EMBO/sd-character-level-ner huggingface dataset:
Upon heat shock or proteasome inhibition, DRiPs promote amyloidogenesis in NoABs (see also ).Figure 2HeLa cells were left untreated or treated with heat shock (HS) at 42˚C for 3 h, alone or with cycloheximide (CHX; 50 μg/ml), or Actinomycin D (Act. D; 3 µM) followed by staining with the dye Amylo-glo. DRiP labelling in GFP-NCL HeLa Kyoto cells that were either left untreated or treated with HS at 42˚C and OP-puro (25 µM) for 2 h. Where indicated, cells were cotreated with CHX (50 μg/ml). HSPA8 subcellular distribution and recruitment inside nucleoli following exposure to HS at 42˚C for 2 h, alone or combined with CHX, in GFP-NCL HeLa Kyoto cells. DRiP labelling in HeLa cells treated with MG132 (10 µM) alone or with CHX (50 μg/ml) for 8 h; where indicated, OP-puro (25 µM) was added during the last 45 min, prior to cell fixation. Amylo-glo staining of HeLa cells left untreated or exposed to MG132 (10 µM) alone or combined with CHX (50 μg/ml) for 8 or 13 h. Amylo-glo staining of HeLa cells left untreated or subjected to transcriptional stress (MG132 10 µM and Act. D 4 µM) for 6 hrs. Where indicated, translation was concomitantly inhibited with CHX (50 μg/ml). Amylo-Glo staining is also shown in HeLa cells treated with Act.D alone or with CHX for 6 hrs, as control. Amylo-glo staining of HeLa cells exposed to HS at 42˚C for 3 h and let to recover for 3 h in drug-free medium (+ rec. control) or with VER (40 µM; + rec. VER). Where indicated cells were treated with VER alone (VER 3 h). Amylo-glo staining of HeLa cells treated with MG132 (10 µM) for 13 h and let to recover for 5 h in drug-free medium (+ rec. control) or with VER (40 µM; + rec. VER). Where indicated cells were treated with VER alone (VER 5 h). HeLa cells were transfected with vectors encoding for GFP, GFP-GR50 or RPL23a-GFP. 24 hrs post-transfection cells were fixed and stained with Amylo-glo. The percentage of transfected cells with Amylo-Glo positive nucleoli is shown. Number of cells counted/condition: GFP, 412; RPL23a-GFP, 482; GFP-GR50, 359, in three independent experiments; statistical significance via One-way ANOVA; p < 10-7, ± s.e.m.
Toxicity studies of RK-33 in mice.Figure 5Following injection of 20 mg/kg of RK-33, twice a week for 7 weeks, extensive histopathological examination was carried out following necropsy. Identical patterns were observed both in the control and in the treated mice (n = 2). Samples were stained with H&amp;amp;E. Scale bar is 50 μm. Pharmacokinetics of RK-33 in SCID mice at various time intervals. Results are mean ± SD from 5 mice. LC-MS/MS method was used to determine concentration of RK-33 in mouse plasma and tissue. Liquid chromatography-mass spectrometry (LC-MS/MS) analysis was performed to determine different metabolites of RK-33. RK-33 and metabolites characterized by LC-MS/MS in human liver microsomes using the scan mode function of the LC-MS/MS. HAPI cells were treated with RK-33 (10 μM), HA14-1 (25 μM), or DMSO vehicle, followed by oligomycin (oligo, 0.5 μg/ml), FCCP (3 μM), and antimycin A (AA, 1 μM) while oxygen consumption rate (OCR) was measured. Pyruvate (10 mM) was added in combination with FCCP to ensure that substrate supply was not rate-limiting for maximal OCR. Data are mean ± SD from 2 to 3 wells and representative of independent experiments performed with two different HAPI passages. OCR is baseline-normalized to the point prior to drug or vehicle addition. HAPI microglial cells were incubated for 1 h in glucose-free XF24 assay medium that was supplemented with 2-deoxyglucose (50 mM) and pyruvate (10 mM). RK-33 (10 μM), oligomycin (0.5 μg/ml), or vehicle control was additionally present as indicated. Results are mean ± SD from 12 replicates pooled from experiments using two consecutive passages. Significance was assessed by two-sided, unpaired t-test.
Figure 5(A-B) Proteins and RNA samples of colon tissue were extracted from mice with the indicated treatments. (A) Cytokine levels of IL-6, IL-1β and INF-λ were determined by ELISA (n=4-7). Results represent two or more independent experiments. (A-B) Proteins and RNA samples of colon tissue were extracted from mice with the indicated treatments. (B) mRNA expression levels of IL-6, IL-12p35 and IL-23p19 were assessed by quantitative RT-PCR and normalized to TBP (n=4-6). Results represent two or more independent experiments.
Impact of pathway mutations on overall survival across treatment groupsA-E) Overall survival of patients with tumors containing mutations in pathways A) WNT signaling, B) Hedgehog signaling, C) NOTCH2 signaling, D) p53 independent DNA damage repair, E) TGFβ signaling, stratified by groups receiving no systemic treatment (n=336), endocrine therapy only (Endo only; n=1,579), endocrine and chemo therapy (Endo + Chemo ± any; n=914), as well as HER2 treatment with any other treatment or none (HER2 ± any; n=348).Figure 6A-E) Overall survival of patients with tumors containing mutations in pathways A) WNT signaling, B) Hedgehog signaling, C) NOTCH2 signaling, D) p53 independent DNA damage repair, E) TGFβ signaling, stratified by groups receiving no systemic treatment (n=336), endocrine therapy only (Endo only; n=1,579), endocrine and chemo therapy (Endo + Chemo ± any; n=914), as well as HER2 treatment with any other treatment or none (HER2 ± any; n=348). Specific treatments in these groups are detailed in Table EV4. In each Kaplan-Meier plot, wildtype (wt) cases are plotted in blue, mutated (mut) cases are plotted in red, the log-rank P value is given, and the hazard ratio (HR) for mutation is given with a 95% CI and after uni-variable and multi-variable (MV) Cox regression adjustment. Covariables included in the MV analysis were age at diagnosis, lymph node status, tumor size, and the variables denoted by the following symbols: ¶, ER, PgR, HER2, and NHG; ¤, ER, PgR, and NHG; $, HER2 and NHG. Abbreviations: ER, estrogen receptor; PgR, progesterone receptor; HER2, human epidermal growth factor receptor 2; NHG, Nottingham histological grade.
Inhibition of Gli‐dependent MB tumor cell growth A, B Ex vivo cell cultures from Ptch1+/− mice MBs were treated with GlaB (5 μM), GANT61 (10 μM) or DMSO only.Figure 5A, B Ex vivo cell cultures from Ptch1+/−mice MBs were treated with GlaB (5 μM), GANT61 (10 μM) or DMSO only. After the indicated times, a trypan blue count was performed (A) to determine the growth rate of viable cells. A, B Ex vivo cell cultures from Ptch1+/−miceMBs were treated with GlaB (5 μM), GANT61 (10 μM) or DMSO only. After the indicated times, Gli1 mRNA expression levels were determined by qRT-PCR (B) normalized to endogenous control (β2‐microglobulin and HPRT). C-F GlaB inhibits MB‐SCs' self‐renewal and proliferation. (C) Suspension of single MB‐SCs isolated from Ptch1+/−mice. MBs were cultured in stem cell medium to allow the formation of primary neurospheres. Primary neurospheres were dissociated and treated with increasing concentrations of GlaB or DMSO only. After 7 days of treatment, the number of secondary neurospheres derived from a known number of single cells was counted. The self‐renewal MB‐SCs' capability is expressed as percentage of neurosphere‐forming cells (left). Representative bright field images of tumor neurospheres after GlaB treatment are also shown (right). C-F GlaB inhibits MB‐SCs' self‐renewal and proliferation. (D, E) MB‐SCs isolated from Ptch1+/−miceMBs were treated for 48 h with GlaB (5 μM) or DMSO only. qRT-PCR analysis show Hh, proliferation and stemness target mRNA expression levels. For qRT-PCR, results were normalized to endogenous control (β2‐microglobulin and HPRT). C-F GlaB inhibits MB‐SCs' self‐renewal and proliferation. (D, E) MB‐SCs isolated from Ptch1+/−miceMBs were treated for 48 h with GlaB (5 μM) or DMSO only. Western blot analysis show Hh, proliferation and stemness target protein expression levels. C-F GlaB inhibits MB‐SCs' self‐renewal and proliferation. (F) BrdU assay in MB‐SCs treated with GlaB (5 μM) for 24 or 48 h and plated on polylysinated chamber slides. Inhibition of cell proliferation was measured as percentage of BrdU incorporation in comparison with DMSO‐treated sample. G TUNEL assay in MB‐SCs. MB‐SCs isolated from Ptch1+/−miceMBs were treated with GlaB (5 μM) and compared to DMSO‐treated sample. Bottom panel shows a Western blot of caspase‐3 in GlaB‐treated versus control cells.
Figure 3A) Gene ontology enrichment analysis showing immune system process (GO:0002376) pathways for genes with >1.5 fold-change expression between IL-15 and IL-2. The color of the nodes is determined by the significance of the enriched term assessed by 2-sided hypergeometric test with Bonferroni's step-down method for P value correction. B) Volcano plot showing genes compared after two-days cytokine activated NK cells. Genes with significant changes (p value<0.05) and >1.5 log2 fold change between IL-2 and IL-15 activated NK cells are labeled in red. Genes belonging to cell surface (GO:0009986) are annotated with gene name. C) Quantification of CD25+ CD54+ population changes after two days treatment (n=4, biological replicates). D) Representative flow cytometry graphs for CD25 and CD54 staining in IL-2 and IL-15 NK cells in the presence or absence of PGE2. E) Quantification of the number of cell clusters. NK cell clusters were defined as cell aggregates occupying an area at least 2000 μm2 and an eccentricity of <0.8). (n=4, biological replicates). F) Representative phase contrast image of IL-2 and IL-15 activated NK cells in the presence or absence of PGE2. Scale bar 50 µm. G) Chromium-release cytotoxicity assay by two-day cytokine activated NK cells in the presence or absence of PGE2 (1µM) and anti-CD54 (20 µg/ml) (n=4, biological replicates), E:T ratio=5:1.
Figure 7(A). Maximum intensity z-stack projections of confocal IF images (DNA stain, indirect IF for tubulin and TPX2) in untreated oocytes (control), oocytes expressing RanT24N or injected with importin β(71-876). The representative of at least 5 experiments is shown. (B-D) Maximum intensity z-stack projections of IF images in control and importin β(71-876)-injected cells were used to quantify the spindle vs. whole-cell area fraction (B) and the ratios of the average signal in the spindle vs. cytoplasm for the tubulin (C) and TPX2 (D) staining. Means ± SDs, with t-test in (B, C) and Mann-Whitney test in (D), oocyte numbers indicated in brackets. (E) Ratios of the spindle-associated fractions of TPX2 vs. tubulin were calculated in maximum intensity z-stack projections of IF images. Means ± SDs, Mann-Whitney test, oocyte numbers indicated in brackets. (F) Maximum intensity z-stack projections of IF images were used to quantify the number of ectopic MT asters. Means ± SDs, ANOVA with Kruskal-Wallis and Dunn's tests, oocyte numbers indicated in brackets. (G) Maximum intensity z-stack projections of confocal IF images stained for DNA, pericentrin, and tubulin (directly labeled primary antibody). A representative example of at least 2 experiments is shown. Scale bar, 20 µm. (H-I) The number and size of pericentrin foci were quantified in maximum-intensity z-stack projections of IF images stained as in (G) in at least 2 separate experiments. Means ± SDs, ANOVA with Kruskal-Wallis and Dunn's tests. (J) Proposed model: The suppression of importin β by RanGTP is required for MT nucleation and assembly, enabling the assembly and function of the MI spindles. Through separate mechanisms, RanGTP opposes the role of importin β in preventing the coalescence of pericentrin-containing
Figure 6(A) Pictures of the nude mice and their tumors after inoculation was shown in two subgroups. (B) Tumor volumes of each group during the tumor growth process. (C) Tumor weight measured from the xenograft model. Data information: All results were expressed as mean ± SD of three independent experiments (n ≥ 3 per experimental condition). Statistical significance was assessed with a two-tailed unpaired Student's t-test. **P<0.01. (D) Pictures of livers in nude mice and their respective representative images of the tissues by hematoxylin-eosin (HE) staining. Data information: All results were expressed as mean ± SD of three independent experiments (n ≥ 3 per experimental condition). Statistical significance was assessed with a two-tailed unpaired Student's t-test. **P<0.01.
Correlative light-electron microscopy of cells containing a GFP-tagged subgenomic replicon.Figure 2(A) Epifluorescence microscopy of live cells containing a subgenomic replicon with a GFP-tagged NS5A. Huh7-Lunet cells were transfected with replicon RNA and seeded onto carbon-patterned sapphire discs. Twenty-four hours later cells were analyzed by fluorescence microscopy and immediately processed for EM. (a) Fluorescence image; (b) enlarged fluorescence image of the cell of interest; (c) merge of bright field and fluorescence images. Coordinates etched onto the surface of the sapphire disc were used to record the position of the selected cells. White squares in a and c enclose the cell shown in b. (B) EM micrograph of the cell boxed in panel Ab overlapped with the fluorescence image. Areas marked with a green dotted line indicate regions of intense fluorescence. Note that the images do not match perfectly because the fluorescence image corresponds to the complete cell whereas the EM image represents one 60 nm ultrathin section of the same cell. (C) Higher magnification images of the two different regions, labeled 1 and 2 in panel B, corresponding to regions with intense fluorescence (1) or a region corresponding to the intersection of high to low fluorescence (2). Region 1 (top panel) corresponds to a DMV-containing area residing in close proximity of the ER; region 2 (bottom panel) corresponds to a LD-enriched area containing DMVs in very close proximity. Areas marked with white squares in the left images are magnified in the corresponding right panels. LD, lipid droplet; ER, endoplasmic reticulum; DMV, double membrane vesicle; m, mitochondrium; if, intermediate filaments.
Transplantable and actionable murine mesothelioma models using KPM cells.Figure 8A Kaplan-Meier survival plot with survival table. B Data summary of pleural effusion volume and total cells (n = 10, 12, 10, 9, and 9 mice/group, respectively, from left to right). C Images of the chest before and after opening, showing effusion (dashed lines), visceral (vpt) and parietal (ppt) pleural tumors on the costophrenic angle (ca), the diaphragm (d), and the chest wall (cw, arrows). t, tumors; l, lungs; h, heart. D May-Gruenwald-Giemsa-stained pleural cells (macrophages, MΦ: black arrow; lymphocytes, LΦ: purple arrow; neutrophils, NΦ: green arrow; eosinophils, EΦ: orange arrow). E Effusion cytology and biochemistry data summary (total n = 10 mice; n = 4, 3, and 3 effusions from mice injected with KPM1, KPM2, and KPM3 cells, respectively, were analyzed and shown are pooled data). F, G C57BL/6 mice received pleural KPM1 cells followed by a single intrapleural injection of liposomes containing 1% DMSO or 15 mg/Kg deltarasin in 1% DMSO at day 9 post-tumor cells. Shown are data summaries of MPE volume (n = 8 and 7 DMSO and deltarasin-treated mice/group, respectively) and pleural fluid nucleated cells at day 19 post-KPM1 cells (F), as well as representative images of pleural effusions (dashed lines) and tumors (t in (G)).
Figure 1A. 7SK was divided into 6 non-overlapping fragments and in vitro transcribed in the presence of 32p-UTP for use in an in vitro modification assay. U250 in fragment 5 is highlighted in red. B. In vitro modification reactions containing the six fragments and HeLa NE were performed. After nuclease P1 digestion, the products were analyzed by 1D-TLC with pU and pψ as controls. C. WT and mutant 7SK fragment 5 containing U250G were analyzed as in B. D. Outline of the biotin-facilitated extraction and analysis of the modification status of site-specifically labeled nucleotides in 7SK by 1D-TLC. E. 7SK RNA, generated by either in vitro transcription (IVT) or purified from HeLa total RNA and then site-specifically labeled at the indicated positions by the procedure in D, was analyzed by 1D-TLC. F. Cyber Gold staining of 7SK obtained by either IVT or immunoprecipitation (IP) with the indicated antibodies. G. 2D-TLC analysis of 32p-labeled mononucleotides derived from IVT or CDK9-bound 7SK RNA, with the first panel depicting a schematic migration pattern of the indicated 5'-phosphorylated mononucleotides. H. 7SK RNA, obtained by either IVT or anti-CDK9 IP and then site-specifically labeled at the indicated positions by the procedure in D, was analyzed by 1D-TLC.
Figure 3A HeLa cells were transfected with control or TIAR siRNAs for 48 hours prior to treatment with 0.4 µM APH. Cells were fixed at regular time intervals, and p(S10)-H3-positive cells were quantified by flow cytometry (mean ± SE, n = 3). B HeLa cells transfected with control or TIAR siRNAs were treated with APH for 24 hours. Pan-nuclear γH2AX signals were quantified by HTM (n = 3, 2000 cells examined per experiment and condition). Each dot represents the signal from one cell, horizontal lines indicate mean values, and the blue area delineates cells above an arbitrarily chosen threshold. Data information: statistical significance was determined by Wilcoxon rank-sum test. ; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. C Chromatin-bound RPA2 signals were quantified by HTM HeLa cells transfected with control or TIAR siRNAs (n = 3, 600 cells examined per experiment and condition). D HeLa cells were transfected with control or TIAR siRNAs for 72 hours prior to treatment with ATRi (5 μM.) alone or together with Ro3306 (5 μM) for 12 hours. Pan-nuclear γH2AX signals were quantified by HTM (n = 3, 1000 cells examined per experiment and condition). Data information: statistical significance was determined by Wilcoxon rank-sum test. ; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. E HeLa cells were transfected with control or TIAR siRNAs were treated with ATRi for 48 hours, and the sub-G1 population was quantified by flow cytometry following propidium iodide staining (mean ± SD, n = 3). Data information: statistical significance was determined by unpaired Student's t-test; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. F HeLa cells transfected with control or TIAR siRNAs were treated with APH, alone or in combination with Gö6976 (1µg/ml) or UCN-01 (300 nM). After 24 hours, p(S10)-H3-positive cells were quantified by flow cytometry, and expressed relative to the values in the untreated condition (mean ± SE, n = 3). Data information: statistical significance was determined by unpaired Student's t-test; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.
E-cadherin saturates with mutant β-catenin over time.Figure 4Immunoprecipitation (IP) of E-cadherin from VilCreER Catnblox(ex3)/lox(ex3) mice, 24 h (day 1, top) and 48 h (day 2, bottom) after induction. The ratio of wild-type (WT, top lane) and mutant exon 3 β-catenin (Δex3, bottom lane) bound to E-cadherin (IP:Ecad) was calculated for each tissue. Graphs show average of experiments (N = 3 mice for each genotype and time point); error bars represent s.e.m. IP of E-cadherin from VilCreER Catnblox(ex3)/+ mice at day 4 and day 8. Graphs show average of experiments (N = 3 mice for each genotype and time point); error bars represent s.e.m.
Figure 4A, Western blot analysis of USP33 proteins in HT1080 cells treated with additional NaCl (+17, 34 and 51 mM) for 12 hrs. Data information: Data are representative of at least two biological replicates B Western blot analysis of USP33 proteins in THP1 cells treated with additional NaCl (+17, 34 and 51 mM) for 12 hrs. Data information: Data are representative of at least two biological replicates C Western blot analysis of USP33 levels in the spleen tissues of mice fed an NSD or HSD for 7 days or 30 days as indicated. Data information: Data are representative of at least two biological replicates D Immunoprecipitation analysis of Viperin ubiquitination in HEK293T cells co-transfected with Flag-Viperin, HA-Ub and shUSP33, then treated with additional NaCl (+34 mM) for 12 hrs. Data information: Data are representative of at least two biological replicates E Western blot analysis of Viperin levels in 2fTGH cells transfected with shCtrl or shUSP33 and then infected with VSV (MOI=1.0, 12 hrs) immediately after addition of NaCl (+34 mM). Data information: Data are representative of at least two biological replicates F Western blot analysis of Viperin in the splenocytes of Usp33+/+ or Usp33-/- mice treated with mIFNβ (500 IU/ml, 12 hrs) immediately after addition of NaCl (+34 mM). Data information: Data are representative of at least two biological replicates G RT-qPCR analysis of VSV RNA levels in 2fTGH cells transfected with shCtrl or shUSP33 and then infected with VSV (MOI=1.0, 24 hrs) immediately after addition of NaCl (+34 mM). Data information: Data (G) represent mean and SD of three biological replicates, and p values were calculated using two-tailed unpaired Student's t-test For all statistical testing: NS, not signiﬁcant (p > 0.05). ***p < 0.001. H Mouse splenocytes of Usp33+/+ or Usp33-/- mice were infected with VSV (MOI=1.0, 24 hrs) immediately after addition of NaCl (+34 mM). VSV-G protein levels were analyzed by Western blot. Data information: Data are representative of at least two biological replicates I Survival curves of 8-week-old Usp33-/- mice fed an NSD (0.5% NaCl) or HSD (4% NaCl) for 7 days and then infected with VSV (1x108 PFU per gram body mouse, n=10). Data information: ; Data (I) show mean and SEM of ten biological replicates, and p value was calculated using logrank (Mantel-Cox) tests. For all statistical testing: NS, not signiﬁcant (p > 0.05). ***p < 0.001.
Figure 2A Z score for AUC and IC50 of 37 different drugs (shown along the horizontal axis) identified as hits in 13 of 17 samples screened. A drug hit is defined as z score of less than -2 for both AUC and IC50. Each dot in a column represents a sample screened for that drug. The size of the dot corresponds to the IC50 z score for that sample (the larger the dot, the smaller the IC50). Dots below the black horizontal line represent sample with AUC z score of less than -2. Dots are color coded for drug hit types. All color dots below the black line represent a hit for the corresponding drug. B Plots of AUC z score against IC50 z score for each of the drugs screened in the 13 samples with drug hits.
Figure 4(A) Sphere formation assay of MGC-803 and MKN-45 cells incubated with 20 μg/mL sEVs from MGC-803 or LSD1 knockout (KO) MGC-803 cells for 7 days (n = 3 biological replicates; mean ± standard error of mean (SEM); **P = 0.0090 (MGC-803) and **P = 0.0012 (MKN-45); two-tailed unpaired Student's t-test; scale bar = 100 µm). (B) In vitro limiting dilution assays performed using MGC-803 (upper panel) and MKN-45 (bottom panel) cells incubated with 20 μg/mL sEVs from MGC-803 or LSD1 KO MGC-803 cells for 14 days (the solid line represents the sphere formation ability curve, while the dashed line represents the 95% confidence interval. The circles and triangles represent data from different groups). (C) Expression levels of LSD1, OCT4, and SOX2 in MGC-803 and MKN-45 cells incubated with 20 μg/mL sEVs from MGC-803 or LSD1 KO MGC-803 cells for 48 h. (D) Quantification of the results of (C) (n = 3 biological replicates; mean ± SEM; *P = 0.0385 (LSD1), *P = 0.0160 (OCT4), and ***P = 0.0006 (SOX2) for MGC-803; *P = 0.0299 (LSD1), *P = 0.0147 (OCT4), and *P = 0.0258 (SOX2) for MKN-45; two-tailed unpaired Student's t-test). (E) Expression level of CD44 in MGC-803 and MKN-45 cells incubated with 20 μg/mL sEVs from MGC-803 or LSD1 KO MGC-803 cells for 48 h. (F) Sphere formation assay results of MGC-803 (upper panel) and MKN-45 (bottom panel) cells incubated with 20 μg/mL sEVs from HEK293T cells (left panel), WT-LSD1 sEVs (middle panel), and LSD1 K661A sEVs (right panel) for 7 days. Scale bar = 100 µm (n = 3 biological replicates; mean ± SEM; **P = 0.0021 (MGC-803) and **P = 0.0020 (MKN-45); two-tailed unpaired Student's t-test).