EMBO/sd-character-level-ner huggingface dataset:
Figure 4A. DIS3L2-bound uridylated mRNA reads map to the 5' and 3' UTRs. B. Most 3' UTR U+ reads match the stem-loop structure of histone mRNAs. Heat map representation of the uridylation position. C. The uridylated aberrant RNAs are stabilized in cells with nonfunctional DIS3L2. Small RNAs were modified with 3' end linker, and linker-specific primer was used for cDNA synthesis. Uridylated RNAs were PCR amplified in a semi-quantitative way with the same amount of input RNA used for RT reaction. We used a combination of the gene- and linker-specific primers, -RT ctrl is RT-PCR control, where no reverse transcriptase was added; PCR control is a reaction with no cDNA added. The graph on the right summarizes the percentage of uridylated and nonuridylated 5' fragments of OAT mRNA in HEK293T-Rex cells (Ctrl) and cells overexpressing mutant DIS3L2 (D391N). D. DIS3L2-bound uridylated RNAs are degraded by DIS3L2 in vivo. RT-PCR amplification of 5'mRfs of OAT and RPL12 genes and 3' extended forms of U12 snRNA (RNU12 ext) from HEK293T cell lines with modified expression of DIS3L2 (as marked on top) was performed as described in C. Regions of mature 5S rRNA and 7SL RNA were used as loading controls. DIS3L2 KO is a DIS3L2 knock-out cell line. DIS3L2 rescue is DIS3L2 KO cells with stably integrated mutant (D391N) and wild type (WT) DIS3L2 expressing constructs, respectively. Bands marked with * are unspecific primer-dimer PCR products. E. Metagene analysis of the position of DIS3L2 D391N clipped U+ reads peak maximum around mRNA transcription start sites (TSS) and of the ChIP-seq data for RNAP II. F. U+5'mRfs and aberrant forms of U12 snRNA are highly enriched in the cytoplasm. RT-PCR detection of 5'mRfs of the OAT and RPL12 and aberrant form of U12 snRNA was performed as described in C with RNA isolated from nuclear and cytoplasmic fractions, respectively. The RT-PCR amplification of the body of 5S rRNA was used as a positive control for RNA isolation and RT reaction in both fractions. The identity and uridylation status of cytoplasmic OAT U+5'mRfs was confirmed by sequencing (shown on the bottom). In red are untemplated nucleotides, in green region corresponding to the first intron of OAT pre-mRNA. The efficiency of the subcellular fractionation was monitored by western blot (right panel, WB). DDX5 is nucleoplasmic, ACO2 is mitochondrial, and DIS3L2 and TUBa are cytoplasmic markers.
Figure 5Expected and observed number of mesodermal (Flk1+) cells of the E7.0 mouse embryo expressing Nanog and selected mesodermal or hematopoietic gene expression, based on single cell RNA-seq data (Scialdone et al., 2016). Statistical significance was calculated with a hypergeometric test. PCA showing the distribution of Flk1+ E7.0 mesoderm cells expressing Nanog (green) or Tal1 (red). The single cell expressing both genes is shown in yellow and indicated by an arrow. E6.5 Nanogtg embryos after 8 hours ex-utero culture in the presence (+dox) or absence (-dox) of doxycycline. Scale bar, 100 µm. RT-qPCR quantification of the relative expression of Nanog, Tal1, and Klf1 in individual untreated embryos (-dox) or treated embryos (+dox) (n=5). **P < 0.005, ***P < 0.0005; Student's t-test. Horizontal line represents mean values and error bars SD. Whole mount in situ hybridization of Tal1 in E7.5 untreated (-dox) or in utero treated (+dox) Nanogtg embryos. Scale bar, 100 µm. UCSC browser view of the Tal1/Stil1 region (mm9; chr4:114,705,753-114,756,741), indicating the presence of the NANOG binding peak, determined by ChIP-seq, in EpiLCs (2 replicates are shown) but not in ES cells (Murakami et al., 2016); the binding peak was deleted by CRISPR/Cas9 genome editing (scissors). RT-qPCR determination of relative expression in wild type and CRISPR-deleted embryos (n=5) of Tal1 (wt, n=19; deleted, n=13), Klf1 (wt, n=3; deleted, n=6), Gfi1b (wt, n=10; deleted, n=8), Runx1 (wt, n=13; deleted, n=5) and Stil (wt, n=19; deleted, n=13). **P < 0.005, Student's t-test. Horizontal line represents mean values and error bars SD. Experimental design for ES to EpiL cell differentiation of Nanogtg cells and two independent clones (Nanogtg;dTal1del#1 and Nanogtg;dTal1del#2) where the binding site for NANOG distal to Tal1 has been deleted (left). On the right, relative expression of Tal1 determined by RT-qPCR for each ES cell line (ESC; n=9 for all three lines) and EpiL cells without (EpiLC; Nanogtg and Nanogtg;dTal1del#1, n=8; Nanogtg;dTal1del#2, n=6) or with dox treatment (EpiLC +dox; n=9 for all three lines). The genotype of the cell lines is indicated below. Values were normalized to Nanogtg ESC. *P < 0.05, **P < 0.01, ns = not significant; ANOVA with Fisher post-test. Horizontal line represents mean values and error bars standard error of the mean (SEM).
Figure 7Wild type, Traf3ip3-/- and Mavs-/- mice (three littermates per group) were infected intravenously with VSV at a MOI of 2×108. The spleens were collected 4 hours or 8 hours after infection and Ifna4 (A), Ifnb (B), Isg54 (C) and Il6 (D) inductions were measured respectively by qPCR. All data are presented as the mean values based on three independent experiments, and error bars indicate s.d. P values were determined by unpaired two-tailed Student's t-test. *P<0.05 and **P<0.01. NS indicates no statistically significant difference. Wild type, Traf3ip3-/- and Mavs-/- mice (three littermates per group) were infected intravenously with VSV at a MOI of 2×108. The spleens were collected 4 hours or 8 hours after infection and VSV RNA levels were measured by qPCR (E). All data are presented as the mean values based on three independent experiments, and error bars indicate s.d. P values were determined by unpaired two-tailed Student's t-test. *P<0.05 and **P<0.01. NS indicates no statistically significant difference. Wild type, Traf3ip3-/- and Mavs-/- mice (three littermates per group) were infected intravenously with VSV at a MOI of 2×108. The sera were collected from the treated mice and used for the measurement of IFNβ production by ELISA (F). All data are presented as the mean values based on three independent experiments, and error bars indicate s.d. P values were determined by unpaired two-tailed Student's t-test. *P<0.05 and **P<0.01. NS indicates no statistically significant difference. G Wild type, Traf3ip3-/- and Mavs-/- mice (n=6 each) were injected with VSV via tail vein injection at 5×107 pfu per mouse, and the survival rates were monitored for 15 days. P values were determined by unpaired two-tailed Student's t-test. *P<0.05
Figure 1GFP-ATG8a expressing seedlings in Murashige & Skoog (MS) growth medium or 30 min after treatment with MS containing ACC, ABA, ATP, BL, 6-BA, Flg22, NAA or PEP1. (A) Representative maximum intensity projection images of 10 Z-stacks per image. Scale bar: 10 µm. GFP-ATG8a expressing seedlings in Murashige & Skoog (MS) growth medium or 30 min after treatment with MS containing ACC, ABA, ATP, BL, 6-BA, Flg22, NAA or PEP1. (B) Quantification of GFP foci per 0.0025 mm2. Values are presented as mean ± standard deviation of the mean and were calculated from at least three independent experiments with 3 individuals per replicate. Bars marked with an asterisk (*) are statistically significant (P<0.05) according to the T -test. GFP-ATG8a expressing seedlings in Murashige & Skoog (MS) growth medium or 30 min after treatment with MS containing ACC, ABA, ATP, BL, 6-BA, Flg22, NAA or PEP1. (C) GFP-ATG8a cleavage immunoblot for plants exposed to the same treatments as in (A). Numbers below the blots represent ratio for given sample normalized to input and relative to non-treated control. Experiments were repeated minimum 3 times with similar results. GFP-ATG8a expressing seedlings in Murashige & Skoog (MS) growth medium or 30 min after treatment with MS containing ACC, ABA, ATP, BL, 6-BA, Flg22, NAA or PEP1. (D) NBR1 immunoblot for atg2-2 samples for given treatments. Numbers below the blots represent ratio for given sample normalized to input and relative to non-treated control. Experiments were repeated minimum 3 times with similar results.
Validation of progestogen pellets.Figure 4Progesterone plasma levels in control (n=79) and progesterone-treated (n=20) mice at sacrifice as measured by LC-MS, shown as means ± SD. Progestin plasma levels in treated mice (n=10, 14, 18, 7, 6, 10) and in women (n=20, 11, 3, 4, 6, 8) on hormonal contraceptives measured by LC-MS, shown as means ± SD, as well as values reported in the literature (Cmax). The active form of DSG, 3-ketodesogestrel was measured. Progestin plasma levels in treated mice (n=10, 14, 18, 7, 6, 10) and in women (n=20, 11, 3, 4, 6, 8) on hormonal contraceptives measured by LC-MS, shown as means ± SD, as well as values reported in the literature (Cmax). The active form of DSG, 3-ketodesogestrel was measured. H Violin plots showing plasma levels of 17-β-estradiol, progesterone, and testosterone in individual xenografted NSG females measured by LC-MS; n= 7-106 per treatment. Dotted line indicates lower limit of detection. Statistical significance was tested by non-parametric Kruskal-Wallis test, followed by Dunn's test. * P <0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001,.
Figure 5Structure of the chr7 piRNA cluster. Top gray bars represent a putative piRNA precursor predicted by H3K4me3 ChIP-seq, RNA-seq, and small RNA-seq. Only uniquely mapped reads were used for displaying the sequencing result. For RNA-seq and small RNA-seq, only the results of minus strand are shown, because plus strand does not express appreciable level of RNA. A promoter region deleted is shown above the putative piRNA precursor. RPM, Read Per Million. Low expression level of piRNAs from chr7 cluster in chr7 piRNA clusterΔpromoter/Δpromoter testes. Unchanged expression of piRNAs from chr10 cluster (bottom) shows chr7 cluster is specifically affected. Only uniquely mapped piRNAs are shown. DNA methylation status of the Rasgrf1 differentially methylated region (DMR) in spermatogonia from chr7 piRNA clusterΔpromoter/Δpromoter. See Figures 4A and B for information about the region examined.
W300A mutation impairs PNRC1 interaction with DCP1α and its nucleolar translocation A.Figure 5A. Co-immunoprecipitation performed on HeLa cells expressing HA-PNRC1WT, HA-PNRC1W300A or HA-PNRC1ΔNLS with a specific anti-HA antibody and probed with anti-NPM1 and anti DCP1α antibodies. B-C. Co-immunoprecipitation performed on HeLa cells expressing HA-PNRC1W300A mutant with a specific anti-HA antibody and probed with antibodies specific for PNRC1-interacting proteins. The white arrow indicates the specific DCP2 band. D. Confocal microscopy images of HeLa cells silenced for the endogenous DCP1α and expressing an shRNA-resistant GFP-DCP1α in combination with RFP, RFP-PNRC1WT or RFP-PNRC1W300A mutant (green: GFP, red: RFP, blue: DAPI, scale bar: 5µm). E. Confocal microscopy images of RFP, RFP-PNRC1 or RFP-PNRC1W300A-expressing cells stained for DCP1α, DDX6 and LSM1. RFP-PNRC1WT or RFP-PNRC1W300A-transfected cells are indicated with arrowheads. Scale bar: 10µm F. Quantification of HeLa cells expressing RFP-PNRC1W300A classified according to the P-body localization of the three stained proteins. The average of 3 independent experiments ± SD is reported. G. Co-immunoprecipitation performed on HeLa cells expressing LacZ control, HA-PNRC1WT, HA-PNRC1W300A or HA-PNRC1ΔNLS constructs with a specific anti-DCP1α antibody and probed with anti-PNRC1 and anti-NPM1 antibodies.
Figure 4A. Analysis of SCAI-interacting proteins in Xenopus egg extracts. Volcano plot shows enrichment of proteins in both SCAI-C and SCAI-N immunoprecipitates (IPs) compared to an IgG control, plotted against the P-value (n=4 independent experiments; permutation-based FDR<0.01, s0=2). B. SCAI, REV7, REV1 and REV3 immunoprecipitates (IP) in NPE were immunoblotted with the indicated antibodies. Asterisks denote antibody cross-reactivity. C. Whole proteome MS analysis of mock- versus SCAI-depleted egg extracts. The volcano plot shows the difference in abundance of proteins between the mock reaction and SCAI-depleted samples (with either N or C antibodies) (x-axis), plotted against the p-value resulting from two-tailed Student's t-test (y-axis). Proteins significantly down-regulated (FDR<5%) in SCAI-depleted extracts are highlighted in red (n=4 biochemical replicates; FDR<5% corresponds to a permutation-based FDR-adjusted q-value of <0.05). D. GFP IPs from U2OS or U2OS/GFP-SCAI cells transfected with indicated siRNAs were immunoblotted with indicated antibodies. Note that a small fraction of REV7 is still immunoprecipitated with GFP-SCAI in REV3 knockdown cells. This could be due to incomplete knockdown, and/or additional interactions of SCAI with the REV1-Polζ complex that are independent of REV3. E. Schematic of SCAI interaction with the REV1-Polζ complex. The SCAI-interacting region of human REV3 and its alignment with a homologous region in human AHDC is indicated. NTD, N-terminal domain; PCD, positively charged domain; Pol, polymerase domain; CTD, C-terminal domain. F. GFP IPs from U2OS cells transfected with indicated REV3-GFP expression constructs were immunoblotted with indicated antibodies. G. As in (F), except that U2OS or U2OS/GFP-SCAI cells were transfected with FLAG-NLS-REV3(995-1194) expression plasmid. H. Mock-, SCAI-, REV1- or SCAI/REV1-depleted egg extracts were analyzed by immunoblotting with indicated antibodies. Asterisks denote antibody cross-reactivity. I. pICLpt was replicated in egg extracts immunodepleted with the indicated antibodies and processed as in Figure 3J,L. J. pICLpt was replicated in egg extracts immunodepleted with indicated antibodies, isolated at various times via plasmid pulldown and associated proteins were analyzed by immunoblotting.
Figure 4A) Cells expressing the indicated GFP-fusions (green) were loaded with Alexa647-Transferrin (red; arrowheads indicate transfected cells) for 15 minutes, fixed and the morphology of the transferrin positive recycling endosomes analysed by confocal microscopy. Scale bars = 10 μm. Arrows in inset indicate the juxtanuclear ERC B) Cells transfected as in (A) were loaded with Alexa-647 transferrin for 15 minutes and the fluorescent transferrin chased out for the indicated time periods. At the end of the timecourse the cells were fixed and the transferrin content of GFP positive cells analysed by flow cytometry. Results are expressed as a percentage of Tfn fluorescence at t=0 and are the mean of 3 independent experiments, ± s.e.m. C) Cells transfected as in (A) were stained for the cis-Golgi marker GM130. Arrowheads depict transfected cells. Scale bars = 10 μm. Bar chart is quantification of fragmented Golgi complex (> 100 cells per condition from three independent experiments, ± s.e.m. ** p < 0.01, one way ANOVA with Sidak's post-test). D) HeLa C1 cells transfected with CFP or CFP-TBR were treated with D/D solubiliser (Clontech) or D/D solubiliser plus 2.5 μg/ml Brefeldin A (BFA) for the indicated time periods, trypsinised, fixed and their GFP content analysed by flow cytometry. Results are expressed as a percentage of GFP levels at t=0, and the mean of 3 independent experiments ± s.e.m.
Figure 7A. Triplicate normalized fitness functions (*) for E. coli BW25113 strains harboring plasmid copies of tet(B)WT, tet(B)K183E,A290V,T326I (variant 6) and tet(B)F151I,F179L,T326A (variant 7) in TET and their corresponding least squares model fits (solid line) where both variants display decreased fitness in TET compared to WT. B. Percent change relative to WT in parameters κ and γ for strains expressing tet(B)K183E,A290V,T326I and tet(B)F151I,F179L,T326A. Parameter values were determined by the least squares model fits in 7A. C. Homology model of TetB using YajR (PDB 3wdo) built using Phyre2 where the periplasmic side is on top and the cytoplasmic side is on the bottom. The A-helices are shown in blue, B-helices are shown in green, C-helices are shown in grey and the interdomain loop is cyan. Spheres indicating variant positions were made using PyMOL (K183 and A290V- red; F151 and F179- red; and T326-black as this residue is altered in variant 6 and 7). The right image depicts the view through central TetB pore from the periplasm into the cytoplasm.
Fertile men with USP26 mutations produce XY aneuploid spermatozoa.Figure 7A, B Quantification of different types of spermatozoa in fertile men with or without USP26 mutations. The spermatozoa from fertile men without USP26 mutations (n = 10 independent experiments); fertile men contained the USP26 c.463C>T mutation (The sample was analyzed in three repeated experiments), the USP26 c.125T>C mutation (n = 2 independent experiments, one sample was analyzed in two repeated experiments), the USP26 c.1044T>A mutation (n = 5 independent experiments), and the USP26 c.370_371insACA/494T>C/1423C>T mutation (n = 10 independent experiments) were collected to perform Chr X and Chr Y FISH. P-values are shown for comparison to fertile men without USP26 mutations. C The fertile men with the USP26 mutated haplotype produced XY and O aneuploid spermatozoa. FISH assay of Chr X (green) and Chr Y (red) was performed in fertile men with or without the USP26 mutated haplotype. Nuclei were stained with DAPI (blue). The arrows indicate the Y chromosome, and the arrowheads indicate the X chromosome. D Squares denote male family members, circles denote female family member; a solid symbol indicates affected KS member, open symbols represent unaffected KS family members. The X labeled with red color indicates the X chromosome with the USP26 mutated haplotype (c.370_371insACA/494T>C/1423C>T). All family members were Sanger sequenced.
Figure 3Schematic representation of the experimental set-up of CARD14E138A induction and MALT1 inhibitor treatment. Cleavage of MALT1 substrates CYLD and BCL10 as analyzed by western blotting of lysates of ear tissue of mice four days after treatment with tamoxifen and MALT1 inhibitor or vehicle. Actin is shown as a loading control. Each lane represents one mouse. Changes in relative ear thickness and body weight in vehicle- and MALT1 inhibitor-treated mice upon CARD14E138A induction. M1 inh= MALT1 inhibitor, WT = K14creERTg/+ Rosa26+/+, ieCARD14E138A = K14creERTg/+ Rosa26LSL-CARD14-E138A/+. Combined results of two independent experiments are shown (WT + vehicle: n=9, WT + M1 inh: n=8, ieCARD14E138A + vehicle: n=9, ieCARD14E138A + M1 inh: n=10). Representative histological sections of ear tissue stained with hematoxylin and eosin measurements of epidermal thickness of tamoxifen-treated mice. Combined results of two independent experiments are shown. Each symbol represents the mean of at least ten epidermal thickness measurements for each ear; the line represents the mean value. IL-6 and IL-17a production by ear draining lymphocytes upon ex vivo restimulation with anti-CD3/anti-CD28 for 3 days (n≥3 biological replicates). Heatmap showing relative mRNA expression levels of the indicated genes in ear tissue normalized to reference genes. Values represent median of each group (n≥3).
Figure 99.Graphical Mechanistic Hypothesis. Cachexia-mediated IL-6 axis activation results in elevated catabolic signaling and disruption of WNT signaling in skeletal muscle. WNT dysfunction represents an apparent blockade of anabolic androgen signaling. Treatment with 10 mg/kg AR-42 modulates IL-6 axis activation and restores WNT responsiveness, promoting anabolism in skeletal muscle.
Parkin and PINK1 affects HCV replication.Figure 6A and B) Inhibitory effect of Parkin, PINKI, and ATG5 silencing on HCV replication. Huh7 cells transfected with Non-Targeting (NT) or gene-specific siRNA pools targeting Parkin, PINK1, and ATG5 genes, respectively, were infected with HCVcc. At day 3 post-infection, the levels of HCV RNA (A) and targeted gene mRNA A (B) were analyzed by real-time qRT-PCR, as described in Materials and Methods (mean ± SD; n = 3; *p<0.01). GAPDH was used to normalize changes in Parkin, PINK1, and ATG5 gene expression. (C and D) Rescue of HCV RNA replication by Parkin overexpression. Huh7 cells stably expressing mock vector (M-KD), Non-target-shRNA (NT-KD), and Parkin-shRNA (P-KD), respectively, were infected with HCVcc. P-KD cells were further transfected with two different concentration of the plasmid DNA encoding wild-type Parkin for 2 days before harvest. At 3 days post-infection, intracellular HCV RNA levels were analyzed by real-time qRT-PCR. GAPDH was used to normalize changes in HCV RNA expression (C) (mean ± SD; n = 3; *p<0.01, **p<0.05). (D) The expression levels of Parkin and HCV core protein were analyzed by immunoblotting with anti-Parkin and HCV core antibodies. Ectopic expression level of HA-tagged wild-type Parkin was detected by immunoblotting with anti-HA antibody. β-actin was used as an internal loading control. P values were calculated by using an unpaired Student's t-test. The relative intensity of HCV core expression normalized to β-actin was analyzed by ImageJ.
Figure 7(A-C) NR2F1 expression in cerebral organoids during neural induction and differentiation in vitro (day 40 and day 70, respectively), together with the NP markers PAX6 (green in A), SOX2 (green in B,B') and DCX as a neural marker (blue in A,B,B''). Quantification of NR2F1 levels in NPs is shown in (C). n ≥ 4 organoids from n = 2 batches. Data information: the pixel intensity or the number of positive cells were quantified in 100µm-width boxes, randomly placed across the cortex/organoid neuroepithelia In graphs, data are represented as means ± SEM. 2-way ANOVA test (*P<0.05, **P<0.01, ***P<0.001). Scale bars: 50µm. (D-G) NR2F1 (red) and PAX6 (green) IF in day 40 organoids. Neuroepithelia with high NR2F1 levels show weak PAX6 expression (E,E'), and vice versa (F,F'). Quantification by pixel intensity analysis (G). n ≥ 4 organoids from n = 2 batches. Data information: Nuclei (blue stained with DAPI. the pixel intensity or the number of positive cells were quantified in 100µm-width boxes, randomly placed across the cortex/organoid neuroepithelia In graphs, data are represented as means ± SEM. 2-way ANOVA test (*P<0.05, **P<0.01, ***P<0.001). Scale bars: 50µm. (H) Schematic representation of an organoid electroporation. PX458 plasmids were injected in ventricular-like cavities, organoids electroporated and processed after 7 days of in vitro culture. (I-J) NR2F1 (red) and GFP (green) IF in day 40 human brain organoids upon electroporation of the PX458-αNR2F1 plasmid. In (I,I'), GFP+NR2F1- cells (empty arrowheads) and a GFP+NR2F1+ cell (white arrowhead) are shown; quantification in (J). n ≥ 6 organoids from n = 2 batches. Data information: Nuclei (blue stained with DAPI. the pixel intensity or the number of positive cells were quantified in 100µm-width boxes, randomly placed across the cortex/organoid neuroepithelia In graphs, data are represented as means ± SEM. 2-way ANOVA test (*P<0.05, **P<0.01, ***P<0.001). Scale bars: 50µm. (K-M) TUJ1 (red; differentiating neurons) and GFP (green) IF 7 days after electroporation of control (K) and PX458-αNr2f1 plasmids (L). Co-expression of GFP with SOX2 or TUJ1 distinguishes neural progenitors (NPs) from neurons (Ns), as quantified in (M). Arrowheads in (K,L) point to TUJ1+ GFP+ cells. n ≥ 6 organoids from n = 2 batches. Data information: Nuclei (blue stained with DAPI. the number of positive cells was normalized over the total number of GFP+ cells. In graphs, data are represented as means ± SEM. 2-way ANOVA test (*P<0.05, **P<0.01, ***P<0.001). Scale bars: 50µm. (N) Red and green arrowheads in (N) point to a SOX2+ (red) progenitor and a TUJ1+ (blue) neuron, respectively. Scale bars: 50µm.