate organoid lines, upon acute A83-01 removal (48 hours) or long-term adaptation (n = 6 biological replicates). Two-way ANOVA, Sidak's test, p-value *** (<0.001), **** (<0.0001). B Representative stereoscopic images of C57#1 and C57#3 DLP mouse prostate organoid lines adapted to the absence of A83-01 and subsequently treated with either Tgfb1 (500 ng/mL, 6 days) or Activin A (50 ng/mL, 6 days). Scale bar = 1 mm C Western blot analysis in the C57#1 DLP mouse prostate organoid line upon acute removal (24 hours) or long-term adaptation to A83-01 removal, in the presence or absence of Activin A (50 ng/mL, 24 hours).