EMBO/sd-character-level-ner huggingface dataset:
expressionA) HSF1 was identified as a strong occupier of the PROM2 promoterB) MCF10A cells were treated with either 25uM 4HNE or 5uM RSL3 for times that ranged from 0 to 120 minutes.Figure 3HSF1 was identified as a strong occupier of the PROM2 promoter MCF10A cells were treated with either 25uM 4HNE or 5uM RSL3 for times that ranged from 0 to 120 minutes. mRNA was isolated from each time point and HSF1 expression was quantified by qPCR. Shown is an average of three independent experiments with standard deviation (n=3 experiments per time point). P‑values were obtained by unpaired Student's t‑test with *P<0.05, **p<0.01, ***p<0.005. Exact p‑values are reported in the Appendix Table S1. MCF10A cells were pretreated for 15 minutes with either DMSO or 10uM BIRB, followed by 60 minutes with either DMSO, 25uM 4HNE, 5uM RSL3, 4HNE and BIRB or RSL3 and BIRB. mRNA was isolated and HSF1 expression was quantified by qPCR. Shown are three independent experiments with standard deviation (n=3 experiments per group). P‑values were obtained by unpaired Student's t‑test with *P<0.05, **p<0.01, ***p<0.005. Exact p‑values are reported in the Appendix Table S1. MCF10A cells were pretreated for 15 minutes with either DMSO, EtOH, or 10uM BIRB, followed by 60 minutes with either DMSO, EtOH, 25uM 4HNE, 5uM RSL3, 4HNE and BIRB or RSL3 and BIRB. Isolated protein was assessed by immunoblotting for prominin2, phospho-HSF1 (S326), total HSF1, phospho-p38 (Thr180/Tyr182), total p38 and β-actin expression. Shown is one replicate of three independent experiments (n=3). MCF10A cells were plated on slides pre-coated with laminin. Cells were then pretreated for 15 minutes with either DMSO, EtOH, or 10uM BIRB, followed by 60 minutes with either DMSO, EtOH, 25uM 4HNE, 5uM RSL3, 4HNE and BIRB or RSL3 and BIRB. Cells were stained for total HSF1 and counterstained with DAPI. Images were taken at 20x magnification. Scale bar is 50 microns. Shown is one replicate of three independent experiments (n=3). HSF1 expression was diminished in MCF10A cells for 48 hours using siRNA prior to treatment with either DMSO, EtOH, DMSO, 25uM 4HNE or 5uM RSL3. Isolated protein was assessed by immunoblotting for prominin2, phospho-HSF1 (Ser326), total HSF1, phospho-p38 (Thr180/Tyr182), total p38, and β-actin expression. Shown is one replicate of three independent experiments (n=3). HSF1 expression was diminished in MCF10A cells for 48 hours using siRNA prior to treatment with either DMSO or RSL3 at the range of concentrations shown. Cells were assessed for viability after 24 hours. Absorbance was normalized to DMSO control. Shown are three independent replicates with standard deviation (n=3 experiments per group). P‑values were obtained by unpaired Student's t‑test with *P<0.05, **p<0.01, ***p<0.005. Exact p‑values are reported in the Appendix Table S1.
Figure 3Feature weight prediction in M. pneumoniae. Weights of the different features considered in the classification by RanSEPs. Bars indicate the global averaged variance that each feature explains by itself along with its associated standard deviation (black line). Method accuracy comparative. Receiver operating characteristic curve for RanSEPs (orange) and five additional tools (blue gradient). The closer a curve to the left-hand border, the more accurate the tool. The area under the curve (AUC) associated with each method is presented, with values closer to 1 indicating a more accurate method. The dashed grey line represents a classifier that assigns the coding class randomly. Boxplot representing the relationship between RanSEPs positive ('RanSEPs+', score≥0.5) and negative ('RanSEPs-', score<0.5) SEPs predictions and associated RCV (ribosome profiling ratio coverage, in log2) in E. coli. Only annotations ≤300 nucleotides in length were included. As positive and negative controls, we considered annotated SEPs ('Annotated') and non-coding RNAs ('ncRNAs'), respectively. Annotations within RanSEPs+, RanSEPs- and ncRNAs overlapping with known annotated genes were excluded. Annotations with RCV=0.0 are filtered out and the number within the box represents the percentage of values in that class that are kept in the comparative. Along the top, p-values computed by Mann-Whitney rank test are indicated.
a) Proteolysis in HEK293 cells stably transfected with an empty plasmid (Ctr) or plasmids expressing WT or G/S LRRK2 under the control of a tet promoter, treated with doxycycline to activate protein expression and then labeled with [3H]leucine for 48 h.figf4(a) Proteolysis in HEK293 cells stably transfected with an empty plasmid (Ctr) or plasmids expressing WT or G/S LRRK2 under the control of a tet promoter, treated with doxycycline to activate protein expression and then labeled with [3H]leucine for 48 h. After extensive washing, protein degradation at the indicated times was measured, in cells maintained either in the presence (+) or absence (-) of serum, as the amount of acid-precipitable radioactivity (amino acids and small peptides) released into the media. Values are expressed as percentage of radioactivity in proteins (acid precipitable) at time 0 (n = 4 independent experiments, each in triplicate). (b) The same cells, transiently transfected with a KFERQ-PA-mCherry1 fluorescent reporter for CMA activity. After photoactivation, cells were maintained for 16 h in the presence or absence of serum. Left: representative micrographs; right column shows higher magnification images of outlined regions. Arrows denote puncta. Right: average number of puncta per cell in each condition, measured in >150 cells. (c) Cultured ventral midbrain dopaminergic neurons from Ctr and from WT-LRRK2 and G/S-LRRK2 transgenic mice, transduced with the same reporter as in b and co-stained with antibody to tyrosine hydroxylase (TH). Nuclei are highlighted with DAPI (4′,6-diamidino-2-phenylindole) in blue. Left: representative images. Right: average number of puncta per cell in >50 TH-positive cells. Scale bars, 10 μm. All values are mean + s.e.m.; differences with Ctr (*) and between WT LRRK2 and G/S LRRK2 (§) were significant at P 0.05; unpaired t-test in a and ANOVA and Bonferroni test in b,c.
Mesenchymal-like PDAC cells deactivate PSCs.Figure 3(A) Gene expression level of stromal activation markers COL1A1, ACTA2, SPARC, FN1 and FAP in PS-1 cells which were exposed for 72 hrs to CM from control (CM of PS-1 cells ), epithelial (blue) or mesenchymal (red) PDAC cell lines using qPCR. Values were normalized to control and n=6 per group, 3 biological replicates for each cell line. Data information: data are represented as mean±SD, Student's t-test. (B) Heatmap representation of COL1A1 and ACTA2 relative gene expression levels measured with qPCR in PS-1 cells after treatment using a large cohort of PDAC cell lines and primary cell cultures 67 and 53m. Values were Log2 transformed and normalized to Suit-2 expression. Data information: data are represented as mean±SD, Student's t-test. (C) Brightfield images of PS-1 cells exposed to 10 days of treatment with 1 and 10 µM all-trans retinoic acid (ATRA). Scale bar represents 100 µm. (D) Gene expression levels of COL1A1 and ACTA2 in PS-1 cells after treatment using qPCR, values were normalized to control. n=3 technical replicates per group. Data information: data are represented as mean±SD, Student's t-test. (E) Brightfield images of primary human CAFs exposed to 72 hrs of treatment with CM from control (CM of primary CAFs) or mesenchymal PDAC cell lines. Scale bar represents 100 µm. (F) Relative gene expression levels of COL1A1 and ACTA2 in primary CAFs after treatment using qPCR, values were normalized to control. n=3 technical replicates per group. Data information: data are represented as mean±SD, Student's t-test.
Figure 6A. Intravenously injected Cy5-labeled 2'-O-MOE-PSoligonucleotide is distributed to intracranialGBM8tumor. In the normal brain, the signal is observed in blood vessels but not within brain parenchyma. "T" - tumor, "B" - brain tissue. Each image is representative of three mice analyzed. B-D. Systemic inhibition of miR-10b markedly reduces tumor burden. Uncomplexed 2'-O-MOE-PSmiR-10b inhibitor (miR-10b-i) or non-targeting control of the same chemistry were injected at 80 mg/kg through the tail vein at the days indicated by arrows.B. The left panels illustrate tumorimages of representative animals at day 34, and average signals (photons/sec) are indicated below the images. The bars represent average signal ratios for each group at day 34, relative to day 6 B-D. Systemic inhibition of miR-10b markedly reduces tumor burden. Uncomplexed 2'-O-MOE-PSmiR-10b inhibitor (miR-10b-i) or non-targeting control of the same chemistry were injected at 80 mg/kg through the tail vein at the days indicated by arrows.B. The left panels illustrate tumorimages of representative animals at day 34, and average signals (photons/sec) are indicated below the images. The bars represent average signal ratios for each group at day 34, relative to day 6.C. Each mouse was sacrificed when the tumor-generated signal reached 5X107 photons/sec, and Kaplan-Meier survival plots were built retrospectively.D. Growth curves of individual tumors based on the ratios of bioluminescence signals to the baseline signals at day 6. E. The efficacy of miR-10b inhibition in intracranial tumors was assessed by qRT-PCR analysis of the resected tumor tissues, with miR-10b expression levels normalized to miR-125b. F. qRT-PCR analysis demonstrates that miR-10b targets were de-repressed in orthotopic GBM8 upon systemic administration of the miR-10b inhibitor. Seven tumors per condition, and two specimens per tumor have been analyzed. mRNA expression levels were normalized to GAPDH. G. Inverse correlation between miR-10b levels and expression of its mRNA targets in resected GBM8 tumors.
Figure 1A. Frequencies and total numbers (mean +SEM) of CD11b+myeloid cells isolated from the spleen and peripheral LN of naïve non-immunized mice. B. Total numbers (mean +SEM) of CD11b+ cells expressing IL-1β shown in (A) and restimulated with indicated stimuli for 4 h. C. Quantification (mean +SEM) of myeloid cell populations expressing IL-1β, isolated from LN and restimulated with GM-CSF shown in (B). D. Frequencies and total numbers (mean +SEM) of CD11b+myeloid cells isolated from the spleen of immunized mice. E. Analysis of IL-1R1 expression by CD11b+cells isolated from dLN of mice treated as in (D). Data are representative FACS histogram overlays, gated on VD-CD11b+cells with mean frequencies per group. F. Quantification (mean +SEM) of IL-1β expression by CD11b+ cells shown in (E). G. Analysis of IL-1β expressing CD11b+cells shown in (E). Data are representative FACS plots, gated on VD- CD11b+IL-1β+cells with mean frequencies per group.H. Quantification (mean +SEM) of myeloid cell populations expressing IL-1β shown in (G).
Fig.Figure 2A Experimental scheme of retroviral injection paradigm. Adult mice were stereotactically co-injected with the MMLV CAG-dnLEF-IRES-GFP (dnLEF) and CAG-RFP (control) and were sacrificed 17dpi. B Representative images of transduced adult-born neurons at 17dpi. CAG-dnLEF-IRES-GFP (dnLEF, green) and CAG-RFP (control, red) double transduced cells (arrows) and CAG-RFP single transduced cells (arrowheads). Scale bar= 20 µm C Representative reconstructions of control and dnLEF neurons. Scale bar= 20 µm D Analysis of morphology showed a reduction in dendritic length in dnLEF transduced neurons [p<0.0001], while the number of branch points remained comparable [p=0.7914]. Sholl analysis displayed a reduction in dendritic complexity in dnLEF transduced neurons and indicated decreased growth of terminal dendritic branches [p<0.0001] (control: n= 20 cells from 3 animals, dnLEF: n= 38 cells from 6 animals). E DnLEF neurons displayed basal dendrites [p<0.0001] (control: n= 20 cells from 3 animals, dnLEF: n= 38 cells from 6 animals). F Schematic representation of the conditional alleles of the Ctnnb1(ex3) fl (β-catex3) mouse model and the retroviral paradigm used to analyze canonical Wnt signaling gain of function in neural progenitors. Control animals harbor the wildtype allele for Ctnnb1. G Representative images depicting CAG-GFP-IRES-Cre transduced adult-born neurons in control and β-catex3 mice at 17dpi. Scale bar= 20µm H Representative reconstructions of control and β-catex3 neurons. Scale bar= 20µm I Quantification showed decreased dendritic length of β-catex3 neurons [p<0.0001]; no difference was apparent in number of branch points [p=0.3256]. Sholl analysis displayed a less complex dendritic tree of β-catex3 neurons [p<0.0001] (control: n= 20 cells from 5 animals, β-catex3: n= 20 cells from 5 animals). J The number of basal dendrites was increased in β-catex3 neurons [p=0.0003] (control: n= 20 cells from 5 animals, β-catex3: n= 20 cells from 5 animals).
Figure 8(A) Western blotting of livers isolated from the indicated mice at 3 months of age using the indicated antibodies. (B) Quantification of band intensity. Values are average ± SD (n = 3 mice for each genotype). Significance was calculated using ANOVA with post-hoc Tukey: *p<0.05, **p<0.01, ***p<0.001.
Figure 6Monocyte recruitment in the draining lymph node requires collective DC activation (A) Representative dot plots showing the percentage of monocytes (CD11b+ Ly6Chigh cells, black box) present in popliteal dLN of WT:Irf3‑/‑Irf7‑/‑ chimeric mice injected with poly(I:C) (middle and right) or with PBS (left). CD11b+ Ly6Clow/int cells corresponded to neutrophils and were not included in the analysis. Monocyte recruitment in the draining lymph node requires collective DC activation (B) Flow cytometric quantification of monocyte numbers in the lymph nodes of mice injected with poly(I:C) expressed as a fold change over PBS controls. Data are pooled from 2 independent experiments. (C‑D) Activation of NK cells in the draining lymph node requires collective DC activation. (C) Representative histograms showing granzyme B and CD69 expression by NK 1.1+ Ly6Clow cells in dLN of 90:10 (dark blue) and 5:95 (grey) WT:Irf3‑/‑Irf7‑/‑ chimeric mice injected with poly(I:C) or with PBS (black line). (D) Quantification of granzyme B intracellular content (left) and CD69 expression (right) in NK1.1+ Ly6Clow cells of chimeric mice injected with poly(I:C), expressed as a fold change in gMFI over PBS‑injected controls. Data are pooled from 2 independent experiments. (E‑F) Crosspriming of CD8+ T cells requires DC activation and type I IFN. (E) Representative dot plots showing the percentage of CD44high Kb‑OVA tetramer+ CD8+ T cells in the draining lymph node of WT and Irf3‑/‑Irf7‑/‑ mice injected with PBS, OVA or a mixture of OVA plus poly(I:C). (F) Flow cytometric quantification of CD44high H‑2Kb‑OVA tetramer+ CD8+ T cells in the draining lymph node of WT mice injected with PBS (grey dots), OVA (black dots), OVA plus poly(I:C) (blue dots) and in draining lymph node of Irf3‑/‑Irf7‑/‑ mice injected with OVA plus poly(I:C) (orange dots). Data are pooled from 2 independent experiments. Collective DC activation is essential for the generation of T cell responses. (G) Representative dot plots showing the percentage of CD44high tetramer H‑2Kb‑OVA+ CD8+ T cells in the draining lymph node of chimeric mice injected with PBS or a mixture of OVA+poly(I:C). Collective DC activation is essential for the generation of T cell responses. (H) Flow cytometric quantification of CD44high H‑2Kb‑OVA tetramer+ CD8+ T cells in the draining lymph node of 90:10 and 5:95 (WT:Irf3‑/‑Irf7‑/‑) chimeric mice injected with PBS or OVA+poly(I:C). Data are pooled from 2 independent experiments.
Figure 1(A) MOG1 interacts genetically with the H2B deubiquitination machinery. Ten-fold serial dilutions of the indicated strains were spotted on YPD and incubated for 2 days at the indicated temperatures (B) Expression levels of total H2B and H2Bub1 were analyzed in wild-type (WT), mog1Δ, ubp8Δ and ubp8Δmog1Δ whole cell lysates by western blotting using anti-total H2B and anti-H2Bub1 antibodies. Bar graphs of H2Bub1 levels after H2B normalization show the mean and standard deviations of at least three independent experiments. The P-value was calculated using Student´s t-test. Error bars represent SD. The P-value was calculated using Student´s t-test (*, P = 0.01-0.05; **, P = 0.001-0.01; ***, P < 0.001) Ten-fold serial dilutions of the indicated strains were spotted on YPD and incubated for 2 days at the indicated temperatures (C), (D and were then spotted on YPD or YPGal and incubated for 2 days Ten-fold serial dilutions of the indicated strains were spotted on YPD and incubated for 2 days at the indicated temperatures (E and were then spotted on YP and incubated for 2 days
Ongoing autophagy is essential for the continued survival of old HSCs.Figure 5a, qRT-PCR analyses of pro-autophagic genes in old HSCs (n = 3-5). Results are expressed as log2 fold expression compared to young HSCs (set to 0). Apoptosis levels in cultured young and old HSCs (n = 3). Results are expressed as percent caspase activation compared to +cytokines young HSCs. Hatching indicates -cytokines conditions. 2-NBD glucose uptake in cultured young and old HSCs (n = 3). Percent colony formation in young and old WT HSCs cultured for 32h ± BafA and methylpyruvate (M-Pyr) (n = 3). Colonies were counted at day 10 and normalized to -BafA conditions. Data are means ± s.d. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.
a) Wild-type, ubp3Δ or bre5Δ strains containing either a control plasmid (pRS315) or plasmids expressing Ubp3p or catalytically inactive Ubp3p-C469A from the endogenous promoter were spotted in serial dilutions onto YPD plates with (left) or without (right) 5 nM rapamycin.figf4(a) Wild-type, ubp3Δ or bre5Δ strains containing either a control plasmid (pRS315) or plasmids expressing Ubp3p or catalytically inactive Ubp3p-C469A from the endogenous promoter were spotted in serial dilutions onto YPD plates with (left) or without (right) 5 nM rapamycin. The plates were photographed after 2 days at 30 °C. (b) Induction of a GCN4-LacZ reporter was measured 0, 2 and 4 h after amino-acid starvation, and is plotted as Miller units. (c) Polysome profiles of exponentially growing wild-type and ubp3Δ cells were determined in rich medium or 3 h after nitrogen starvation. The arrows mark the 60S peak. (d) Wild-type, atg7Δ and ubp3Δ cells containing either RPL25-GFP, control GFP (HOG1-GFP) or RPS2-GFP were analysed as in Fig. 1c. One of three representative experiments is shown. (e) Ribosomes and associated proteins were immunopurified from starved wild-type and ubp3Δ cells containing (RPL25-GFP) and (9 × MYC-UBI) as indicated. The GFP immunoprecipitates (IP; left) and total extracts (TCA, right) were analysed by immunoblotting with anti-Myc antibodies to reveal ubiquitinated proteins (top). Ub, ubiquitin. Bottom: immunoblotting with anti-GFP antibodies controls for the presence of Rpl25p-GFP, and anti-Rpl1p antibodies confirm equal loading of the extract. The asterisk marks protein G on the beads recognized by the secondary antibody.
core complex.Figure 2A, B Native PAGE separation reveals distinct detergent-sensitive PIN1-complexes. Plasma membrane preparations from dark grown Arabidopsis cell suspension cultures were solubilized with (a) Complexiolyte 27 (a mixture of ionic and non-ionic detergents), and (b) Complexiolyte 47 (lower stringency, non-ionic detergent), (both Logopharm); first dimension BN-PAGE, second dimension SDS-PAGE, blots stained with anti-PIN1 antibody revealing distinct PIN1 complex populations at the indicated positions. Values are given in KDa. c) Molecular abundance (abundancenormspec values calculated as summarized peptide PVs divided by the number of MS-accessible protein specific amino acids) of PIN subunits and ABCB19 in anti-GFP affinity purification of CL47-solubilized PIN1-GFP-expressing roots with a GFP-specific DARPin. Note the significant heteromerization of several endogenous PINs with PIN1-GFP and the absence of other abundant interaction partners (ABC19B shown as an example).
Figure 2(A) Mass spectrometry (MS) analysis of aryl hydrocarbon receptor (AhR) pulldown protein from Mulberry bark derived exosome-like nanoparticles (MBELNs) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) treated MC38 cell lysate. Data from three technical replicates are shown. (B) Co-immunoprecipitation of COP9/COP9 Constitutive Photomorphogenic Homolog Subunit 8 (COPS8) bound AhR from MC38 cell lysate treated with MBELNs. Data are mean ± SEM from three biological replicates, **P<0.01 using Student's t-test. (C) Western blot analysis showing expression of COPS8 in mice ileum crypts. Data are mean ± SEM of three biological replicates, **P<0.01 using Student's t-test. (D) Representative images showing effect of MBELNs with and without TCDD (1 nm)/ carbobenzoxy-Leu-Leu-leucinal (MG132; 20 µM) on co-expression of AhR and COPS8 using immunofluorescence. Scale bar 10 μm, images are from three biological replicates. (E) Western blot showing MBELNs, TCDD and MG132 effect on co-expression of AhR and COPS8. The data represent as mean ± SEM from three biological replicates **P<0.01, ***P<0.001 using one-way ANOVA. (F) Heat map showing MS analysis of AhR pulldown protein from MC38 cells treated with MBELNs in combination with proteasome inhibitor, MG132. Data are represented from three technical replicates. (G) Expression of Cullin 1 (CUL1) and neddylated CUL1 (N-CUL1) following MBELN treatment using Western blot. Data are mean ± SEM from three biological replicates. **P<0.01 using Student's t-test. (H-I) Western blot showing expression of COPS8 in wildtype (Wild) and AhR-KO MC38 cells and expression of AhR in wild and COPS8-KO MC38 cells. MC38 cells were KO using CRISPR/ CAS9 KO plasmid transfection. Data represents mean ± SEM from three biological replicates, **P<0.01, NS- nonsignificant, using one-way ANOVA.
Fig.Figure 7A: Impaired y-H2AX and 53BP1 accumulation in Ubc9 D100A cells. Stable U2OS cells were transfected with siRNA against endogenous Ubc9 for 72 h. Cells were treated with 125 µM H2O2 and immunofluorescence microscopy was performed. Left panel: representative immunofluorescence images. Right panel: quantification of cells with γ-H2AX and 53BP1 foci. Error bars represent S.E.M, n=3 independent experiments * p < 0.05. B: H2O2 exposure leads to prolonged DNA damage in Ubc9 wt and Ubc9 D100A cells. Cells were treated as described in A. Stable U2OS cells were transfected with siRNA against endogenous Ubc9 for 72 h. Cells were treated with 125 µM H2O2 and immunofluorescence microscopy was performed. An alkaline comet assay was performed to evaluate the DNA damage. Representative images are shown. Error intervals represent S.E.M, n=4 independent experiments.
Figure 5 (A, B) Representative immunoblots (A) and quantification of Hrs, TSG101, CHMP2b, and Rab35 protein levels (B) from 14 DIV neurons treated with either DMSO (CON) or glucocorticoids (GC). GC treatment selectively decreases Rab35 protein levels without affecting levels of Hrs, TSG101, or CHMP2b (n=9-10/condition, unpaired student's t-test, *p=0.0221)   (C) Rab35 mRNA levels are decreased by GC in 14 DIV hippocampal neurons and N2a cells (for neurons, n=9/condition, unpaired student's t-test, **p=0.0036; for N2a cells, n=6/condition, unpaired student's t-test, *p=0.0138)   (D) Schematic diagram of GC and vehicle control (CON) treatment in rats for 15 days  (E, F) Representative immunoblots (E) and quantification of levels of different Rab proteins (F) in hippocampus of GC-treated and CON animals. Protein levels of Rab35, but not other Rabs, are decreased in GC-treated animals compared to CON ones (n=5 animals/condition, unpaired student's t-test, *p=0.0257)   (G, H) Immunofluorescence staining of Rab35 (green) and DAPI (blue) (G) showing that Rab35 fluorescence intensity is reduced in hippocampal area (CA1) of GC-treated aniamls (H) (n=15 slices/condition; unpaired student's t-test, ***p=0.0003). Data information: All numeric data represent mean ± SEM