EMBO/sd-character-level-ner huggingface dataset:
Figure 3(A) Schematic showing presence of tumor cells capable of optimal and non-optimal apoptotic activation by DR5 agonists constitute heterogeneous tumors. ApoEVs isolated after IgG1 and KMTR2 treatment (OVCAR-3) were blotted against CD63 and PD-L1 in dot blots. ApoEVs isolated after IgG1 and tigatuzumab treatment (MDA-MB-436) were run on western and blotted against CD63 and PD-L1. ApoEVs isolated after IgG1 and lexatumumab (D) treatment (MDA-MB-436) were run on western and blotted against CD63 and PD-L1. (E) Details of experimental data (F) ApoEVs isolated from DR5 sensitive tumor cells grown in Met-HPG+ media were added on to DR5-KO cells (growing in regular media). After 48 hrs flow cytometry analysis was carried out with HPG catalyzing dye. HPG incorporation from flow cytometry data confirms ApoEVs transfer from cells growing in Met-HPG+ (DR5-WT) to DR5-KO cells. (G) ApoEVs isolated from DR5-WT OVCAR-3 cells (KMTR2 treated) were added on to DR5-KO (MDA-MB-231) cells for indicated times (24-96 hrs) to analyze PD-L1 transfer kinetics from ApoEVs after addition of MDA-MB-436 derived ApoEVs (treated with combination of KMTR2+lexatumumab DR5 agonists or IgG1) on to DR5-KO MDA-MB-231 cells, total lysates were immunoblotted for PD-L1 after 24 hrs. KMTR2+lexatumumab (n=3), IgG1 (n=2). (I) Left: PD-L1 surface histogram shows direct antibody treatment on DR5-KO cells. Right: PD-L1 surface histogram from DR5-KO cells after treatment (24 hrs) with ApoEVs isolated from DR5 sensitive cells after either Lexa or KMTR2 treatment. ApoEVs treated tumor cells were analyzed in PD-1 reporter co-culture assays for luciferase signal (K) MDA-MB-436 cells were treated with KMTR2 for 6 hrs and along with ZDEVD for indicated times. - ZDEVD shows final time of KMTR2 exposure to cells in absence inhibitor. Lysates were immunoblotted for cleaved caspase-3, 8, PARP, PD-L1, S5a, ROCK1 and GAPDH (n=3). u.c: uncleaved, c: cleaved, n.s: non-specific band (L) Relative caspase-8 and caspase-3 activity assays caspase-8 maintained steady activity, while caspase-3 activity required at least 40 (+) mins of DR5 agonist treatment without ZDEVD (n=3). (M) After indicated treatments with KMTR2 (DR5 agonist) and ZDEVD (caspase-3 preferred inhibitor) , cells were allowed to grow 24 hrs, followed by cell viability analysis.
Figure 1(A) Outline of the experimental setup to assess the immunomodulatory effects of HIV-1 NEF on the generation of humoral immune responses. Antigen-specific interactions are established using HEL-OVA as model antigen with HEL-specific SWHEL B cells (BHEL) and OVA-specific OT-II CD4 T cells (CD4OT-II). (B, C) Levels of anti-HEL IgG in serum as measured by ELISA at the indicated days post immunization with HEL-OVA/CFA following adoptive transfer of BHEL cells without CD4OT-II cells (no T cell control) or with control CD4OT-II cells or NEF-expressing CD4OT-II cells. (B) NEF WT of HIV-1SF2 (C) patient-derived NEF variants. Shown are relative levels of HEL IgG production with the mean of IgG levels in mice receiving control cells between days 12 - 14 post immunization arbitrarily set to 1. Each dot represents one animal. Bars represent mean values with SD from 2-10 mice analyzed from 3 (B) and 2 (C) independent experiments. Statistical significance was assessed by Mann Whitney U test. ***P ≤0.001, *P ≤0.05
a) HeLa cells transfected with plasmids encoding GFP or GFP–IRGMd were stained with MTR and imaged by live-cell confocal microscopy.figf6(a) HeLa cells transfected with plasmids encoding GFP or GFP-IRGMd were stained with MTR and imaged by live-cell confocal microscopy. Scale bars, 5 μm. (b-d) Quantification of mitochondrial morphologies in cells transfected with IRGMd, IRGMdS47N or IRGMb GFP fusions (control cells express GFP only). (e)HeLa were transfected with the indicated siRNAs and vector encoding GFP-IRGMd, and labelled with MTR. The percentage of cells with mitochondrial morphologies ranging from normal, punctiform (dots) and elongated were quantified. (f) HeLa were transfected with the indicated siRNAs and vector encoding GFP-IRGMd (or vector encoding GFP only, as a control) and labelled with MTR. Percentages of GFP+ cells with MTR staining at 48 h were quantified. (g) Confocal microscopy images of HeLa cells transfected with vector encoding GFP-DRP1 (A) or GFP-IRGMd (B-G) and labelled with MTR (A-D) or antibodies against COX-IV (F). A, steady-state DRP1 distribution; B-D, translocation of IRGMd from the cytosol to mitochondria at indicated times post-transfection; E-G, co-localization of IRGMd and COX-IV (G is merged from E and F). Scale bars, 5 μm. (h) HeLa cells, co-transfected with GFP-IRGMd, and control, BECN1 or ATG7 siRNA, were labelled with MTR 48 h post-transfection, imaged by live-cell microscopy, and percentages of GFP+ cells that were also MTR+ were quantified. (i) Cells from Atg7 wild-type (WT) or Atg7−/− MEFs transfected with vectors encoding GFP-IRGMd for 48 h were stained with MTR, imaged, and percentage of GFP+ cells that were also MTR+ cells was quantified. (j) Wild-type W2 (Bax/Bak+/+) or mutant D3 (Bax/Bak−/−) BMK cells were transfected with vectors encoding GFP-IRGMd and after 48 h were labelled with MTR. Cells were imaged by live-cell microscopy, and percentages of GFP+ cells that were also MTR+ were quantified. (k) HeLa cells were transfected with vectors encoding GFP-IRGMd, treated with z-VAD as indicated, and after 48 h stained with MTR. Cells were imaged by live-cell microscopy, and percentages of GFP+ cells that were also MTR+ were quantified. Data are means ± s.e.m. (n = 3). Dagger indicates P ≥ 0.05, asterisk indicates P 0.05 and double asterisks indicate P 0.01 (t-test) for the indicated data.
Figure 7ROC curves showing the ability to determine infectivity (as assayed by viral culture in Vero E6-TMPRSS2 cells) using the CT values of WHO-E, Wölfel-sgE, and Mills-sgE for each of the samples for which these values were determined. For each test, the true-positive rate (chance of correctly predicting samples to be culturable at a given or lower CT) was plotted against the false-positive rate (chance of incorrectly predicting samples to be culturable at a given or lower CT) as a step function line. The lines show performance of the tests as the (CT) threshold for a positive result is increased; the lines are solid for detectable values and dashed when a primer set returned an undetectable value. Each line is labelled with the lowest CT value that surpassed a 95% sensitivity threshold (the horizontal dotted line) for that marker.
Figure 4Purified RPA and Rtt105-RPA complex were resolved on 15% SDS-PAGE gels and analyzed by silver staining. EMSA assay was performed to analyze ssDNA binding ability of RPA or Rtt105-RPA complex. Cy3-labeled 30nt ssDNA was used as the substrate, and reaction mixtures were resolved on native PAGE gels. Quantification of the RPA-bound ssDNA is shown in (C). Recombinant Rtt105-RPA complex was mixed with a 30nt-ssDNA in a 2:1 ratio, and the reaction product were subjected to gel filtration chromatography. Equal volume of fraction 5-10 were resolved on SDS-PAGE for detecting proteins by Ruby staining (upper panel) or were resolved by native agarose gel for detection of Cy3-ssDNA signal (lower panel). GST-RPA pull down Rtt105 with increased amount of 30nt ssDNA, and the bound proteins were resolved on SDS-PAGE and visualized by silver staining.
Beclin‐1 depletion reduces targeting of several outer kinetochore proteins.Figure 4(A) CENP‐E and CREST. Two asterisks indicate significant results, P=0.0053. (B) CENP‐F and CREST. Three asterisks indicate significant results, P=0.0006. (C) ZW10 and CREST. Three asterisks indicate significant results, P=0.0007. (D) MAD2 and CREST. Two asterisks indicate significant results, P=0.0032. (E) HEC1 and CREST. (F) KNL‐1 and CREST. On the right, magnified areas and quantification of the fluorescence intensity of the protein of interest normalized to the fluorescence intensity of CREST staining are shown. Graphs represent three independent experiments (error bars=s.d.). Scale bar, 10 μm. (G) Western blot of whole‐cell lysates from control and Beclin‐1 siRNA‐treated cells. siRNA, small interfering RNA.
Figure 2(A‑B) BMDCs were plated at different densities and stimulated with poly(I:C) for 24h or left unstimulated. DC activation was evaluated based on surface expression of CD86 and CD40 molecules as measured by flow cytometry. (A) Representative dot plots showing CD86 and CD40 expression. (B) Quantification of CD86 and CD40 upregulation as a fold change in geometric mean fluorescence intensity (gMFI) over unstimulated controls. Data are pooled from 3 independent experiments. (C) MutuDCs were plated at different cell densities and stimulated with poly(I:C) or left unstimulated. The graph shows the upregulation of CD86 as a fold change in gMFI over unstimulated cells. Data are pooled from 4 independent experiments. (D) Flow cytometric quantification of CD86 and CD40 upregulation on WT (blue) or Ifnar‑/‑ (green) BMDCs expressed as a fold change in gMFI over unstimulated controls. (E) CD86 and CD40 expression was quantified on BMDCs plated at high density (2x105 cells/cm2) and stimulated with poly(I:C) in the presence (red dots) or absence (blue dots) of anti‑IFNAR1 blocking antibody. Data are pooled from 2 independent experiments. (F) CD86 and CD40 expression was quantified on BMDCs plated at low density (2x103 cells/cm2) and stimulated with poly(I:C) (dark blue dots), IFN‑α (light blue dots) or a combination of both (green dots). Data are representative of 2 independent experiments. (G) IFN‑α (left) and IFN‑β (right) production was quantified by ELISA for BMDCs plated at different densities and stimulated or not with poly(I:C). Dotted lines represent the ELISA threshold of detection. Data are pooled from 2 independent experiments.
Autophagy inhibition blocks the HTTex1 72Q aggregate-reducing effect of TBK1: (A) Representative immunoblot of a filter retardation of HTT (ab-MAB5492) from the insoluble cellular fraction; HEK293T cells co-expressed HTTex1 72Q eGFP and TBK1 or TBK1 KD for 48 hour, and for the last 16 hour, they were treated with the indicated proteasome inhibitor (MG132, 5 µM) or autophagy inhibitor (Baf A1, 200 nM, NH4Cl, 10 mM, 3-MA,5 mM).Figure 6(A) Representative immunoblot of a filter retardation of HTT (ab-MAB5492) from the insoluble cellular fraction; HEK293T cells co-expressed HTTex1 72Q eGFP and TBK1 or TBK1 KD for 48 hour, and for the last 16 hour, they were treated with the indicated proteasome inhibitor (MG132, 5 µM) or autophagy inhibitor (Baf A1, 200 nM, NH4Cl, 10 mM, 3-MA,5 mM). (B) Quantification of the fold change in HTT aggregates compared to TBK1 KD treated with DMSO from the blots like in A (n=3). (C) Immunoblot of LC3 (ab48394) from soluble HEK293 cellular fractions overexpressing TBK1 or TBK1 KD for 24 hour; for the last hour, Baf A1 (500 nM) was added as indicated. (D) Fold change in LC3-II levels (lower band) relative to the respective untreated kinase dead mutant normalized to actin from the blots like in C (n=3). (E) Immunoblot of LC3 (ab48394) from soluble rat primary neuronal cells overexpressing lentivirus-mediated TBK1 and TBK1 KD for 96 hour. For the last 1 or 4 hour, Baf A1 (500 nM) was added as indicated. (F) Quantification of the fold change in LC3-II levels (lower band) compared to the kinase dead mutant, which was untreated, normalized to GAPDH from the blots like in E (n=3).
Bosutinib improves cognitive performance in a parkin-dependent manner*Significantly different to DMSO or as indicated, Mean ± SEM, ANOVA with Neumann Keuls multiple comparison.Figure 6(A) Represents results of Morris water maze test in lentiviralAβ1-42-injected ± Bosutinib WT (N = 12) and parkin−/− (N = 10) mice, and (B) heat maps for each group.Graphs represent total number of entry into platform area and distance travelled. Represents results of Morris water maze test in Tg-APP ± Bosutinib (N = 12) mice.Graphs represent total number of entry into platform area and distance travelled.
Model depicting the dual function of VcdRP in balancing carbon metabolismVcdRP is a dual-function regulator that represses CTX production in V.Figure 7VcdRP is a dual-function regulator that represses CTX production in V. cholerae. Additionally, CRP and cAMP act on the regulator at the transcriptional level and suppress the production of VcdRP. The sRNA element (VcdR) acts via a conserved stretch of four consecutive cytosines to base pair with the translation initiation sites of PTS-specific and non-specific sugars, resulting in the inhibition of carbon uptake. The peptide VcdP binds to and increases the activity of citrate synthase enzyme, thereby modulating the levels of metabolites within the cycle. Thus, VcdR and VcdP together synchronize the carbon uptake and its subsequent utilization, thereby balancing carbon metabolism.
Fig.Figure 2A: Upper panel: Strategy for the identification of oxidation resistant Ubc9 variants. Ubc9 was mutagenized by PCR under conditions that introduced 1 - 3 random mutations. Ubc9 variants were expressed in bacteria and the bacterial lysates of individual clones tested for E2 activity in the presence or absence of 1 mM H2O2. For this purpose, we used a FRET-based in vitro SUMOylation assay with recombinant SUMO E1, YFP-SUMO and CFP-RanGAPtail and ATP. Lower panel: Ubc9 W103R is H2O2 resistant. Bacterial lysates containing Ubc9 wt (left panel) or Ubc9 W103R (right panel) were tested as described. B: Ubc9 W103 mutants are H2O2 resistant but not fully active. Left panel: Recombinant Ubc9 W103R, W103A and W103F were purified (embedded panel). Resistance against oxidation was tested under conditions of limiting E1 enzyme: 21 nM Aos1/Uba2 and 73 nM Ubc9 were incubated with H2O2 prior to the addition of 160 nM each of YFP-SUMO1 and CFP-RanGAPtail. Right panel: To compare specific activities of wt and mutants in the absence of H2O2, sumoylation assays were carried out using limiting Ubc9 concentration. Reactions contained 35 nM Aos1/Uba2, 11 nM Ubc9, 85 nM each of YFP-SUMO1 and CFP-RanGAPtail and 1 mM ATP.
Fig.Figure 7Current model illustrating the role of mitochondria as chromophore-assembly stations for phytochrome.
reactivates latent HIV-1 in the primary CD4+ T model of latency.Figure 6A. Outline of latency establishment in the primary CD4+ T cells. Human primary CD4+ T cells were activated and expanded with α-CD3/CD28 beads at day 1. The α-CD3/CD28 beads were removed at day 3. Cells were then infected with HIV-1 NL4.3-nanoluc at 3rd day after expansion and maintained over 7 days with a decreasing concentration of IL-2 to establish latency until day 12. At day 12, cells were infected with CRISPR/Cas9 and PEBP1-sgRNA lentivirus. B. Transcription of HIV-1 in the primary CD4+ T cells was determined by nanoluc luciferase assays during HIV-1 infection. C. After infection with VSV-G pseudotyped HIV-1 NL4.3-nanoluc, the mRNA expression of PEBP1 was measured by qPCR. Jurkat CD4+ T cells were treated with IFN-α, IFN-β and IFN-γ for 24 h. The mRNA expression levels of PEBP1 were measured by qPCR Jurkat CD4+ T cells were treated with IFN-α, IFN-β and IFN-γ for 24 h. The protein expression levels of PEBP1 were measured by Western blot. F. PEBP1 knockout enhanced HIV-1 transcription in the primary CD4+ T cell model of latency. The expression of HIV-1 was measured by nanoluc after gene knockout where α-CD3/CD28 stimulation served as a positive control. G. PEBP1 gene deletion after Lv-PEBP1-sg1 knockout in the primary CD4+ T cells. The PCR products of PEBP1 were cloned and then sequenced. PEBP1-sg1 target gene sequences are shown in red letters. Dashes indicate deleted bases relative to the wild-type sequence.
Mitochondrial respiratory deficiency impairs PAS recruitment of the Atg1–Atg13 complex under amino‐acid starvation.Figure 6(A)ATG8 induction is independent of the Atg1-Atg13 complex and autophagic flux. Wild‐type, rho0, Δatg1, Δatg7, Δatg9, and Δatg11 cells harbouring prATG8‐GFP‐ATG8 were analysed as described in Figure 1A. The means and s.d. of four (n=4) independent experiments are indicated. (B) Atg1 and Atg13 recruitment to the PAS depends on mitochondrial function. Wild‐type and rho0 cells harbouring prATG8‐GFP‐ATG8 and prATG1‐ATG1‐mCherry (upper panels) or prATG13‐ATG13‐mCherry (lower panels) were exposed to amino‐acid starvation medium supplemented with galactose for 3 h. Wild‐type cells were treated with antimycin A (AA 30′) after 2.5 h of starvation for 30 min or with oligomycin (O) for 3 h of starvation. Arrowheads indicate the position of GFP‐Atg8 puncta. Transmission and fluorescence light microscopy images were superimposed to visualize cellular boundaries. Scale bar represents 1.5 μm. (C) Steady‐state levels of Atg1‐ and Atg13‐mCherry during amino‐acid starvation. Wild‐type, rho0, and Δatg7 cells expressing prATG1‐ATG1‐mCherry (upper panels) or prATG13‐ATG13‐mCherry (lower panels) were exposed and treated as described in (B) and analysed by whole cell extraction and western blot analysis using α‐dsRed and α‐Cdc11 antibodies.
Figure 1(A) Growth curve of cells cultured at high densities (n=3 pairs). (B) Results of clonal assays at 14 days after plating. Representative images are shown on the left, and quantification of multiple assays on the right (n=3 pairs). (C) Distribution of colony size (i.e., number of cells per colony) at 3 days after plating (n=3 pairs) (D) Heat map of genes differentially expressed (p<0.05) in control and Ovol2 SSKO NBPKs. Plotted values are log10(FPKM) (E) GO analysis of the top up- or downregulated gene sets (F) GSEA analysis of RNA-seq data. NES, normalized enrichment score. FDR, false discovery rate (G) RT-qPCR of the indicated genes normalized to Gapdh (n=3 pairs)
α1S subunit knockdown and RyR1 expression.Figure 6(A) Lysates from TAΔDHPR (ΔDHPR) and TACtrl (c) were immuno‐blotted for RyR1 or α‐actin. (n=4). Graph depicts mean±s.e.m. of relative expression of RyR1 determined by densitometry and nomalized to the α‐actin expression for each muscle. Results were expressed in protein levels of ΔDHPR in TAΔDHPR normalized to TACtrl for each mice, *P⩽0.005, n=4. (B) Longitudinal cryo‐sections of TAΔDHPR (ΔDHPR) and TACtrl (c) were stained with anti‐α1S subunit (red), anti‐RyR1 (green) antibodies and imaged by confocal microscopy. Bars=20 μm.
Figure 4 ELISA plates were coated with purified soluble TREM2-Fc, and then incubated with various concentrations of Aβ42 monomer Binding of Aβ42 monomers with soluble TREM2-Fc shows a dose dependent signal. Results were expressed as the OD450 averaged ± standard error (n =3; * p<0.05, ** p<0.01, *** p<0.001, **** p < 0.0001, Two-way ANOVA, Bonferroni multiple comparisons, see Appendix table S2 for p value)  ELISA plates were coated with purified soluble TREM2-Fc, and then incubated wit 1 µM Aβ42 oligomers Binding of 1 µM Aβ42 oligomers with soluble TREM-Fc family members. Results were expressed as the OD450 averaged ± standard error (n = 4, * p < 0.05, **** p<0.0001, ANOVA, Bonferroni multiple comparisons, see Appendix table S2 for p value)
Kinetochore individualization in meiosis I is required for step-wise cohesin removal A) On monopolar spindles, as on bipolar spindles, chromosomes segregate in meiosis I, and sister chromatids in meiosis II.Figure 7A) On monopolar spindles, as on bipolar spindles, chromosomes segregate in meiosis I, and sister chromatids in meiosis II. B) Model of how separase-dependent kinetochore individualization primes bivalents for deprotection of centromeric cohesin. If separase is absent in meiosis I, arm cohesin and not centromeric cohesin is removed in meiosis II. Under these conditions, sister kinetochores individualize in meiosis II instead of meiosis I, demonstrating that step-wise cohesin removal (first arm cohesin, then centromeric cohesin) depends on the kinetochore structure. Without kinetochore individualization, centromeric cohesin cannot be cleaved in the subsequent division.
Ubiquilins colocalize with autophagosomes.Figure 3(A) Immunolocalization of transfected Myc‐UBQLN (1 or 2) and GFP‐tagged microtubule‐associated protein 1 light chain 3 (GFP‐LC3) was analysed by microscopy. Inset: magnification of UBQLN1 vesicular staining (arrow), which, after acquisition of Z‐stacks, was found in all UBQLN structures analysed. (B) Cells were starved for 2 h, with (top row) or without BAF A1 (bottom row). Cells were stained for endogenous UBQLN2 (magenta) and LC3 (green). (C) Control cells (untransfected, right panel) or cells overexpressing Myc‐UBQLN2 and GFP‐LC3 (left panel) were transferred into starvation medium for 2 h. Localization of Myc‐UBQLN2 and GFP‐LC3 was analysed by immunoelectron microscopy using Myc (arrow) and GFP (arrowhead) antibodies coupled to 10 and 15 nm gold particles, respectively. Double‐membrane vacuoles (autophagosomes) in transfected cells (left panel) show increased staining by both antibodies compared with autophagosomes in untransfected cells (right panel). Scale bar, 0.5 μm. BAF A, bafilomycin A1; GFP, green fluorescent protein; UBQLN, ubiquilin.
Figure 4A) Metaphase II oocytes from mice carrying a conditional, oocyte-specific invalidation of separase (Separaseflox/flox Zp3 Cre+, or Sep-/-) and oocytes from litter mates (Separaseflox/flox, or Sep+/+) were obtained after superovulation. In vivo matured control (Sep+/+) and Sep-/- oocytes are found in oviducts at comparable numbers, enclosed by cumulus cells. Example of cumulus enclosed oocytes from the oviduct is shown below. In (A), n indicates the number of mice and a mean number of oocytes per mouse of each genotype, error bars are + s.e.m. B) Whole mount immunofluorescence to stain cold stable microtubule fibers after in vivo maturation. Microtubules were stained with anti-tubulin antibody (green), centromeres with CREST serum (red) and DNA with Hoechst (blue). Bivalents are attached in a monopolar fashion. Approximate number of z-sections used for overlays to visualize the whole spindle (on the left) or individual chromosomes (magnifications on the right) are indicated.D) Metaphase-to-anaphase transition of meiosis II in oocytes of the indicated genotype cultured in vitro and injected with the separase constructs as indicated, in GV. The schemes indicate the chromosome figures observed upon activation. Chromosome spreads are stained with CREST serum (green), and Rec8 antibody (red). DNA was stained with DAPI (blue). In (D) n indicates the number of oocytes with the shown phenotype (e.g., upon successful activation) and the total number of oocytes analyzed.