EMBO/sd-character-level-ner huggingface dataset:
Figure 5A. Mice were co-colonized with E1 and either N1 WT (n=4) or N1 ΔtssC (n=3). Five days post-inoculation, fecal RNA was extracted and tested for BFT expression via qRT-PCR. B. Four weeks after co-colonization with E1 and either N1 WT or N1 ΔtssC (n= 4 mice per group), the sera were collected, tested via ELISA for anti-BFT IgG and end point titer calculated. C-F. Mice pre-treated with DSS were inoculated with no organisms (sham), E1 only, or E1 competed with N1 WT or N1 ΔtssC. Five days post-inoculation, the ceca were weighed (C) and fixed for histopathological examination after sham (D), E1 only (E) and E1-N1 WT (F) colonizations. Scale bars denote 100μm (main image) and 200μm (inset).
Kinetics of morphological changes in vacuoles and peroxisomal GFP-SKL during microautophagic degradation of P.Figure 4Kinetics of morphological changes in vacuoles and peroxisomalGFP-SKL during microautophagic degradation of P. pastoris in glucose adaptation. At least 350 vacuoles or 350 clusters of peroxisomes were counted for each time point. Cells were counted in three randomly selected fields, and standard deviations are shown as vertical bars.
Figure 1A U2OS cells were transfected with Flag-NAT10 or control vectors. Forty-eight hours later, cells were harvested and whole cell extracts were immunoprecipitated with Flag-antibody affinity resin. The NAT10-binding proteins were resolved by SDS-PAGE and detected by silver staining. B-F U2OS, HCT116 p53+/+ or HCT116 p53-/- cell lysates were immunoprecipitated with control IgG, anti-NAT10 (B and F), anti-p53 (C) and anti-Mdm2 (D and E) antibodies. The immunoprecipitates were subsequently immunoblotted with the indicated antibodies. G Purified NAT10 was incubated with GST, GST-p53 or GST-Mdm2 proteins coupled to Glutathione Sepharose 4B. Proteins retained on the Sepharose were then analyzed by western blot using the antibodies as indicated. The amount of GST fusion proteins are shown in the lower panel. H Full-length GST-p53 fusion protein, its deletion mutants or GST protein was used to pull-down experiments with purified NAT10 protein. The levels of the GST fusion proteins are shown in the left panel. I GST pull-down assay was performed using purified GST-NAT10 deletion mutants or GST protein and overexpressed Flag-p53 or Mdm2 protein in HEK293T cells. Schematic diagram represents the constructs of GST-NAT10 deletion mutants (right panel).
Figure 6A, B Core histones were prepared from E13.5 forebrain explants treated with various concentrations of crotonate for 3 h and subjected to western blotting analysis using anti-H3K9cr (A) and anti-Kcr (B) antibodies. Naïve: isolated forebrains without subsequent culture and treatment.E, F GO analysis (biological process) of genes with increased (E) and decreased (F) expression levels after crotonate treatment in E13.5 forebrain explants. G, H Quantitative analysis of changes in H3K9cr (G, left panel of H) and H3K14la (right panel of H) levels by western blotting among different groups of the embryonic forebrain. Data are presented as mean ± SEM of three independent biological replicates, n = 3. Two-tailed unpaired Student's t-test was used to analyze statistical significance; NS: no significance, P < 0.05, P <0.001. I Quantitative analysis of changes in H3K9cr levels in the developing forebrain. Data are presented as mean ± SEM of three independent biological replicates, n = 3. One-way ANOVA with post hoc Tukey's test was used to analyze statistical significance; NS: no significance, P < 0.05, P <0.01.
circHIPK3:miR-30c:DLL4 dysregulation affects cell cycle(A) qPCR of DLL4 expression in stable expression scr and 2 DLL4 KD TREx cell lines at 0 and 24 hours post-induction.Figure 6(A) qPCR of DLL4 expression in stable expression scr and 2 DLL4 KD TREx cell lines at 0 and 24 hours post-induction. GAPDH was used as a housekeeper (n=3). Data information: data are presented as mean ± SD. *P<0.05, **P<0.01, ***P<0.001 (Unpaired Student's t-test). All repeats are biological. (B) Representative western blot of DLL4 levels in scr and DLL4 KD TREx cells with GAPDH as a loading control, densitometry analysis is n=3. Data information: data are presented as mean ± SD. *P<0.05, **P<0.01, ***P<0.001 (Unpaired Student's t-test). All repeats are biological. (C) Representative western blot of ORF65 levels at 0, 24 and 48 hours post induction in scr and DLL4 KD TREx cells, GAPDH was used as a loading control and densitometry analysis performed on n=3. Data information: data are presented as mean ± SD. *P<0.05, **P<0.01, ***P<0.001 (Unpaired Student's t-test). All repeats are biological. (D) qPCR analysis of ORF57 DNA levels for viral load at 72 hours post induction, with scr and DLL4 KD cells with uninduced cells as a control and GAPDH as a housekeeper (n=3). Data information: data are presented as mean ± SD. *P<0.05, **P<0.01, ***P<0.001 (Unpaired Student's t-test). All repeats are biological. (E) qPCR analysis of ORF57 in HEK-293T cells for reinfection assay (n=3). Data information: data are presented as mean ± SD. *P<0.05, **P<0.01, ***P<0.001 (Unpaired Student's t-test). All repeats are biological.
Figure 2(A) In vitro ubiquitination assay. Purified His-RNF114 was subjected to ubiquitination assays with the indicated components and the samples were analyzed by immunoblotting with anti-FLAG (Ub) antibody. Lanes 1-4 indicate negative controls. The results show that recombinant RNF114 efficiently catalyzed the formation of polyubiquitin chains. (B, C) Purified wild-type and RING mutated His-RNF114 were stained by Coomassie Blue (B). Purified wild-type and RING mutated His-RNF114 were subjected to ubiquitination assays together with E1, Ubc4, FLAG-Ub, and ATP. The results show that mutant RNF114 lacked ubiquitination activity (C). (D) Screening for proteins ubiquitinated by RNF114 on a proteome array. Putative substrates of RNF114 were identified on Ubiquitin Ligase Substrate Identification on ProtoArray® Human Protein Microarrays v5.0. Ubiquitination reactions were performed on quintuplicate arrays using the indicated components, the five sub-array are respectively negative controls, E1+E2 (no E3), WT RNF114 (two arrays, duplicate), and mutated RNF114. (E) A heat map comparing hits between E1+E2, E1+E2+WT RNF114, and E1+E2+mutated RNF114 reactions. Candidate substrates of RNF114 selected for further characterization are indicated in red font; HIP1, indicated by blue font, was considered to be a false positive because it was also detected in the mutated RNF114 reaction. Black font indicates proteins with ubiquitin-binding domains such as UIM, UBA or CUE or proteins without clearly reported functions, which were excluded from further investigation.
c-Maf induction enables ILC2s to respond stronger upon re-challenge.Figure 6(A) Treatment schedule. (B) Representative staining and percentage of IL-13+ (ANOVA: F(2,22)=11.37, p=0.0004) and IL-5+ (ANOVA: F(2,22)=18.05, p<0.0001) ILC2s after 3h in vitro stimulation of cell suspension obtained from the lungs of mice treated as in (A). Each dot represent sample from one individual mouse (*p<0.05, ***p<0.001, ****p<0.0001). Bars represent mean ± SEM. (C) Representative histograms of c-Maf and GATA3 expression in ILC2s from adult PBMC or cord blood cells and percentages of c-Maf+ and GATA3+ ILC2s from adult PBMC or cord blood (n=5 different donors; T test, ****p<0.0001). (D) ILC2 expression of the indicated markers. Each dot represent sample from one donor. Bars represent mean ± SEM. (E, F) c-Maf quantification by qPCR (E) and cytokine concentration (F) in the supernatants of adult and cord blood ILC2s unstimulated, stimulated for 3h with PMA-Ionomycin (3h stim) and stimulated for an overnight with IL-33 and then 3h with PMA-Ionomycin (o.n. stim). ILC2s were sorted from 3 different donors. (E) Multiple T tests, unstimulated: p=0.000101; 3h stim: p=0.0036. (F) Multiple T tests, IL-13: p=0.022; IL-5: p=0.015 (*p<0.05). Bars represent mean ± SEM.
Figure 8(A) Experimental plan. Mice were injected with 4T1 cells in the mammary fat pad. When the tumor reached 80-100mm3 size, mice were treated by gavage with glufosinate (10 mg/kg) or vehicle for 14 days. Evaluation of 4T1 tumor growth over the time (B) , in vehicle and glufosinate (10mg/kg) treated mice (pool of 2 independent experiments; 10 mice per condition in total). mice body weight (C) in vehicle and glufosinate (10mg/kg) treated mice (pool of 2 independent experiments; 10 mice per condition in total). number of metastatic nodules (D), in vehicle and glufosinate (10mg/kg) treated mice (pool of 2 independent experiments; 10 mice per condition in total). Total metastatic area (E) in vehicle and glufosinate (10mg/kg) treated mice (n=8). Six images per lung were analysed. Scale bar: 2mm. number of lung metastatic lesions per size by H&E staining (F) with representative images (G) in vehicle and glufosinate (10mg/kg) treated mice (n=8). Six images per lung were analysed. Scale bar: 2mm. (H, I) Quantification of F4/80+ macrophage density (H), of F4/80+ CD206+ cells infiltration in the lung metastasis and representative images (I), in vehicle and glufosinate-treated mice (n=6). Six images per lung were analysed. Scale bar: 20µm. (J) Experimental plan for intravasation setting. Mice were injected with 4T1-GFP+ cells in the mammary fat pad and immediately treated by gavage with glufosinate (10mg/kg) or vehicle for 14 days (neoadjuvant therapeutic regimen). After 14 days of treatment, at the time of tumor resection, blood was collected and analyzed for GFP+ circulating tumor cells (CTCs). Survival studies were performed until the natural death of the post-surgical mice. GFP+ CTCs amount in blood, expressed as GFP expression levels, was determined by qPCR (n=8) (K). Blood was drawn from treated and untreated tumor-bearing mice at the time of tumor resection and plated in culture dishes. The formation of tumor cell colonies was traced over time. Graph shows the number of GFP+ tumor cell colonies (n=8) (L). After treatment and tumor resection overall survival studies were performed until the natural death of the post-surgical mice (n=11) (M). (N) Experimental plan for extravasation setting. GFP-expressing 4T1 were injected intravenously into Balb/c mice pre-conditioned with glufosinate (10mg/kg) or vehicle for 2 days. After 2 days of treatment, the overall lung metastatic breast cancer cell burden was determined. (O) GFP+ cancer cells amount in lungs, expressed as GFP expression levels, was determined by qPCR in vehicle and glufosinate (10mg/kg) treated mice (n=8). The results were normalized to the vehicle group.
a) Relative CK2α protein concentration over time following cytoplasmic microinjection of REF52 cells with the HA-tagged semisynthetic proteins bearing different modifications using immunocytochemistry with HA-specific antibody.figf2(a) Relative CK2α protein concentration over time following cytoplasmic microinjection of REF52 cells with the HA-tagged semisynthetic proteins bearing different modifications using immunocytochemistry with HA-specific antibody. Data represent mean values ± s.d. (b) Relative CK2α protein concentration following injection of β subunit together with CK2α proteins. Data represent mean values ± s.d. (c) Relative CK2α protein concentration following injection of proteasome inhibitor (1 μM MG132) together with unmodified CK2α protein. Data represent mean values ± s.d. (d) Western blot for total CK2α protein in HeLa cells following treatment with Cdk inhibitors. Numbers indicate relative quantification normalized for loading control. Mean values (n = 3) shown ± s.d. (e) Western blot for total CK2α protein in HeLa cells following treatment with nocodazole to arrest cells in mitosis. Full gels corresponding to those in panels d and e are in Supplementary Figure 8.
C/EBP functions in the presence of inhibitory cytokines A Real-time qRT-PCR analysis of C/EBPβ mRNA in CD4+ naïve T cells inactivated (naive) or cultured for 24 hr under conditions as indicated with DMSO or ATRA (all-trans retinoic acid).Figure 1Real-time qRT-PCR analysis of C/EBPβ mRNA in CD4+ naïve T cells inactivated (naive) or cultured for 24 hr under conditions as indicated with DMSO or ATRA (all-trans retinoic acid). Data are representative of two independent experiments with consistent results and normalized with β-actin (mean and s.e.m. of quadruplicates) Immunoblot analysis of C/EBPβ in the nuclear fraction of naïve CD4+ T cells cultured for 2 d under conditions as indicated. The immunoblot was quantified using Image J Flow cytometry of intracellular Foxp3 staining in CD4+ naïve T cells transduced with control retrovirus (MigRI) or retrovirus encoding A-C/EBP or C/EBPζ and cultured for 2 d under conditions as indicated. Representative experiments are shown in the left panel and pooled data with mean value from 8(C are shown on the right Dot plots are gated for CD4+GFP+ and numbers indicate percent Foxp3+ cells in the gate Flow cytometry of intracellular Foxp3 staining in CD4+ naïve T cells transduced with control retrovirus (MigRI) or retrovirus encoding A-C/EBP or C/EBPζ and cultured for 2 d under conditions as indicated. Representative experiments are shown in the left panel and pooled data with mean values fro 6(D are shown on the right. Dot plots are gated for CD4+GFP+ and numbers indicate percent Foxp3+ cells in the gate Flow cytometry of intracellular Foxp3 staining in CD4+ naïve T cells transduced with control retrovirus (MigRI) or retrovirus encoding A-C/EBP or C/EBPζ and cultured for 2 d under conditions as indicated. Representative experiments are shown in the left panel and pooled data with mean values fro E 7(F are shown on the right. Dot plots are gated for CD4+GFP+ and numbers indicate percent Foxp3+ cells in the gate
Model of potential roles of autophagosome-amphisome in PV production and cell exit.Figure 8We have demonstrated that initiation of autophagy is required for viral RNA replication, while vesicle acidification is required for maturation of virus particles. In our model, by 2-3 h post-infection processed 3A protein integrates into single-membraned structures resembling secretory vesicles, which become the initial sites of viral RNA replication. 3-MA either inhibits formation of these single-membraned vesicles which act as the site of RNA replication, or inhibits the progress of these vesicles into double-membraned vesicles, which act as replication sites. As the peak of viral genome replication (3 h) passes, the autophagic machinery is engaged to change the single-membraned vesicles into double-membraned vesicles. As a result, some of RNA replication complexes and capsid proteins are trapped within the double-membraned vesicles, while some are cytosolic. As the amphisomes acidify, particle maturation is more efficient inside the vesicles. Maturation of the amphisomes can be inhibited using NH4Cl or bafilomycin A1. The mature virions, surrounded by two membranes, have a topological problem in exiting the cell. We propose three possible mechanisms for exit. [1] The outer membrane fuses with the plasma membrane, releasing a short-lived labile single-membraned vesicle containing virus, which eventually is released into the extracellular milieu. [2] The double-membraned vesicles lose one membrane, as occurs during maturation of cellular autophagosomes, and the single-membraned vesicles fuse with the plasma membrane. [3] Virus exits the vesicle into the cytoplasm due to breakup of the vesicle or another mechanism. Virus then exits the cell when lysis occurs at approximately 8 h post-infection.
Vps24 is required for efficient clearance of Htt inclusions.Figure 9.Vps24 is required for efficient clearance of Htt inclusions. HeLa cells expressing HttQ65-mCFP (A) or HttQ103-mCFP (B) and N2a cells expressing HttQ103-mCFP (C) were transfected with control or Vps24 siRNA for 2 d and then incubated in the absence or presence of doxycycline for 3 d to turn off expression of HttQ65/Q103-mCFP. Vps24 depletion was determined by Western blot analysis. (A and C) Clearance of SDS-insoluble Htt inclusions was analyzed by filter-trap assays. (B) Alternatively, aggregate clearance was analyzed by confocal quantification of the number of HeLa HttQ103 cells having visible inclusions after 3 d in the absence or presence of dox. 300 cells from three independent experiments were counted for each condition. Error bars = SD.
Figure 1A HeLa-H2B/tub cells were transfected with control or TIAR siRNAs for 48 hours and synchronized by double thymidine (TT) block. After release from the block, mitotic cells were counted by fluorescence microscopy (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. B Time from S- to M-phase was measured in si-RNA transfected HeLa-H2B/tub cells by time-lapse microscopy following release from TT block (4 repeat experiments; all measurements depicted). Data information: Statistical significance was determined by unpaired Student's t-test; *, p < 0.05; **, p < 0.01; ***, p < 0.001. Western blot analysis of unsynchronized HeLa-H2B/tub cells was carried out to monitor expression of p(S10)-H3, total H3 and TIAR in control and TIAR-depleted cells (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. D HeLa cells were transfected with control or TIAR si-RNAs, alone or together with Cdc25B siRNA. 72 hours after transfection, p(S10)-H3-positive cells were quantified by flow cytometry (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. HeLadox-YFP-TIARr cells were transfected with control or TIAR siRNAs, and 24h hours later cell were cultured in the abscence or presence of 1 μg/ml doxocycline for 48 hours. The expression of p(S10)-H3, total H3 and TIAR was measured by Westen blot analysis. The graph shows the quantification of p-H3 levels normalized to total H3 (mean ± SD, n = 4). Data information: Statistical significance was determined by unpaired Student's t-test; *, p < 0.05; **, p < 0.01; ***, p < 0.001. F HeLadox-YFP-TIARr-RRM123m cells were analyzed as in panel (E) (mean ± SD, n = 4). Data information: Statistical significance was determined by unpaired Student's t-test; *, p < 0.05; **, p < 0.01; ***, p < 0.001. G HeLadox-YFP-TIARr-dQRD cells were analyzed as in panel (E) (mean ± SD, n = 4). Data information: Statistical significance was determined by unpaired Student's t-test; *, p < 0.05; **, p < 0.01; ***, p < 0.001.
Compromising Hsc70 binding potentiates expression of a heat shock reporter under non-stress and heat shock conditions in HeLa cells.Figure 7A Schematics of the used system. HeLa cells were stably transfected with mTag-BFP expressed under the control of the HSPA6 promoter and subsequently transiently transfected with plasmids that expressed HSF1wt or mutant variants in an artificial operon with hrGFP, which is translated from an IRES element. Exemplary flow cytometry data plotting BFP fluorescence versus GFP fluorescence, indicating the gates used for analysis. Numbers indicate the fraction (%) of cells (forward scatter singlets) in each gate. Color indicates the cell density from dark blue (low) to red (high). NS, cells grown at 37°C; HS, cells heat shocked at 43°C for 1 h with a recovery period of 3 h at 37°C. C-H Geometric mean of BFP (C, E) and GFP fluorescence (D, F, H) in the GFPhigh (C, D), GFPlow (E, F), and GFPlow-BFPhigh43 (H) gates. Of note, the number of data points show the number of experiments in which a significant number of cells was found in this gate and missing bars indicate that cells did not fall within this gate in more than two out of four experiments. (G) Geometric mean of BFP fluorescence weighted by the relative number of cells in the GFPlow-BFPhigh43 gate (fluorescence * fraction forward scatter singlets). Note that the GFPlow gate also comprises the GFPlow-BFPhigh37 and that the GFPlow-BFPhigh37 gate also comprises the GFPlow-BFPhigh43 gate. Hsf1-202SSS, Hsf1-I202S,L203S,V205S; Hsf1-209SSS, Hsf1-I209S,L211S,L213S; Hsf1-427SSSS, Hsf1-M427S,L429S,L432S,L436S; Hsf1-465SSS, Hsf1-L465S,V466S,Y468S; Hsf1-473SSSS, Hsf1-L473S,F474S,L475S,L476S
Figure 1A schematic depiction of our GPS random peptide assay. The GPS reporter relies on the expression of two fluorescent proteins from a single promoter, enabled by an internal ribosome entry site (IRES). A 12-residue peptide library was fused to the C-terminus of GFP, whereas RFP serves as an internal control. The GFP/RFP ratio represents protein stability. The GPS peptide library was delivered into cells as a single copy so that each reporter cell expressed a unique GFP-peptide fusion construct. The "% degradation", i.e. the % of library cells displaying a decreased GFP/RFP ratio, reflects the % of peptides within the library that promote GFP degradation. The % degradation of random peptide libraries terminating in the indicated amino acids or C-terminal motifs labeled at bottom. X represents any amino acid residue. To illustrate the reliability of this approach, libraries terminating in each specific amino acid or motif were independently constructed three times and each independent library was analyzed by FACS in triplicate (three random samplings with 105 cells each time). Data are presented as mean ± standard deviation from the nine replicates described above. Since the sample space of all 12-residue peptides far exceeds the size of random sampling, the probability of retrieving repeated peptides in random samples is extremely low. Thus those random samples are expected to carry distinct peptides and should be considered as biological replicates. P values were calculated by unpaired t-test. GPS reporter cells expressing peptide libraries ending with a random (X) or Gly (G) residue were treated with dominant-negative (DN) cullins and analyzed. Data are presented as mean ± standard deviation from nine replicates. GPS reporter cells carrying indicated libraries were treated with shRNAs against various BC-box proteins and analyzed. VHL and FEM1C serve as unrelated BC-box protein controls. Data are presented as mean ± standard deviation from nine replicates. The relative % degradation of indicated libraries in cells with or without assigned BC-box proteins. A value of 1 indicates no difference. BC-box proteins were depleted by shRNA-mediated knockdown and FEM1C served as an irrelevant BC-box protein control. Amino acids with different biophysical properties are marked with different colors, i.e. small aliphatic, hydrophobic, basic, acidic and amide residues are orange, green, blue, magenta and crimson, respectively. Libraries terminating in each specific motif were independently constructed twice and each independent library was analyzed in triplicate. Data are presented as mean ± standard deviation from six replicates. GPS reporter cells expressing indicated diGly-ending full-length human proteins were treated with or without an shRNA against KLHDC2 and analyzed by FACS.
Figure 6(A) (Top) Low magnification view showing organoids embedded in Matrigel (left) next to the liver parenchyma (right) two weeks after engraftment. H&amp;amp;E, hematoxylin eosin staining. (Bottom) Immunofluorescence staining for GFP expression to mark the engrafted organoids and DAPI counterstaining of the cell nuclei. The liver parenchyma corresponds to the right half of the images. Scale bar: 200 m. (B) Characterization of engrafted organoids by immunofluorescence (IF). Almost no organoid cell expressed Ki67 (the one positive cell shown in the image was an exception), in contrast to the high levels of cell proliferation and Ki67 expression observed in organoids cultured in vitro. Scale bar: 25 m.