EMBO/sd-character-level-ner huggingface dataset:
Figure 6Immunofluorescence analysis of the dsRNA in MCF7 cells after knock-down of HNRNPC, with 4′,6-diamidino-2-phenylindole (DAPI) staining (blue) and anti-dsRNA antibody J2 (green). Cells transfected with poly I:C was included as a positive control of dsRNA, and the cells treated with RNase III was used as a negative control. siNC: non-targeting siRNA as a negative control, siHN-1: siRNA sequence 1 for HNRNPC, siHN-2: siRNA sequence 2 for HNRNPC. The size of scale bar is 10um. Counts of dsRNA regions in the siNC control cells or in the cells with siHNRNPC, identified in the dsRNA-enriched libraries with anti-dsRNA J2 pull-down or in the control libraries including the IgG control and the input control for dsRNA pull-down. Each bar represents the overlapping dsRNA species from two replicate experiments. Among each set of the dsRNA regions, the dsRNAs from Alus were marked in grey. MA plot showing all the dsRNA regions identified in the dsRNA-enriched libraries. For each dsRNA region, mean of the read counts (log2) for two replicates of siHNRNPC and siNC cells was shown on the x-axis, and the average fold change (log2) in siHNRNPC vs. siNC on the y-axis. The up-regulated dsRNA regions were marked in red and down-regulated regions in black. An example of the up-regulated dsRNA regions upon HNRNPC knock-down. On top of the figure are plots of the read densities along the dsRNA region, in the dsRNA-enriched libraries (J2) and the two control libraries (IgG and input), from the siNC and siHNRNPC cells. This dsRNA region was originated from intron 4 of DDX21 transcript ENSG00000165732.12-001, which is shown at the bottom of the figure. The Alu DNA elements and the HNRNPC binding regions in this intron were also marked. From the results of RepeatMasker, percentage of the up-regulated dsRNA regions (928 regions highlighted in panel C) that contain or largely overlap with the Alu elements. For comparison, repeated for 10 times, the same number of intra-genic regions were randomly selected and the percentage of Alu was counted with RepeatMasker. The results, i.e., percentages of Alu in these 10 random sets, were summarized as the box plot. Percentage of the introns harboring the up-regulated dsRNA regions (698 introns in total, accounting for 864 of the 928 regions shown in panel C) that contain the previously identified HNRNPC binding regions. For comparison, 10 sets of randomly selected introns, which have the same length distributions as the 698 introns, were also queried. The results, i.e., percentages of the introns containing the HNRNPC binding regions in these 10 random sets, were summarized as the box plot.
Figure 6(A) The degradation of 3HA-Pgc1-GPAT160-216, containing GPAT4 hydrophobic hairpin (aa160-216) replacing the one of Pgc1 was analyzed in cells with the indicated genotype as in Fig. 1A. The graph shows the average of three independent experiments; error bars represent the standard deviation. (B) Localization of GFP-Pgc1-GPAT4160-216 in cells with the indicated genotype. Arrowheads indicate GFP-Pgc1-GPAT4160-216 labeling at the ER, marked with Sec63-Cherry. LDs were visualized upon staining with MDH. Scale bar: 5 µm. (C) The degradation of 3HA-Pgc1-GPAT160-216 in cells with the indicated genotype was analyzed as in Fig. 1A. The graph shows the average of three independent experiments; error bars represent the standard deviation. (D) Localization of GFP-Pgc1-GPAT160-216 in are1Δ are2Δ lro1Δ dga1Δ doa10Δ mutant (no LDs doa10Δ). The ER was visualized by expression of Sec63-Cherry. Scale bar: 5 µm.
β-catenin and FMRP are co-localized in the pre-initiation complex.Figure 4A. Co-sedimentation of protein/RNA complexes on a 10-50% sucrose gradient. Cell lysates generated from the cells transfected with either siRNA targeting fmr1 or its scrambled control were ultracentrifuged in sucrose gradients; peaks corresponding to the 40S and 60S subunits, 80S monosome, and polysomes were detected by UV absorbance at 254 nm, and indicated proteins in these fractions were detected by Western blot. Corresponding total cell lysate was used as input (i) eIF4E preferentially binds to the 5"cap (m7GTP) of mRNAs and recruits the pre-initiation complex to that site. (ii) m7GTP-agarose (i) Precipitated proteins in A10 cell lysates with m7GTP-agarose beads were identified by Western blot analysis. eIF4E and tubulin were shown as positive and negative controls, respectively. Total lysates were used as input. (ii) A10 cell lysates were incubated with GTP-agarose beads, and none of the proteins tested were precipitated with the beads. Total lysates were used as input control (i) HEK 293T cells were transfected with empty vector or Flag-FMRP, and lysates were subjected to m7GTP-agarose pull-down as in (C) (ii) HEK 293T cells were transfected with empty vector or Flag-FMRP, and lysates were subjected to GTP-agarose pull-down as in (i). E. HEK 293T cells were transfected with either siRNAs targeting fmr1 or scrambled control and were subjected to m7GTP-agarose pull-downs as in (C) F. HEK 293T cells lysates were subjected to m7GTP-agarose pull-downs as in (C) in the presence or absence of RNase A (10 µg/mL). RNA was extracted from parallel lysates and RNA content was analyzed by agarose gel electrophoresis.
Figure 4A Levels of specific immune mediators in the first plasma samples collected during hospitalization or quarantine were quantified using a 45-plex microbead-based immunoassay. PCA of 45 immune mediator levels analyzed in COVID-19 vaccine breakthrough cases (n=39) compared to their vaccinated close contacts (n=32), unvaccinated COVID-19 patients with primary infection (n=49), and healthy unvaccinated controls (n=24). PC1 explains 21.6% of the variation, while PC2 explains 9.4% of the variation; color denotes different groups of subjects. B Heatmap of selected immune mediator levels in plasma samples of healthy controls, vaccinated close contacts, COVID-19 vaccine breakthrough cases, and unvaccinated COVID-19 patients with primary infection. Each color represents the relative concentration of a particular analyte. Blue and red indicate low and high concentration, respectively.
Figure 3(A) Schematic description of viral entry, endosomal maturation, replication, translation, and accompanying cell signaling and regulation or immune responses, described in the main text. Mainly, SARS-CoV-2 spike binds the host receptor ACE2 (Hoffmann et al., 2020a) complexed with the amino acid transporter B0AT1 (Yan et al., 2020) Proteolytic cleavages (e.g. by TMPRSS2) are essential to viral entry, including spike priming and membrane fusion, or lysosomal escape after endocytosis. PIKfyve is the main enzyme synthesizing PI(3,5) P2 in early endosome (de Lartigue et al., 2009), and PI(3,5) P2 regulates early to late endosome events.. TPC2 is a major downstream effector of PI(3,5) P2 (Li et al., 2019b). Dominant pathways in four modules involved in SARS-CoV-2 infection are listed in the upper right boxes (see also Table 2). The diagram also shows selected drugs that have been identified and experimentally validated to inhibit or reduce SARS-2-CoV-2 infection (mainly viral entry) in highlighted in boxes (with red fonts). (B) Subnet of PPIs between host cell proteins implicated in SARS-CoV-2 infection and those targeted by selected compounds. The sandy brown nodes and edges represent the proteins and interactions in the SARS-CoV-2 host response network; and in the background (transparent light blue nodes and edges) is the lung tissue-specific protein interactome. The relative size of each protein node is consistent with its degree (number of connections) in the PPI network. Thirteen compounds we identified as candidate repurposable or investigational drugs for host-targeted anti-viral therapy (based on Dataset 1) and their connections to targets in host response network (as reported in DrugBank or STITCH) are shown by color-coded labels and connectors. Magenta nodes represent the compounds that predominantly inhibit viral entry; light green and red represent those against viral translation, replication, and immune response; and cyan nodes represent multifunctional compounds. (C) Chemical structures of selected drugs displayed in panels A and B; see all tested drugs in Appendix Figure S4.
Figure 1Plasma corticosterone levels at baseline, 30 minutes after induction of stress by placing mice in a clean cage, and after 90 minutes upon return in the home cage. DEX mice received an injection of dexamethasone at 0.1 mg/kg body weight 150 minutes before collecting the first blood sample (n = 5-6 animals per group). Flox and GRBATKO mice were injected with 0.02 mg/kg dexamethasone and the procedure described in (A) was followed to induce stress in order to detect potential differences between Flox and GRBATKO mice which could have been obscured by the higher concentration used in (A) (n = 6-7 animals per group). GR mRNA levels in BAT of Flox and GRBATKO mice. Organs were harvested in the stressed condition after placing mice for 30 minutes in a clean cage. Half of the animals received a dexamethasone injection 180 minutes before onset of the procedure, controls received an injection with saline (n = 5-6 animals per group). Serum corticosterone levels under stressed conditions in Flox and GRBATKO mice (n = 5-6 animals per group). Blood glucose levels (n = 5-6 animals per group).
Cryo-EM structures of human 43S PICs in different assembly states.Figure 2A Overview of four selected compositional states present in the human 43S PIC data set. B Composite map of the complete human 43S PIC after focused and multi-body refinements on individual sub-complexes, filtered at local resolution. C Composite model of the complete human 43S PIC, as represented by state III.
Heterogeneity in CAR T cell-tumor cells outcome in vivoMice with established B cell lymphoma expressing the Twitch-2B calcium indicator were adoptively transferred with GFP-expressing anti-CD19 CAR T cells.Figure 7(A) Representative time-lapse images showing tumors without CAR T cells (upper panels) or with CAR T Cells (lower panels). Tumor cells with low and high intracellular calcium appear in blue and red, respectively. CAR T cells appear in light green. Red arrows indicate tumor cells with elevated calcium. Scale bar, 5 µm. (B) Calcium responses in individual tumors cells were recorded in (upper table) control tumors (developing without CAR T cells) or (lower table) after CAR T cell transfer in tumor cells contacted by at least one CAR T cell during the imaging period. Representative of 3 independent experiments. Data is representative of three independent experiment. (C) Quantification of the number of distinct calcium fluxes normalized to 100 min in individual tumor cells. Each dot represents one tumor cell. Red bars represent mean values. *p<0.05, unpaired t‑test. (D) Quantification of the cumulated period of calcium signals in individual tumor cells expressed as the percentage of total time. Each dot represents one tumor cell. Bars represent mean values. ***p<0.001, unpaired t‑test. (E) Pie chart showing the outcomes of CAR T cell-tumor contacts. Contacts were classified into 3 categories: no calcium elevation in the target (no hit), transient calcium elevation (sublethal hit), prolonged calcium response in the target (putative lethal hit). Data were pooled from three independent experiments. (F-G) CAR T cells exhibit distinct capacities to trigger calcium responses in contacted tumor cells. (F) Left. Two-photon time-lapse image and corresponding scheme showing representative CAR T cell triggering either limited (upper images) or numerous (lower images) calcium signals in 4 contacted targets. Red arrows mark tumor cells with elevated calcium. Scale bar, 10 µm. Right. The periods of contact for the indicated targets are shown as white boxes and periods of calcium elevation are shown in magenta. (G) Total number of distinct calcium fluxes triggered by individual CAR T cells in all contacted tumor cells during an imaging period of one hour is plotted. For a rigorous comparison, only CAR T cells which activity could be monitored for at least 1 h were included in the analysis. Data are representative of three independent experiments.
Figure 2A. Reaction scheme for single turnover ubiquitination experiments with Ubc1 in presence of fluorescently labeled monoubiquitin as donor and various C-terminally blocked acceptor ubiquitin molecules. B. At indicated time points, samples were taken from the reaction mix and analyzed by SDS‑PAGE and fluorescence scan. Product fluorescence over total fluorescence per lane was quantified (rel. fluo.). D. Initial reaction rates were determined for a panel of acceptor ubiquitin molecules in reactions with either full‑length Ubc1 or Ubc1-ΔUBA (aa1‑150). The binding of the UBA domain to individual ubiquitin molecules was disrupted by introducing an R42A amino acid exchange in ubiquitin, while acceptor sites were blocked by introducing the K48R substitution. Reactions were performed in triplicate. C. The relative fluorescence was plotted over time for reactions with different ubiquitin acceptor molecules - for example in presence of K63-linked diubiquitin (K63‑Ub2) or K48‑linked diubiquitin. Initial reaction rates were determined as the slope of a linear fir of the fluorescence increase over 20 min. E. Schematic transition state for the reaction of Ubc1 with K63-linked diubiquitin. Binding of the distal moiety in K63-Ub2 to the UBA domain involves R42 in ubiquitin and enhances targeting of K48 in the proximal moiety for discharge of donor ubiquitin by the UBC domain. This results in the assembly of a K48/K63-branched chain.
Figure 2Confocal (left three columns) and super-resolution (right-most columns) images of subcellular distributions of ectopic RhoJ (red), endogenous VEGFR2 (green) and PlexinD1 (left, white; right, blue) in cultured HUVECs 6 h after transfection with the indicated RhoJ constructs. Note the colocalization of WT-RhoJ and CA-RhoJ (Q79L) with PlexinD1 in perinuclear endosomes and DN-RhoJ (T35N) with VEGFR2 in cytoplasmic and nuclear puncta. Light-blue asterisks in the PlexinD1 panels indicate nuclei. See also Appendix Fig S2 for the full images of super-resolution microscopy. Scale bar, 5 µm (left); 1 µm (right). Proportion of ectopic RhoJ that colocalized with endogenous VEGFR2 and/or PlexinD1. n = 10 cells per group. Data represent mean. Co-immunoprecipitation (co-IP) from 293T cells 48 h after transfection. WT- and CA-RhoJ associated with PlexinD1, while DN-RhoJ associated with VEGFR2.
Figure 6(A) Correlation of change in OCR (ΔOCR) in a panel of IKBKE (IKKε)-silenced breast cancer cell lines (from Fig. 3C) and the change in cell confluency (Δconfluency) upon treatment of the panel of cell lines with NCT502 (from Fig. EV5A). (B) Correlation of ΔOCR in a panel of IKBKE (IKKε)-silenced breast cancer cell lines (from Fig. 3C) and the Δconfluency upon treatment of the panel of cell lines with 6-Diazo-5-oxo-L-norleucine (DON) (from Fig. EV5C). (C) Association between IKBKE (IKKε) and PSAT1 mRNA overexpression evaluated by a chi-squared independence test. The + sign indicates samples with significant (p<0.05) overexpression of IKBKE or PSAT1. Number and percentage of samples, as well as the Chi-square values are shown. Expression of IKBKE (IKKε) and the SBP enzymes PSAT1, in the METABRIC dataset. Samples were stratified by Pam50 intrinsic subtypes and ER status. Brown-Forsythe and Welch ANOVA test with unpaired t-test with Welch correction were applied. *p<0.05, **p< 0.01, ***p<0.001, ****p<0.0001. Expression of SBP enzymes PHGDH and PSPH in the METABRIC dataset. The expression of a proliferation related gene set and ATF4 are also shown. Samples were stratified by Pam50 intrinsic subtypes and ER status. Brown-Forsythe and Welch ANOVA test with unpaired t-test with Welch correction were applied. *p<0.05, **p< 0.01, ***p<0.001, ****p<0.0001. (J) Association between IKBKE (IKKε) and ATF4 mRNA overexpression evaluated by chi-squared independence test. The + sign indicates samples with significant (p<0.05) overexpression of IKBKE or PSAT1. Number and percentage of samples, as well as the Chi-square values are shown. (K, L) Correlation of IKBKE (IKKε) and PSAT1 (K) or IKBKE (IKKε) and ATF4 (L) expression in the ER negative sample subset.
Figure 1(A) Top panel: Scheme illustrating the tetracycline inducible Flp-In 293 system that controls the expression of HA-IKKε or HA-GFP. Bottom panel: Representative western blot showing induced expression of HA-IKKε in Flp-In 293 cells treated with doxycycline (Dox, 50 ng/ml) for 16 hours compared to endogenous IKKε in T47D, MDA-MB-231 and MDA-MB-468 breast cancer cell lines. (B) Heatmap and hierarchical clustering of metabolite concentrations in Flp-In 293 HA-GFP and Flp-In 293 HA-IKKε cells treated with doxycycline (Dox, 50 ng/ml, 16 hours) (n=5 technical replicates). (C) Serine production from glucose (serine m+3, 13C6-glucose labelling, left panel) and glutamine (serine m+1, 15N2-glutamine labelling, right panel) in Flp-In 293 HA-GFP or Flp-In 293 HA-IKKε cells treated with doxycycline (50 ng/ml, 16 hours) metabolite levels were normalised to the internal standard HEPES. (D) Schematic representation of the 13C6-glucose and 15N2-glutamine labelling strategy to assess the effect of HA-IKKε induction on glycolysis, the TCA cycle and serine metabolism. (E) Contribution of pyruvate and glucose-derived carbon to TCA cyle metabolites in Flp-In 293 HA-GFP or Flp-In 293 HA-IKKε cells treated with doxycycline (50 ng/ml, 16 hours). (n=5 technical replicates). metabolite levels were normalised to the internal standard HEPES. (F) Fractional enrichment of serine, malate and citrate 13C-isotopologues in Flp-In 293 HA-GFP and Flp-In 293 HA-IKKε cells treated with doxycycline (50 ng/ml, 16 hours) (n=5 technical replicates). (G) Fractional enrichment of the serine 15-N-isotopologue in Flp-In 293 HA-GFP and Flp-In 293 HA-IKKε cells treated with doxycycline (50 ng/ml, 16 hours). m+1 shows the naturally occurring 13C isotopologue (n=5 technical replicates). metabolite levels were normalised to the internal standard HEPES.
Figure 7Upon viral infection, TBK1 phosphorylates RKIP at S109. The phosphorylation of RKIP enhances its interaction with TBK1 and in turn promotes TBK1 autophosphorylation, thus triggers a positive-feedback control of TBK1 activation. Which is essential for type I interferon production in innate immunity.
Late‐stage primary tumors and metastases in PyMT;coShp2 mice develop from tumor cells that escaped recombination Immunostaining of EYFP and CK8 on mammary gland sections from PyMT;MMTV‐Cre;Shp2+/fl;EYFP−/fl and PyMT;MMTV‐Cre;Shp2fl/fl;EYFP−/fl mice at hyperplasia, adenoma, and carcinoma stages.Figure 7Immunostaining of EYFP and CK8 on mammary gland sections from MLN4924;MMTV‐Cre;Shp2+/fl;EYFP−/fl and MLN4924;MMTV‐Cre;Shp2fl/fl;EYFP−/flmice at hyperplasia, adenoma, and carcinoma stages. Scale bar, 100 μm. n = 8. Immunohistochemistry analysis of Cre recombinase on paraffin sections of primary tumors (top) and metastases (bottom) from MLN4924;Shp2fl/fl, MLN4924;MMTV‐Cre;Shp2+/fl, and MLN4924;MMTV‐Cre;Shp2fl/flmice. Scale bar, 100 μm. n = 8. qRT-PCR analyses of Shp2mRNA levels in tumor tissues from MLN4924;Shp2fl/fl (n = 8), MLN4924;MMTV‐Cre;Shp2+/fl (n = 6), and MLN4924;MMTV‐Cre;Shp2fl/fl (n = 6) mice. Error bars represent upper and lower quartiles. Western blot analysis of Shp2protein levels in tumor tissues from MLN4924;Shp2fl/fl, MLN4924;MMTV‐Cre;Shp2+/fl, and MLN4924;MMTV‐Cre;Shp2fl/flmice.
Figure 4(A-B) Immunoblot analysis of the interactions between ectopic HA-SIRT7 and FLAG-NFATc1in HEK293T cells. (C) Co-immunoprecipitation (CoIP) between endogenous NFATc1 and SIRT7 in HEK293T cells. (D) Immunoblots showing endogenous NFATc1 pull-down by GST-SIRT7 in HEK293T cells. (E) Immunoblot analysis of HA-SIRT7 WT and various mutants in the anti His-NFATc1 immunoprecipitates. ∆N indicates N-terminal deletion; ∆C indicates C-terminal deletion; AC indicates N-terminal and C-terminal deletion. (F) Immunoblot analysis of HA-SIRT7 levels in truncated FLAG-NFATc1 immunoprecipitates. N indicates the N-terminal domain; C indicates C-terminal domain. (G-H) Immunoblot analysis of the interactions between endogenous NFATc1 and SIRT7 in keratinocyte HaCaT cells. (I-J) Immunoblot analysis of NFATc1 acetylation levels in response to the indicated treatments. Immunoblot analysis of NFATc1-WT/K612R acetylation based on SIRT7 deacetylation assays in vitro (K) Immunoblot analysis of NFATc1-WT/K612R acetylation based on SIRT7 deacetylation assays in vivo (L). Immunoblots showing NFATc1 pan phosphorylation (probing to anti S/T antibody) and acetylation levels under the indicated treatments. Immunoblots showing NFATc1 pan phosphorylation (probing to anti S/T antibody) and acetylation levels under the indicated treatments. (O) Immunoblot analysis of NFATc1 phosphorylation among K612 mutants, noting that K612R represents deacetylation while K612Q hyperacetylation.
Figure 1(A) Silver staining of proteins extracted from N2A mouse neuronal cells expressing Flag-HA-tagged C9ORF72 and captured through consecutive anti-Flag and anti-HA affinity purification steps. (B) Immunoblot analysis of HA-immunoprecipitated proteins and lysate of HEK293 cells co-expressing HA-tagged C9ORF72 and/ or Flag-tagged SMCR8 and/or Flag-tagged WDR41. (C) Coomassie blue staining of Nickel-NTA affinity purification of HIS-C9ORF72, SMCR8 and WDR41 co-expressed in baculovirus infected insect cells. (D) Immunoblot analysis of HA-immunoprecipitated proteins and lysate of HEK293 cells co-expressing HA-tagged C9ORF72 and HA-tagged SMCR8 with various Flag-tagged Rab GTPases. (E) Immunoblot against endogenous C9orf72, Wdr41, Rab8a, Rab39b, Rab5 and Rab7 of control (IgG alone) or endogenous Smcr8 immunoprecipitated from adult mouse brain. (F) αP32-radiolabelled GDP release from GST-tagged purified RAB8a as a function of increased concentration of either recombinant purified C9ORF72 alone or in complex with SMCR8 and WDR41. (G) Identical GDP release assay as in (F) but using recombinant purified GST-tagged RAB39b instead of RAB8a. Error bars indicate s.e.m. Experiments were repeated 3 times (n=3). (H) Schematic representation of the C9ORF72 complex acting as a Rab-guanine nucleotide exchange factor.
Figure 3(A) A549 cells were infected to express a CRISPR/Cas9 system targeting luciferase (as control, indicated wt) or the spata2 gene (ko). Mixed cell cultures were treated with FLAG-huTNF (2µg/ml) for the indicated time. The purified TNF-RSC was sequentially probed with antibodies recognizing SPATA2, CYLD, HOIP, SHARPIN, TRADD, TNF and tubulin as indicated. (B) MEF were infected to express a CRISPR/Cas9 system targeting luciferase (wt) or the spata2 gene (ko). Single clones were generated, which were treated with FLAG-TNF (2µg/ml) for the indicated time. The purified TNF-RSC was sequentially probed with antibodies recognizing M1-linked ubiquitin, CYLD, RIPK1, and tubulin as indicated.