EMBO/sd-character-level-ner huggingface dataset:
Figure 7(A) qRT-PCR analysis with total RNA isolated from Irgm1+/+ and Irgm1-/- mouse BMDMs transfected with control siRNA or cGAS siRNA or RIG-I siRNA or cGAS siRNA and RIG-I siRNA as indicated. (B) qRT-PCR analysis with total RNA isolated from HT-29 cells transfected with control siRNA or IRGM siRNA or doubly transfected with IRGM siRNA and cGAS siRNA or IRGM siRNA and RIG-I siRNA or transfected with three siRNAs as indicated. (C) qRT-PCR analysis with total RNA isolated from HT-29 cells transfected with control siRNA or IRGM siRNA or doubly transfected with IRGM siRNA and STING siRNA or IRGM siRNA and MAVS siRNA or transfected with all the three siRNAs as indicated. (D) qRT-PCR analysis with total RNA isolated from HT-29 cells transfected with control siRNA or IRGM siRNA or doubly transfected with IRGM siRNA and MDA5 siRNA as indicated. (E) qRT-PCR analysis with total RNA isolated from HT-29 cells transfected with control siRNA or IRGM siRNA or doubly transfected with IRGM siRNA and IRF3 siRNA as indicated. (F) qRT-PCR analysis with total RNA isolated from HT-29 cells transfected with control siRNA or IRGM siRNA or doubly transfected with IRGM siRNA and STAT1 siRNA or IRGM siRNA and STAT2 siRNA as indicated.
Infection with multiple influenza strains in human Airway Chips and resultant immune responses.Fig. 2.(A) Replication kinetics of influenza H1N1 virus strains WSN (square), PR8 (triangle), NL/09 (diamond) (left graph), and of influenza H3N2 virus strains HK/68 (square) and Pan/99 (triangle) (right graph), when infected at MOI = 0.001 in human Airway Chips. (B) Neutrophil responses to influenza infection in human lung Airway Chip. Top, sequential immunofluorescence micrographs showing time-dependent recruitment of neutrophils (white) to the apical surface of the endothelium (unlabeled) within a human Airway Chip infected with influenza PR8 (H1N1) virus (bar, 50 μm). Bottom, immunofluorescence micrographs showing time-dependent recruitment of neutrophils (white) to the epithelium (unlabeled) and clearance of clustered epithelial cells infected with GFP-labeled PR8 (H1N1) virus (green) (bar, 50 μm). (C) Graph showing numbers of neutrophils recruited to the epithelium in response to infection by H1N1 or H3N2. (D) Virus titers of human Airway Chips infected with WSN (H1N1) or HK/68 (H3N2) in the presence (+) or absence (-) of added neutrophils (PFU, plaque-forming units). (E) Production of indicated cytokines and chemokines in the human Airway chip at 48 h post-infection with different clinically isolated influenza virus strains, including NL/09 (H1N1), Pan/99 (H3N2), and HK/97 (H5N1) (MOI = 0.1). *, P<0·05; **, P<0·01; ***, P<0·001.
Figure 3A. Hierarchical clustering created on the 1.010 most significantly differentially expressed (DE) genes between embryonic (Emb) F4/80hi and CD11bhi cells. Heat map displays z-transformed log2-expression values from red to blue via white. B. Hierarchical clustering based on the 755 most significantly differentially expressed genes between adult F4/80hi and CD11bhi cells. Heat map shows z-transformed log2-expression values from red to blue via white. C. Scheme describing the compared samples of F4/80hi (microglia, liver, kidney) and CD11bhi (liver, kidney) tissue MΦ populations for analysis shown in D-I. D. Venn diagrams overlaying the genes which are jointly expressed in F4/80hi or in CD11bhi cells at similar levels irrespective of tissue origin ('non-differentially expressed [non-DE] genes') defined by a false discovery rate (FDR) adjusted p-value > 0.95 for both embryonic and adult cells separately. Numbers in parentheses indicate the amount of all genes for each MΦ population. E. Venn diagram shows the differentially expressed (DE) genes between adult Langerhans cells (LC) and adult F4/80hi or CD11bhi cells (functional connectivity |FC| > 1.5, FDR adjusted p-value < 0.05). F. Number of up- and down-regulated genes in F4/80hi versus CD11bhi populations (|FC| > 1.5, FDR adjusted p-value < 0.05) from genes fulfilling the criteria in (D) i.e. non-DE genes between organs for each group. G. Hierarchical clustering of embryonic F4/80hi and CD11bhi cells based on 43 DE-genes in embryonic cells visualized in F. Heat map exhibits z-transformed log2-expression values from red to blue via white. H. Hierarchical clustering of adult F4/80hi and CD11bhi cells based on 209 DE-genes in adult cells visualized in F. Heat map presents z-transformed log2-expression values from red to blue via white. I. Quantitative RT-PCR of differentially expressed genes depicted in G-H. Analyses of differentially expressed genes in embryonic (Hbb-y and Hba-x) or adult (Rnf135, March8, Slc7a11, Ptgs2, Flt3, Ccr2, Maf and Csf1r) tissue macrophages. Data are normalized to β-actin and GAPDH and expressed as ratio of the mRNA expression compared to microglia and show mean ± s.e.m. Three mice per group were analyzed.
Figure 6HEK293T cells transfected with HA-E2F1 Wild Type (WT) and HA-E2F1 lacking a CY motif (∆RxL) harvested for immunoprecipitation using HA antibodies. Immunoprecipitates were immunoblotted as indicated. HEK293T cells were cotransfected with MYC-tagged ubiquitin and HA-E2F1 or HA-E2F1 ∆RxL lacking the CY motif as indicated (+). HA-E2F1 was immunoprecipitated with an anti-HA resin under denaturing conditions and immunoblotted. HeLa cells stably expressing HA-E2F1 WT and HA-E2F1 ∆RxL under the control of a retroviral promoter were treated with Cycloheximide (CHX) at 50 μg/ml for the indicated minutes (min) Quantification of E2F1 half-life HeLa cells stably expressing an Empty Vector (EV), HA-E2F1 WT and HA-E2F1 ∆RxL under the control of a retroviral promoter were treated with Chk1i for the indicated hours (h). Indicated protein were resolved by SDS page and detected by WB. H3 was used as a loading control. Relative survival (%) of HeLa cells stably expressing an Empty Vector (EV), HA-E2F1 WT and HA-E2F1 ∆RxL under the control of a retroviral promoter measured using rezasurin and compared to EV treated with Chk1i at the indicated concentrations.
Figure 4Anti-RFP immunoprecipitates from mCherry-NDP52 and mCherry-NDP52 ∆SKICH-transfected cells were subjected to SDS-PAGE followed by silver staining. Four bands specific for mCherry-NDP52 are numbered and were identified by mass spectrometric analysis. Asterisk (*) and double asterisk (**) indicate mCherry-NDP52 and mCherry-NDP52∆SKICH bands, respectively Anti-RFP immunoprecipitates from mCherry-NDP52 and mCherry-NDP52 ∆SKICH-transfected cells were subjected to SDS-PAGE followed by silver staining. Four bands specific for mCherry-NDP52 are numbered and were identified by mass spectrometric analysis The summary of mass spectrometric analysis is shown in (B) (C) HeLa-GFP-Parkin cells expressing mCherry-NDP52 mutants were incubated with 1 µM valinomycin and 100 nM bafilomycin A1 for 3 h. Cell lysates were subjected to immunoprecipitation using anti-RFP magnetic beads. Immunoprecipitates were analyzed by western blotting. Data are representative of three independent experiments (D) HeLa-GFP-Parkin cells transfected with siMTPAP were incubated with 1 µM valinomycin for the indicated times. Western blotting was performed using the indicated antibodies. Data are representative of three independent experiments (E) Mitophagic flux in cells transfected with siNDP52, siMTPAP and combinations of siRNAs. Cells were incubated with valinomycin for 3 h. Results are the summary of three independent experiments. Values are the means ± SEM. **p < 0.01 vs scrambled oligo-transfected cells; NS: not significant, determined with one-way ANOVA followed by the Tukey-Kramer post hoc test (F) Images of MTPAP KD cells expressing mCherry-NDP52 under valinomycin treatment. The images are from Movie EV 7. Cyan, GFP-Parkin; magenta, mCherry-NDP52. Images are representative of two independent experiments. Scale bars, 10 µm
Proteome analysis reveals Orm2 as an endogenous Dsc complex substrate.Figure 2(A) Vacuolar proteolysis-deficient pep4∆ and tul1∆ pep4∆ cells were labeled to saturation with heavy 13C6,15C2-L-lysine and then chased for 3 hours in presence of light L-lysine, following quantitative proteome analysis by LC-MS (two biological replicates). To analyze Dsc complex-dependent protein turnover, the ratio of H/L ratios of tul1∆ pep4∆ over pep4∆ cells of 2511 proteins quantified in both strains was plotted, and proteins with a ratio of H/L ratios >2 were selected. Red squares: regulated proteins with at least one predicted transmembrane (TM) domain; black triangles: regulated proteins without TM domains. (B) Schematic representation of the Dsc ubiquitin ligase complex (a top hit from the SGA screen) and the SPOTS complex (a putative Dsc substrate). SPT, serine-palmitoyltransferase; LCB, long chain bases. SDS-PAGE and Western blot analysis with the indicated antibodies of total yeast lysates from WT cells and the indicated mutants SDS-PAGE and Western blot analysis with the indicated antibodies of total yeast lysates from WT cells and the indicated mutants SDS-PAGE and Western blot analysis with the indicated antibodies (E) of input and elution (with FLAG peptide) from denaturing FLAG-Orm2 immunoprecipitations (IP) from WT cells and the indicated mutants. Control cells expressed untagged Orm2 SDS-PAGE and Western blot analysis with the indicated antibodies (F) of input and elution (with FLAG peptide) from native FLAG-Orm2 co-immunoprecipitations from WT cells and the indicated mutants. The control strain expresses untagged Orm2.
Figure 4Live-cell Airyscan image of mitochondrion in HeLa cell, showing different membrane potentials in different mitochondrial regions. Membrane potentials were calculated based on TMRE FI differences between compartments (FIcomp). Regions of interest from cristae and IBM used for ΔΨm calculations are labeled with arrows on the right-hand side of the mitochondrion, using the cytosol as the reference compartment. Labeled with forks on the left-hand side of the mitochondrion are the calculations of ΔΨm between individual cristae and their neighboring IBM. Nernst equation used to calculate different voltages. Scale bar = 500 nm. LUT of mitochondrion in HeLa cell shown in A, color-coding TMRE FIs on scale of white (most intense) to blue (least intense): LUT scale shown in lower left-hand corner. Arrowheads indicate cristae. Scale bar = 500 nm. Note that most-intense pixels (white) only associate with cristae. Quantification of ΔΨm (mV) at cristae and IBM relative to the average ΔΨm of the whole mitochondrion, calculated as in A. Note: ΔΨm at cristae is significantly higher than ΔΨm at IBM, indicating electrochemical boundaries separate these two regions of the IMM. N = 3 independent experiments. Live-cell Airyscan imaging of mitochondria in cultured primary mouse hepatocytes stained with TMRE. Arrowheads denote cristae. D. Image showing TMRE-labeled mitochondria. E. LUT color-coding of TMRE FI. F. Quantification of ΔΨm differences using Nernst equation as shown in A. Scale bar = 500 nm. N = 2 independent experiments. Live-cell STED imaging of mitochondria in HeLa cell stained with TMRM. Arrowheads show cristae. Scale bar = 500 nm. G. Image showing TMRM-labeled mitochondrion. H. LUT color-coding of TMRM FI. I. Quantification of ΔΨm differences using Nernst equation as shown in A. Scale bar = 500 nm. N = 4 independent experiments. Note that calculation of ΔΨm differences between the compartments based on images captured by STED and by Airyscan were very similar.
Figure 3A) If the sex pheromone receptor is a GPCR as hypothesized, it could elicit chemoattraction through the signal transduction pathways that control cGMP-gated channels and TRPV channels. Knocking out any key component in the two pathways should reduce or abolish chemoattraction behavior. B) Mutant males of 4 GPCR signaling cascade components failed to respond to C. remanei sex pheromone, suggesting that the sex pheromone receptor is a GPCR and that both aforementioned signaling pathways are involved in pheromone perception. We assayed 400 males from each strain in the sex pheromone chemoattraction assay. Two biological replicates were combined into a single value. Significance was determined using one-way ANOVA with Bonferroni correction: ***P<0.001. Means ± SEM (error bars) are shown. C) In males from both AWA-specific-rescue odr-3 mutant strain and odr-3 rescued mutant strain, the sex pheromone response was significantly restored in single male worm arrival assays. However, males from the CEM-specific-rescue odr-3 mutant strain failed to reach the sex pheromone within 30 min. In odr-3 and him-5 strain experiments, the sample sizes were 12 and 20 worms, respectively; in all other experiments, 40 worms were used.
LRRC8 subunit‐ and osmolarity‐dependent carboplatin/cisplatin transport.Figure 6Carboplatin uptake into control KBM7 cells and two LRRC8D‐deficient clones (n = 6). Hypotonicity‐stimulated cisplatin uptake in HEK WT cells was selectively blocked by 100 μM carbenoxolone (CBX), a non‐specific blocker of VRAC (n = 3). Cisplatin uptake into HEK cells of indicated genotypes using 40 μM cisplatin under long‐term isotonic (C) or 200 μM cisplatin in short‐term hypo‐ and isotonic (D) conditions as function of time (n = 3). Similar results were obtained in HCT116 cells (Fig EV4). Cisplatin uptake (200 μM) into HEK cells of indicated genotypes. LRRC8(B,C,E) −/− and LRRC8(B,D,E) −/− cells express only LRRC8A and LRRC8D, and LRRC8A and LRRC8C, respectively (n = 3 for WT, 6 for LRRC8D −/−, 9 for LRRC8A −/− in E; n = 3 in F). Mean current densities of maximally activated ICl,vol at −80 mV. The number of cells is indicated for each column. Ratio of LRRC8‐dependent swelling‐activated cisplatin uptake (60 min) to mean ICl,vol (as in G) as function of genotype.Data information: Data are presented as mean ± SEM. *P < 0.05; **P < 0.01; and ***P < 0.001 (for (C, E) compared to HAP1−/− cells). Dotted lines in (D) and (F) highlight that there is no significant difference in isotonic 60‐min uptake between the genotypes.
a) Relative body weight changes in mice after bilateral injection with rAAV-R, rAAV-Q and rAAV-HQ at 6–14 weeks of age (n = 11 for all three groups until 12 weeks; at 14 weeks, n = 7 for R/R, n = 10 for Q/Q, n = 11 for HQ/HQ) (*, P = 0.figf6(a) Relative body weight changes in mice after bilateral injection with rAAV-R, rAAV-Q and rAAV-HQ at 6-14 weeks of age (n = 11 for all three groups until 12 weeks; at 14 weeks, n = 7 for R/R, n = 10 for Q/Q, n = 11 for HQ/HQ) (*, P = 0.0401; **, P = 0.0081; ***, P = 0.0182; ****, P = 0.0225). (b) Clasping scores. n = 11 for all three groups (scale: 0, no clasping; 1, clasping of the forelimbs only; 2, clasping of both fore and hind limbs once or twice; 3, clasping of both fore and hind limbs more than 3 times or more than 5 s). (c) Rotarod performance; n = 11 for all three groups (*, P = 0.0055; **, P = 0.002; ***, P = 0.0455; ****, P = 0.0242). (d) Survival curves for mice after bilateral injection with rAAV-R, rAAV-Q, and rAAV-HQ; n = 11 for all groups. Median survival (days): R/R-108; Q/Q-123; HQ/HQ-140. Mean survival (days): R/R-105.5 ± 3.9; Q/Q-122.3 ± 4.8; HQ/HQ-136.2 ± 4.4.Log-rank test, P 0.0001; Wilcoxon test, P 0.0001.
AP characterization of CMs generated from control and RA-treated differentiations.Figure 3A AP illustrating the analyzed parameters. B Representative APs of day 31 CMs from control (CT) and RA-treated (RA) groups at 1 Hz. C-E RMP, APAmax and APAplat (C), dV/dtmax (D) and APD20, APD50 and APD90 of CT and RA CMs (E).F Plot showing all measured APAplat values of CT and RA CMs. G Representative APs of CT and RA CMs at 0.5-4 Hz.H Average APAplat at 0.5-4 Hz. Please note that the AP differences in morphology are present at all measured frequencies.
Figure 5A RNA pull-down was performed using in vitro transcribed biotinylated transcripts and whole cell extracts from Miwi+/-, Miwi-/-, Miwi-/ADH, and Mov10l1f/-;Stra8-Cre testes. Mov10l1f/-;Stra8-Cre mice represent a pachytene piRNA-deficient condition. In vitro transcribed RNAs corresponding to approximately one repeat of major and minor satellite sequences were used as baits. Precipitated protein was visualized by Western blotting using anti-Dicer antibody. B Equal amounts of total RNA isolated from spermatocytes of Miwi+/-;Dicerf/+;Stra8-Cre, Miwi-/-;Dicerf/+;Stra8-Cre, Miwi+/-;Dicerf/-;Stra8-Cre and Miwi-/-;Dicerf/-;Stra8-Cre mice, were resolved on a 1% agarose gel. The gel was subjected to Northern blot analysis using probes specific for the sense and antisense sequences of major and minor satellites. The blot was re-hybridized with a 28S rRNA-complementary probe to demonstrate equal loading (bottom on the left). C in vitro RNA processing assay with nuage-enriched nuclear fraction from Miwi+/-;Dicerf/+;Stra8-Cre, Miwi-/-;Dicerf/+;Stra8-Cre, and Miwi+/-;Dicerf/-;Stra8-Cre mice, and 5'-end radioactively labelled RNA targets corresponding to approximately one and two repeats of sense and antisense major satellite sequences. Arrow indicates the cleavage products of In vitro transcribed sense major satellite RNAs by MIWI-piRNA complex. D Equal amounts of total RNA isolated from spermatocytes of Miwi+/-;Dicerf/+;Stra8-Cre, Miwi-/-;Dicerf/+;Stra8-Cre, Miwi+/-;Dicerf/-;Stra8-Cre and Miwi-/-;Dicerf/-;Stra8-Cre mice, were digested with RNase III (RNase III) or nuclease S1 (S1), and further resolved on a 1% agarose gel. The gel was subjected to northern blot analysis using probes specific for the sense (S) and antisense (As) strands of major (Maj) and minor (Min) satellite transcripts. The blot was re-hybridized with 18S and 28S-complementary probes to demonstrate equal loading and the same efficiency of RNase III or nuclease S1 digestion. E Sequential in vitro RNA processing assay using 5'-end radioactively labelled RNA targets corresponding to approximately one repeat of sense and antisense major satellite sequences. The nuage-nuclear fraction from Miwi+/- testes is indicated as N. Total cellular extracts (T) from Miwi+/- testes were subjected to immunodepletion using anti-Dicer antibodies (α-Dicer), with IgG (IgG
Figure 3(A) Representative confocal images of muscle-6/7 NMJ synapses of abdominal hemisegments A2-A3 for the indicated genotypes labelled with anti-Synaptotagmin (green) and HRP (red) to reveal the synaptic vesicles and the neuronal membrane. Scale bar: 20 μm. (B-D) Quantification of normalized bouton number ((B), total number of boutons/muscle surface area (μm2 × 1,000)), normalized axon length (C) and normalized branching (D) of NMJ 6/7 in A2-A3 of the indicated genotypes. Bars show mean ± s.e.m. Multiple comparisons were performed using one-way ANOVA with a post-hoc Sidak-Bonferroni correction (n.s. = not significant; p<0.05 = *; p<0.01 = **; p<0.001= ***; p<0.0001=****). Data information: In (B-D, , bars are labelled with the number of replicates. (E) Immunofluorescence analysis of adult brains for the indicated genotypes using anti-Fascilin II (FasII) antibody to visualize lobes of the MB. Pink dashed lines highlight the normal and fused β-lobes. Scale bar 50 μm. (F) Quantification of the penetrance of fusion phenotype in the indicated genotypes. Data information: In bars are labelled with the number of replicates. (G) Quantification of normalized bouton number (total number of boutons/muscle surface area (μm2 × 1,000)), of NMJ 6/7 in A2-A3 of the indicated genotypes. Bars show mean ± s.e.m. Multiple comparisons were performed using one-way ANOVA with a post-hoc Sidak-Bonferroni correction (n.s. = not significant; p<0.05 = *; p<0.01 = **; p<0.001= ***; p<0.0001=****). Data information: In G), bars are labelled with the number of replicates.
Long-read sequencing implies tissue specific transposon mobility A Genomic DNA was isolated from pools of 60 guts or 60 heads dissected from the same individuals and subjected to ONT long-read sequencing.Figure 5A Genomic DNA was isolated from pools of 60 guts or 60 heads dissected from the same individuals and subjected to ONT long-read sequencing. If a somatic insertion occurs in a tissue and does not undergo clonal expansion, it will be present in a small fraction of cells within the pool sequenced. Long-read sequencing allows to identify putative somatic integrations as rare TE inserts, fully contained in a single continuous sequencing read and generating a valid TSD. B TE families with tissue-specific, singleton TSD-bearing insertions detected in pooled gut or head DNA. C The target site motif found around (+/- 10bp) gut-specific rover singleton insertion sites identified with the long-read sequencing. (E-value was calculated with MEME software (Bailey et al, 2009), where E-value < 0.05 is considered statistically significant)
Figure 4(A, B) Side view and top view of the Cryo-EM reconstruction (A) and molecular model (B) of the disome stalled on a (CGA-CCG) reporter mRNA. The first ribosome in the disome stalled on the di-codon mRNA is referred to as the leading ribosome, the second as the colliding ribosome (C) Cut top views on the leading and colliding ribosome. The leading ribosome is in a non-rotated POST state containing tRNAs in P/P and E/E states and an empty A site, whereas the colliding ribosome is in a rotated state containing hybrid A/P and P/E site tRNAs. An overlay is shown omitting the 40S subunit for clarity. (D) Cut top view on the disome with focus on the mRNA density highlighted in red. (E) Zoom on the mRNA at the interface of the leading and colliding ribosome. The mRNA exits the leading ribosome close to ribosomal proteins uS11, eS26 and eS28 and enters the colliding ribosome near h16, uS3, uS5 and eS30.
Figure 1(A) HT29 cells were infected with Shigella WT, S325, or ospD deletion mutants, and incubated for 8 h. Aliquots of cellular supernatants were subjected to cytotoxicity assays. *P<0.05 (one-way ANOVA). (B) HT29 cells were infected with Shigella WT or ∆ospD3 and incubated for 8 h. Infected cells were fixed and subjected to TUNEL and PI staining. Percentages of positive cells (TUNEL, green; PI, red) are shown in graph at right. The nuclei were stained with DAPI (blue). Scale bar: 100 μm. n.s., not significant; *P<0.05 (unpaired two-tailed Student's t-test). (C) HT29 cells were infected with Shigella WT, S325, or ∆ospD3 and incubated for 8 h. Infected cells were subjected to Giemsa staining. Arrows indicate cells in which the cytoplasm disappeared. Scale bar: 20 μm.
Biallelic truncating mutations in CENATAC (CCDC84) cause MVA.Figure 1Clinical phenotypes of CENATAC (CCDC84) mutant patients. See also Figure EV1. Schematic representation of CENATAC annotated with zinc fingers (ZF1 and ZF2), predicted coiled coil, and conserved motifs 1-4 (M1-M4). Upper: sequence logos of both zinc fingers and the conserved residues defining motifs 1-4 (underlined). See Appendix Figure S2 for the full-length logo. Lower: C-terminal protein sequences of wild-type and MVA mutant CENATAC. The MVA truncation site is indicated by the red dotted line; the four conserved motifs are outlined in red. CENATAC and tubulin immunoblots of lysates from lymphoblasts of patient 1 and relatives. Wild-type and truncated, mutant proteins are indicated. Wild-type CENATAC (CEN): 38 kDa, Mut1: 34.5 kDa (father), Mut2: 31.1 kDa (mother). Phosphorylated CENATAC is indicated with asterisks: black (wild-type), red (Mut1), or blue (Mut2) (Wang et al, 2019).