EMBO/sd-character-level-ner huggingface dataset:
Figure 8Changes in LAMP2A expression. RT-qPCR was performed to detect actin and LAMP2A mRNA following treatement with siCTRL or siLAMP2A in sNASP-FL and UBR7-FL 293T cells. Grey dotted line represents expression level in siCTRL treatment. Error bar represents mean +/- standard deviation. A two-tailed one sample t-test was used to calculate p-values (italics) associated with group means difference from a hypothetical value of 1.0. Experiment was performed twice. Immunoblot for LAMP2A in parental 293T cells or cells expressing UBR7-FL (left) or sNASP-FL (right) following treatment with siCTRL, siLAMP2A, or MG-132. Immunoblot for total and modified histones following anti-FLAG IP from the soluble fraction of parental 293T cells, or sNASP-FL treated with siCTRL, siLAMP2A, or MG-132. D. Immunoblot for total and modified histones following anti-FLAG IP from the soluble fraction of UBR7-FL 293T cells treated with siCTRL, siLAMP2A, or MG-132. Quantification of data represented in (C, D). Error bars represent mean +/- standard deviation. A two-tailed one sample t-test was used to calculate p-values (italics) associated with group means difference from a hypothetical value of 1.0 (represented by dashed line). Experiments in (C-E) were performed three times.
Figure 1A. Schematic of UPF3B mutagenesis strategies using CRISPR-Cas9. UPF3B locus is in black where rectangles represent exons and horizontal line denotes introns; coding region is shown as wider rectangles. Red arrowheads represent guide RNA targeting sites. Top: two guide RNAs delete the UPF2 binding domain (2BD) of UPF3B protein coding region as shown to create 3BΔ2BD cells. Bottom: a donor template is used to insert puromycin resistant gene (PuroR) and bovine growth hormone (BGH) polyadenylation signal at the cut site to generate UPF3B knockout (3BKO) cells. B. Immunoblot of parental wild-type (WT) and UPF3B mutant cell lines showing levels of proteins on the right. In 3BΔ2BD, a smaller UPF3B protein with deletion of amino acids 20-155 (UPF3BΔ20-155) is expressed. Relative Expression (Rel Exp) of this deletion protein as compared to the full-length UPF3B along with standard error of mean (SEM) are indicated below lane 2. UPF3B antibody recognizes antigen outside the deleted region in 3BΔ2BD. HNRNPA1 is used as a loading control. ND = UPF3B levels not determined. C. Cumulative Distribution Function (CDF) plots of PTC+ isoforms and PTC- isoforms from the same set of genes. X-axis represents fold change in 3BΔ2BD versus WT cells each with control knockdown (siNC). Number of transcripts in each set (n) and p-value from Kolmogorov-Smirnov (KS) test comparing the two distributions are shown. D. Bar plots from isoform specific RT-qPCR analysis showing average fold change (y-axis) of PTC+ and PTC- isoforms from genes indicated on the bottom in WT and the two UPF3B mutant cells identified in the legend on the top right. For each isoform, levels in mutant cells are compared to the levels in WT cells (set to 1). E. Cumulative Distribution Function (CDF) plots of NMD+ isoforms and NMD- isoforms. X-axis represents fold change in 3BΔ2BD versus WT cells each with control knockdown (siNC). Number of transcripts in each set (n) and p-value from Kolmogorov-Smirnov (KS) test comparing the two distributions are shown. F. Cumulative Distribution Function (CDF) plots of PTC+ isoforms and PTC- isoforms from same set of genes. X-axis represents fold change in UPF1 knockdown (siUPF1) versus control knockdown (siNC) in 3BΔ2BD cells. Number of transcripts in each set (n) and p-value from Kolmogorov-Smirnov (KS) test comparing the two distributions are shown. G. Cumulative Distribution Function (CDF) plots of NMD+ isoforms and NMD- isoforms. X-axis represents fold change in UPF1 knockdown (siUPF1) versus control knockdown (siNC) in 3BΔ2BD cells. Number of transcripts in each set (n) and p-value from Kolmogorov-Smirnov (KS) test comparing the two distributions are shown.
Figure 3A. We measured the intensity of fluorescence emitted by the fluorescent reporter (GFP) to estimate: (i) the amount, (ii) the location and (iii) the deposited/diffused state of the model proteins. Accordingly, for each cell analyzed, we measured the fluorescence intensity of its cytoplasm (FTOTALsol for URA3sol and FCYTOagg for URA3agg) and of its foci (FFOCIagg). We used these measurements to estimate the different effects of protein phase separation on cell fitness. The overall fitness effect of forming a protein deposit depends on three factors: foci formation, loss of function and gain of function (Eq.1). In our system, we can distinguish between the cost of forming Ura3p deposits, cost of losing the essential Ura3p activity and the benefit from protection against Ura3p toxicity. Each cost and benefit effect depend on the ratio of deposited or free protein and the specific environment. Accordingly, we can split these cost and benefit effects into two components: one associated with the amount of deposited or free protein (FFOCIagg/FTOTALagg or FCYTOagg/FTOTALsol) and a magnitude of effect defined by the environmental conditions (α, β or γ) (Eq.2). For the non-essential, essential and toxic roles (+Ura, -Ura and +5FOA) of Ura3p, the number and type of effects applicable is different (Eq.3, Eq.4 and Eq.5). B. Plots showing the distribution and relationship between the fractions of deposited or free Ura3p, its magnitude of effect on fitness (α/β/γ) and the selection coefficient (S) in different environments. C. Average of the selection coefficient (left) and average of the cytosolic fluorescence (equivalent to the fractional abundance of the free protein in URA3agg, right). D. Schematic representation highlighting how phase separation of the same protein can result in different fitness effects depending on the environment.
Figure 3MHC staining (red) - performed on 3D bioprinted and bulk structures loaded with Mabs after 15 days of culture - reveal a significant difference in terms of myotube organization between the two hydrogel systems, with the wet-spun constructs greatly outperforming the bulk gels. Such difference - quantified by means of image analysis - is shown in the polar plots. Nuclei were counterstained by DAPI (Blue).
Figure 5In the early stages of infection, the P. syringae deploys histidine kinase RhpS to dephosphorylate P-RhpR and RhpR in a low-phosphorylated state, which allows the activation of downstream hrpRS-hrpL-T3SS cascade. The expression of T3SS genes enhance the ability of bacteria to invade host plants. When infecting the host cells, RhpS directly senses either in vivo or in vitro polyphenols (e.g., TA, PGG, EGCG), which inhibit the phosphatase activity of RhpS. Meanwhile, three non-cognate HKs (PSPPH_3550, PSPPH_5115 and PSPPH_3736) cooperatively keep the phosphorylation state of RhpR at a high level, which in turn suppresses the induction of downstream T3SS genes and the virulence of P. syringae.
Knock out (KO) mice show reduced myofiber size and impaired glucose clearance.Figure 5(A) Blood glucose in 6‐ to 10‐month (mo)‐old mice (n=12-15), and (B) serum insulin levels in 10‐mo‐old fed control (Con) and KO mice (n=6). (C) Glucose tolerance tests in 10‐mo‐old chow diet (RD)‐fed (n=5), and (D) in 10‐ to 12‐mo‐old high‐fat diet (HFD)‐fed Con and KO mice (n=4-9). (E) Insulin tolerance test in 10‐mo‐old RD‐fed Con and KO mice (n=5). (F) Immunoblots for indicated proteins in skeletal muscle from 10‐mo‐old Con and KO mice. (G) Hematoxylin and eosin (HE)‐stained gastrocnemius (GA) sections from 6‐mo‐old Con and KO mice. Myofiber cross‐sectional area (μm2) is shown (n=3). (H) Expression of MuRF‐1 and Atrogin‐1 in GA and TA from 10‐mo‐old Con and KO mice (n=4) (I) myogenic genes in GA from 10‐mo‐old Con and KO mice (n=6). (J) Immunoblots for indicated proteins in tissues from 4‐mo‐old Con and KO mice (n=4). (K) mRNA for IRS1, (L) IRS2 in epididymal white adipose tissue (eWAT) and GA from 10‐mo‐old Con and KO mice (n=6).
a) RT-qPCR analysis of the levels of LINE-1 ORF1 and ORF2 RNA in ovaries of Becn1/Atg6+/− mice compared with wild-type controls (n=4 wild-type, Becn1/Atg6+/− mice).f5(a) RT-qPCR analysis of the levels of LINE-1ORF1 and ORF2 RNA in ovaries of Becn1/Atg6+/− mice compared with wild-type controls (n=4 wild-type, Becn1/Atg6+/− mice). (b) qPCR analysis of relative number of genomic copies of LINE-1ORF2 in ovaries of Becn1/Atg6+/− mice and wild-type controls normalized to LINE-1 5′UTR and 5S rDNA (n=4 wild-type, 6 Becn1/Atg6+/− mice). *P=0.02, t-test. (c) RT-qPCR analysis of the levels of LINE-1 ORF1 and ORF2 RNA in the prefrontal cortex of Becn1/Atg6+/− mice compared with wild-type controls (n=4 wild-type, Becn1/Atg6+/− mice). (d) qPCR analysis of relative number of genomic copies of LINE-1 ORF2 in muscle, liver and prefrontal cortex of Becn1/Atg6+/− mice and wild-type controls normalized to LINE-1 5′UTR and 5S rDNA (n=4 wild-type, 6 Becn1/Atg6+/− mice). *P=0.019 prefrontal cortex, P=0.048 muscle, analysis of variance. All error bars represent s.e. of the mean.
Figure 4A Histochemical stained roots from composite plants transformed with proMtNPF6.5:GUS (left column), proMtNPF6.7:GUS (middle column), proMtNPF6.6:GUS (right column). B Expression of MtNPFs in roots after transfer to media containing different levels of NO3- for 30 minutes. C=KCl control, N=KNO3 (Biological replicates: n = 3 (3 pooled root organs in each replicate), asterisk denotes significance p<0.05, Student's t-test). The means of all samples were compared to that of their concentration-matched KCl controls. Expression is shown relative to Ubiquitin. C Scanning confocal image of MtNPF6.5-GFP (top view) and MtNPF6.7-GFP (side view) expressed in root epidermal cells near the tips of M. truncatula transgenic hairy roots with FM-464 used as a PM marker. The gene fusions were expressed from the Lotus japonicus Ubiquitin promoter. Bars represents 10 μm.
is a positive determinant of HKC and SCC cells proliferative and oncogenic potential.Figure 5(A) Colony forming assays of multiple HKC strains from White and Black donors (passages 2,3) infected with a HSD17B7 expressing lentivirus (HSD-oe) versus empty vector control (ctrl), plated in triplicate dishes at limited density (1000 cells per 60mm dish) at 5 days after infection. A week later cells were fixed and stained by crystal violet and number of colonies (>0.149mm2) was determined. Shown are numbers of colonies +/- SD. n(dishes per condition) =3. *p<0.05. 2-tailed unpaired t-test. (B) Colony forming assays of the indicated SCC cell lines stably infected with a HSD17B7 expressing lentivirus (HSD-oe) versus empty vector control (ctrl), utilizing the same conditions as in the previous panel. Shown are numbers of colonies +/- SD. n(dishes per condition) =3. *p<0.05; ****p<0.0001. 2-tailed unpaired t-test. (C) Proliferation live-cell imaging assays of SCC cell lines plus/minus HSD17B7 overexpression as in the previous panel. Cells were plated in triplicate wells in 96-well plates followed by cell density measurements (IncuCyte), taking four images per well every 2h for 150-160h. n (wells per condition)=3. ****p<0.0001. Pearson r correlation. (D-F) Intradermal tumorigenicity assays with HKCs (269W) stably infected with an HSD17B7 overexpressing lentivirus (HSD17B7-oe) versus control (Ctrl). Experimental conditions were the same as in Fig 2. Mice were sacrificed 15 days after injection. Shown are representative images and quantification of tumor cells density assessed by immunohistochemical staining with anti-pan keratin antibodies (D,) as well as % of Ki67 positive cells (E) and K1 positivity (F) of pan-keratin positive areas. For (D), n(tumors per condition) = 9; for (E and F) n(3 fields per tumor, 5 tumors per condition) = 15. *p<0.05, ****p<0.0001. Horizontal line: median. 2-tail unpaired t-test. Scale bar: 500μm (D), 100μm (F). (G, H) Intradermal tumorigenicity assays with SCC13 cells overexpressing HSD17B7 (HSD17B7-oe) versus control (Ctrl). Mice were sacrificed 15 days after injection. Shown are representative images and quantification of tumor cells density, assessed by immunohistochemical staining with anti-pan keratin antibodies (G) as well as % of Ki67 positive cells in pan-keratin positive areas (H). For (G), n(tumors per condition) = 4; for (H), n(3-5 fields per tumor, 4 tumors per condition) 15, 19. *p<0.05; Horizontal line: median. One-tailed paired t-test. Scale bar in (G, H): 100μm.
21–22nt siRNAs guide AGO6 to target chromatin A Schematic of the ChIP crosslinking between chromatin and the AGO6 protein which is tethered to its targets by a Pol V‐derived RNA scaffold.Figure 3A Schematic of the ChIP crosslinking between chromatin and the AGO6 protein which is tethered to its targets by a Pol V‐derived RNA scaffold. B H3Ac control ChIP from the same chromatin used in the AGO6‐IP for the At1g08200 constitutively expressed gene and the Athila6A TSS. Enrichment of H3Ac at the Athila6A TSS is increased upon transcriptional activation in ddm1 mutants, while enrichment of H3Ac at the gene At1g08200 is consistently high in both wt Col and ddm1. Data are represented as mean ± SD of six biological replicates for each genotype. C-E AGO6‐ChIP qPCR analysis of (C) the gene At1g08200 and the Pol IV‐RdDM target SimpleHat2, (D) the Athila6A TSS in wt Col TE‐silenced epigenomes (left) and ddm1 TE‐active epigenomes (right) and (E) TAS3a. ChIP signals in (D) are normalized to the ago6 or ddm1 ago6 no‐antigen controls. ChIP signals in (C) and (E) are normalized to the ago6 no‐antigen control. In (C-E), each biological replicate data point is shown as a gray dot. The data are represented as mean ± SD. For multiple statistical tests, one‐way ANOVA was performed using Tukey's multiple comparison test. F ChIP qPCR analysis of the FLAG‐tagged POL V protein in wt Col and ddm1. ChIP signals are normalized to the wt Col no‐antigen control.
Identification of blood cell markers.Figure 1Study outline and proteomics workflow. Erythrocytes, thrombocytes, platelet-rich and platelet-free plasma were generated from ten healthy female and male individuals by differential centrifugation and successive purification steps. To generate reference proteomes for each of the blood compartments, the respective protein samples of the 20 study participates were digested to peptides. Proteins and (C) peptides identified for platelets, erythrocytes, platelet-rich and platelet-free plasma. Selection of the most suitable quality marker proteins for platelet contamination (blue dots) and (E) erythrocyte contamination (red dots) based on their abundance, the platelet/erythrocyte to plasma ratio and the coefficient of variation. Proteins that were only detected in platelets or erythrocytes, but not in plasma are aligned on the right side of the graph.
Figure 1 The 3'-UTR sequence of mUcp1 RNA. Canonical CPEs (UUUUA1-2U in bold), the reported BRF1-binding site (UAUUUAU, gray box) and predicted IMP2-binding motifs (AYAYA and YA2YA, underlined) are denoted. Ucp1L and Ucp1S carrying long and short 3'-UTRs, respectively, are resulted from alternative use of two polyadenylation signals (Hex: AAUAAA, open box). ORF, open reading frame. The arrowhead marks the end of Ucp1S sequence. Northern blots of Ucp1 using 5 µg BAT total RNA and the denoted radiolabeled probes   Schematic comparison of mUcp1L and hUcp1. Poly(A) signal (Hex, open box) and CPE (black box) are denoted. The RT-PCR results from human, WT (Ucp1+/+) and UCP1-KO (Ucp1-/-) BAT RNA samples   Dual luciferase reporter assay and RT-qPCR. The reporter plasmids, Fluc-mUcp1S, -mUcp1L or -hUcp1L 3'-UTR, and Rluc were co-transfected into differentiated HIB1B adipocytes. The transfected cells were used for luciferase activity assay and RT-qPCR. Data are mean ± SD from 3 independent experiments. Translation efficiency was defined as normalized activity (Fluc/Rluc) divided by normalized RNA level (Fluc/Rluc). ***p < 0.001 compared with Fluc-mUcp1S, one-way ANOVA
MOAP-1 localizes at the p62 bodies upon exposure to cellular stresses.Figure 1A MOAP-1 co-localizes with p62 in the cytoplasmic inclusion bodies upon proteasome inhibition. HCT116 cells were transfected with plasmid encoding GFP-MOAP-1 or GFP only control. 14 hours later, cells were treated with the proteasome inhibitor MG132 (5 µM) for 8 hours and analyzed by immunofluorescence (IF) with anti-GFP (in green) and anti-p62 (in red) antibodies. Data information: , nuclei were counterstained with DAPI (blue) B Endogenous MOAP-1 resides at the p62 bodies upon proteasome inhibition. HCT116 cells were treated with MG132 (5 µM) for 8 hours and analyzed by IF with anti-MOAP-1 (in green) and anti-p62 (in red) antibodies. Data information: nuclei were counterstained with DAPI (blue) C MOAP-1 is recruited to the p62 bodies upon exposure to cellular stresses. LO2 hepatocytes were transfected with plasmid encoding Myc-MOAP-1. 14 hours later, cells were then treated with MG132 (5 µM), arsenic trioxide (As2O3, 10 µM) or diethylnitrosamine (DEN, 200 µM) for 8 hours each. Cells were then processed for IF analysis with anti-Myc (in green) and anti-p62 (in red) antibodies. Data information: nuclei were counterstained with DAPI (blue) D MOAP-1 spontaneously localizes to the p62 bodies at resting state in the liver cancer cell lines, HepG2, Huh-1, JHH5 and JHH7. The liver cancer cell lines were transfected with the plasmid encoding Myc-MOAP-1. 14 hours later, transfected cells were subjected to IF analysis with anti-Myc (in green) and anti-p62 (in red) antibodies. Data information: nuclei were counterstained with DAPI (blue) E Absence of the aggregated patterns of MOAP-1 in the p62 deficient cells. WT and p62 KO HepG2 cells were transfected with plasmid encoding Myc-MOAP-1 for 14 hours and the cells were subjected to IF analysis as in (D). Data information: , nuclei were counterstained with DAPI (blue) F Western blotting analysis of p62 and Myc-MOAP-1 protein levels in the WT and p62 KO HepG2 cells as described in (E). Actin as loading control.
Figure 2A Schematic diagram for tissues (left panel) and the number of differentially expressed genes (right panel) in osprr73 mutant compared with their respective wild type (DJ). B Venn diagram showing the number of differentially expressed genes in osprr73 mutant with or without salinity stress. C GO (Gene ontology) classification of differentially expressed genes in osprr73 mutant treated by NaCl for 4 h, based on biological process (BP). P-values were generated by Fisher's test. D The number of differentially expressed genes, which belongs to BP cluster in GO analysis, in osprr73 mutant. E Venn diagram showing the overlapped DEGs in osprr73 mutants (pink circle) with previously identified salt-responsive genes which derived from China rice data center (black circle). Heatmap showing 6 common genes, the colored bar represents log2 (fold change). F Heat map of OsHKT2;1, OsHKT1;5 and the genes related to the oxidative stress in GO analysis, showing their transcriptional abundance in osprr73 mutant. The colored bar indicates log2 (FPKM) values.
Figure 6A, B. Representative maximum projections of exposure matched confocal images of immunofluorescence for nucleolin and neurofilament (NF) and the neuronal marker Tuj1 in sciatic nerve axons from WT and GAR+/- mice shown in (A). Upper panels show total nucleolin stain. Middle panels show merged image of nucleolin (gray), NF and Tuj1 (magenta), and DAPI (blue). Lower panels show nucleolin overlaps with NF and Tuj1 signals as the "axon only" signal. B shows quantification of nucleolin immunofluorescence with approximately a 50% reduction in nucleolin in the axons of GAR+/- mice in vivo. n = 3 WT, n = 4 GAR+/-; means ± SEM; * p < 0.05, unpaired Student's t-test. Scale bar - 10 µm. C-F. Representative, exposure matched maximum projection confocal images of FISH for Kpnb1 (C) or mTOR (E) mRNA and NF plus Tuj1 immunostaining from sciatic nerve sections from WT (left) or GAR+/- mice. Upper panels for each show total mRNA signal. Middle panels show mRNA (gray) signals merged with NF plus Tuj1 (magenta) and DAPI (blue). Lower panels show mRNA signal that overlap with NF plus Tuj1 signal (labeled "axon only" signal). Quantification of axonal Kpnb1 (D) and mTOR (F) mRNA signals compared to the negative control, DapB mRNA, show a significant reduction in these axonal mRNAs in the GAR+/- mice. n = 3 WT, n = 3 GAR+/-; means ± SEM; ** p < 0.01, *** p < 0.001, ****p < 0.0001 unpaired Student's t-test. Scale bar - 10 µm.
Function of possible coat proteins in AP-3 vesicle formation.Figure 1(A) Co-localization of c-terminally GFP- or mCherry-tagged Apl5, Sec7 and Vps41 in a vam3 temperature sensitive (ts) background strain. Cells were grown over night at 23°C and where indicated shifted to 37°C for 3 h prior to imaging. White arrowheads indicate co-localization events. Scale bar, 5 µm. Scale bar in inset, 2 µm. (B) Quantification of co-localization events Single events were counted and the percentage of co-localization normalized to the number of Apl5-punctae was plotted (n ≥ 187 cells). Bars show the mean percentage of co-localization ± standard deviation. (C) Re-localization of Apl5 upon Vps41 re-localization. Vps41 was tagged with a C-terminal FRBGFP tag in a strain, where Apl5 carries a C-terminal mCherry and Pma1 a FKBP-tag Cells were grown in either the absence (control) or presence of 10 μM rapamycin, and localization of Vps41 and Apl5 was analysed by fluorescence microscopy. Scale bar, 5 μm. (D) Effect of clathrin deletion on the AP-3 pathway. GFP-tagged Nyv1 with a C-terminal Snc1 transmembrane domain (GNS) was localized relative to FM4-64 stained vacuoles in the indicated strains. Labeling of the cell surface by GFP indicates a defect in the AP-3 pathway (E) Quantification of AP-3 defect from (D). Linear intensity plots were laid over the cells, the AP-3 defect was calculated by dividing GFP-intensities on the vacuolar membrane by the sum of the intensity of vacuolar, and plasma membrane signal (n ≥ 30 cells). Bars show the mean vacuolar signal over total signal ± standard deviation.
(A) Co-immunoprecipitation of the Siwi−Vasa complex in the presence and absence of EDTA.Figure 1(A) Co-immunoprecipitation of the Siwi−Vasa complex in the presence and absence of EDTA. Isolated proteins were subjected to western blotting. n.i.: non-immune mouse IgG. (B) Co-immunoprecipitation and identification of Ago3-associated proteins. Left: The immunopurified Ago3 complex was resolved by SDS-PAGE and silver-stained. Right: DEAD-box helicases identified by mass spectrometry. Proteins identified by co-immunoprecipitation under EDTA-containing conditions only are listed in red. PSM: peptide sequence match. (C) Co-immunoprecipitation using BmN4 cells transfected with the Flag-tagged DEAD-box helicases identified by shotgun proteomics. The Flag-tagged proteins and Ago3 were detected by western blotting. Full-length Flag-tagged proteins are indicated with red asterisks. h.c.: heavy chain. (D) Co-immunoprecipitation of endogenous Ago3 and DDX43 in the presence or absence of EDTA. These factors were detected by western blotting. (E) Pull-down assay using high salt-washed Ago3-conjugated magnetic beads and purified His-DDX43. All samples were subjected to SDS-PAGE and detected by silver staining. (F, G) Analysis of Siwi-piRNA and Ago3-piRNA levels in Siwi and Ago3 complexes with DDX43, respectively. The WB (western blotting) and NB (northern blotting) signals represent the PIWI protein and piRNA levels, respectively (see also Figs EV1A and EV1B). The relative ratios of piRISC were calculated based on the signal intensities of the PIWI proteins and piRNAs.