EMBO/sd-character-level-ner huggingface dataset:
Pharmacological inhibition of BCL-XL in combination with DNA damage-inducing chemotherapeutics or γ-radiation at clinically relevant doses is tolerated in mice.Figure 6(A) GFP-Chimeras were treated with the BCL-XL inhibitor A1331852 (n=8, 5 doses by oral gavage, 25 mg/kg body weight each dose). Control groups (n=8 each group) include untreated GFP-Chimeras and un-irradiated C57BL/6-Ly5.1 (wild-type) mice treated with the BCL-XL inhibitor A1331852 or left untreated. Mice were monitored for up to 200 days post-treatment (dpt). Data are presented as % survival. (B) C57BL/6-Ly5.1 (wild-type) mice (females, aged 10 weeks) were treated with Cyclophosphamide (n=7, 150 mg/kg body weight, 1 dose i.v.) or 5-Fluorouracil (5-FU, n=7, 100 mg/kg body weight, 1 dose i.v.) and after 5 days additionally treated with the BCL-XL inhibitor A1331852 (5 doses by oral gavage, 100 mg/kg body weight each dose). Control groups (n=7 each group) include C57BL/6-Ly5.1 (wild-type) mice (females, aged 10 weeks) treated with A1331852 alone, Cyclophosphamide alone, 5-FU alone or left untreated. Mice were monitored for up to 150 days post-treatment (dpt). Data are presented as % survival. (C) C57BL/6-Ly5.1 (wild-type) mice (females, aged 10 weeks) were treated with the BCL-XL inhibitor A1331852 (n=7, 5 doses by oral gavage, 25 or 100 mg/kg body weight each dose). Total platelet counts were determined by ADVIA at the indicated time points. Data are presented as mean ±SEM. Each data point represents one individual mouse. Statistical significance was assessed using one-way ANOVA analysis with Tukey's multiple comparisons test comparing untreated and drug-treated mice within each group (**p<0.01, ***p<0.001, ****p>0.0001). Total red blood cell count (RBC), platelet count, hemoglobin (HGB) content and hematocrit (HCT) were determined by ADVIA as indicated in the blood of drug-treated GFP-Chimeras or control mice (n=8 each group) at the termination of the experiment Data are presented as mean ±SEM. Each data point represents one individual mouse. Statistical significance was assessed using one-way ANOVA analysis with Tukey's multiple comparisons test comparing untreated and drug-treated mice within each group (*p<0.05, **p<0.01, ***p<0.001, ****p>0.0001). Total red blood cell count (RBC), platelet count, hemoglobin (HGB) content and hematocrit (HCT) were determined by ADVIA as indicated in the blood of in drug-treated C57BL/6-Ly5.1 (wild-type) mice or control mice at the indicated time points (n=7 each group). Data are presented as mean ±SEM. Each data point represents one individual mouse. Statistical significance was assessed using one-way ANOVA analysis with Tukey's multiple comparisons test comparing untreated and drug-treated mice within each group (*p<0.05, **p<0.01, ***p<0.001, ****p>0.0001).
Figure 1A. 293T cells were co-transfected with CMV-renilla, 8X-TBS (eight repeats of the TEAD-binding-sequence) luciferase reporter, Flag-YAP, HA-TEAD2 and wild-type (WT) or catalytically inactive (CS) USP9X-V5. One day after transfection, luciferase activity was measured and normalized with respect to renilla activity. The value for Flag-YAP/HA-TEAD-transfected cells (2nd column) was adjusted to 1. Shown below is a representative Western blot showing that the expression levels were comparable between samples (n=4). Error bars indicate the S.E.M. (*p< 0.05, **p < 0.01; paired Student's t-test). B. 293T cells were transfected with control or USP9X-targeting siRNAs. One day after siRNA transfection, the cells were co-transfected with CMV-renilla, 8X-TBS luciferase, Flag-YAP and HA-TEAD2. Reporter activity was quantified as described in (A) (n=4). Error bars indicate the S.E.M. (***p < 0.001; paired Student's t-test). C. RPE or MCF10A cells were transfected with the indicated siRNAs for 24 hours and then re-seeded to either sparse or dense culture conditions. At 48 hours after siRNA transfection, the cells were harvested and cell extracts were analyzed by Western blotting for the indicated proteins. L.E., long exposure, S.E., short exposure. D. Cells were treated as described in (C), the indicated mRNAs were analyzed with RT-qPCR, and the results were normalized with respect to the β-actin mRNA (n=4). Error bars indicate the S.E.M. (*p< 0.05, **p < 0.01; paired Student's t-test). E. RPE cells were transfected with the indicated siRNAs for 24 hours, and then co-transfected with CMV-renilla and 8X-TBS luciferase. One day after the latter transfection, the cells were re-seeded to either sparse or dense conditions, and reporter activity was measured at 24 hours after re-seeding (n=3). Error bars indicate the S.E.M. (**p < 0.01; paired Student's t-test).
Figure 1(A) The "in-plane" and the "clamp" models for the membrane topology of pore-forming Bax protein. After activation by BH3 proteins, the soluble Bax protein unfolds the α-helical bundle structure and inserts the α9 helix into the MOM. The tail-anchored Bax proteins dimerize via their α2, α3, α4 and α5 helices resulting in an amphipathic structure (shown as Bax (α2-α5) dimer with the protomers in cyan or gray color) with a polar surface containing positive and negative charged α2 and α3 residues (blue and red sticks) and a nonpolar surface containing aromatic α4 and α5 residues (orange rings). According to the in-plane model, the α2-α5 dimer engages the planer cytosolic surface of the MOM with the aromatic α4 and α5 residues projected into the cytosolic leaflet of the lipid bilayer to generate membrane tension thereby inducing a lipid pore with radially arranged lipids at the rim. In contrast, the clamp model positions the α2-α5 dimer at the lipid pore rim with the aromatic α4 and α5 residues intercalated between the radiated lipids to reduce line tension thereby stabilizing the pore. As illustrated, the membrane topologies of other helices are also different between the models. The structures of the soluble Bax protein and the α2-α5 dimer were generated from PDB files 1F16 and 4BDU, respectively, using PyMOL program. The structures of other helices are modeled based on the corresponding structures in the soluble Bax protein. (B) 2D 1H-15N TROSY-HSQC spectra of DMPC/DHPC bicelle-bound Bax (α2-α5) (blue) and soluble Bax (α2-α5) (red) at 600 MHz. The magnified regions highlight the spectral changes for some residues in the bicelle-bound and soluble proteins.
a–e, Cell viability by trypan blue dye exclusion in ADR-senescent control;Bcl2 versus ADR-treated Suv39h1−;Bcl2 lymphoma cells at day 5 compared to the untreated condition in various biochemical, pharmacological or genetic interventional settings in vitro.f2a-e, Cell viability by trypan blue dye exclusion in ADR-senescent control;Bcl2 versus ADR-treated Suv39h1-;Bcl2 lymphoma cells at day 5 compared to the untreated condition in various biochemical, pharmacological or genetic interventional settings in vitro. All results in this figure represent means ± s.d. (n = 5 each). a, Inhibition of glucose metabolism by phloretin, cytochalasin B, and sodium oxamate. b, Exposure to 2DG or switch to glucose-free medium with or without glucose replenishment. c, Lentiviral knockdown of hexokinase 2 (Hk2) by short hairpin RNA (shHk2). d, Inhibition of AMPK by compound C. e, Block of fatty acid oxidation by the carnitine palmitoyltransferase I inhibitor etomoxir. f, Inhibition of the electron transport chain by antimycin A. *P  0.01, except b (n = 8 each) and e, in which *P  0.05.
Characterization of azithromycin-treated PfMev CLD-EcDPCK-mCherry-apt Δdpck parasites under permissive and non-permissive conditions A.Figure 6A.) Attempted PCR detection of the ldh, sufB, and cox1 genes from the nuclear (N), apicoplast (A), and mitochondrial (M) genomes, respectively. We were unsuccessful in amplifying sufB from the PfMev CLD-EcDPCK-mCherry-apt Δdpck parasite line after treatment with 100nM azithromycin (Azith) in the presence of 50μM mevalonate, indicating the disruption of the apicoplast organelle. B.) Live epifluorescence microscopy of the apicoplast-negative PfMev CLD-EcDPCK-mCherry-apt Δdpck line post treatment with 100nM azithromycin in the presence of 50μM mevalonate. This parasite line expresses api-SFG (green) and the CLD-EcDPCK-mCherry construct (red) with DAPI marking the nuclei (blue). Images represent fields that are 10µm long by 10µm wide. C.) Growth curve of the PfMev CLD-EcDPCK-mCherry-apt Δdpck parasite line containing a disrupted apicoplast grown either in the presence of 0.5μM aTc and the absence 0.5μM Shield1 (permissive condition, blue), or the absence of 0.5μM aTc and the presence of 0.5μM Shield1 (non-permissive condition, red). Pantothenate (50nM) and mevalonate (50μM) were supplied in both conditions and the cultures were diluted 1:5 on day 4. Treatment under non-permissive conditions resulted in reduced parasite growth beginning at day 2 (two-way ANOVA, followed by Bonferroni's correction; **, P < 0.01). Error bars represent the standard error of the mean from two independent experiments, each conducted in quadruplicate. D.) Upon apicoplast disruption, DPCK is located within vesicles that contain apicoplast-specific proteins and may still be able to generate CoA.
Figure 1A: Serial dilutions of stationary phase cultures of the indicated strains were spotted on YEPD and incubated at the indicated temperatures. B: Mps1 sequence alignment around S. cerevisiae Ser635. Sc: Saccharomyces cerevisiae; Sp: Schizosaccharomyces pombe; Mm: Mus musculus: Hs: Homo sapiens; Dm: Drosophila melanogaster. Wild type, mps1-3 and mps1-1 mutant cells were synchronised in G1 with α-factor at permissive temperature (25°C) and then released at restrictive temperature (34°C, t=0). Cells were collected at the indicated time points for FACS analysis of DNA contents (C) Wild type, mps1-3 and mps1-1 mutant cells were synchronised in G1 with α-factor at permissive temperature (25°C) and then released at restrictive temperature (34°C, t=0). Cells were collected at the indicated time points for immunofluorescence using anti-tubulin antibodies in order to score metaphase and anaphase spindles (D). Budding and nuclear division were scored on the FACS samples. E: Frequencies of chromosome loss were quantified at permissive temperature (25°C). F: Wild type and mps1 mutant cells bearing the TetO/TetR-GFP system to mark the centromere of chromosome V [43] and expressing the SPBs marker Spc97-mCherry were synchronised as in (C-D) and arrested in anaphase through the temperature-sensitive cdc15-2 allele. At 120 and 150 minutes, cells were fixed for scoring chromosome V segregation (n≥172). Arrowheads indicate the sister chromatids of chromosome V. Error bars: SD. N=3. Representative images of cells for each genotype are shown on the right. Scale bar: 5 μm. G: Wild type and mps1-3 mutant cells carrying the TetO/TetR-GFP markers for CEN5 labelling and expressing mCherry-Tub1 were grown at 25°C and then shifted to 34°C for 1 hour before filming. Cells were filmed at 34°C every 2 minutes by time lapse fluorescence microscopy. DIC: differential interference contrast. Scale bar: 5 μm.
Figure 3Intracellular iron concentration was measured in CT26 cells stably expressing mock, OTUD1 or OTUD1C320S (n = 2 biological replicates) Intracellular iron concentration was measured in wild-type (WT) or OTUD1─/─ NCM460 cells (n = 4 biological replicates, mean ± s.e.m., ***P < 0.001, two-tailed unpaired Student's t-test) (C) The protein levels of IREB2, TFRC and OTUD1 were assessed by western blot in colon tissues from wild-type (WT) or Otud1─/─ mice. (D) Intracellular iron concentration was measured in colon tissues from wild-type (WT) or Otud1─/─ mice with supplementation of ferrous gluconate (n = 3 biological replicates, mean ± s.e.m., ***P = 0.0007, two-tailed unpaired Student's t-test). Mean corpuscular volume (MCV) (n = 5 biological replicates, mean ± s.e.m., ns, not significant (P > 0.05), *P = 0.0472, two-tailed unpaired Student's t-test) in wild-type (WT) and Otud1─/─ mice with or without low-iron diets for indicated times. mean corpuscular hemoglobin (MCH) (n = 5 biological replicates, mean ± s.e.m., ns, not significant (P > 0.05), **P = 0.0048, two-tailed unpaired Student's t-test) in wild-type (WT) and Otud1─/─ mice with or without low-iron diets for indicated times. mean corpuscular hemoglobin concentration (MCHC) (n = 5 biological replicates, mean ± s.e.m., ns, not significant (P > 0.05), **P = 0.0068, two-tailed unpaired Student's t-test) in wild-type (WT) and Otud1─/─ mice with or without low-iron diets for indicated times. , red boold cells (RBCs) (n = 5 biological replicates, mean ± s.e.m., ns, not significant (P > 0.05), **P = 0.0049, two-tailed unpaired Student's t-test) in wild-type (WT) and Otud1─/─ mice with or without low-iron diets for indicated times. hemoglobin (HB) (n = 5 biological replicates, mean ± s.e.m., ns, not significant (P > 0.05), **P = 0.0036, ***P = 0.0002, two-tailed unpaired Student's t-test) (I) in wild-type (WT) and Otud1─/─ mice with or without low-iron diets for indicated times.
Epigenetic immune memory in stimulated macropghages.Figure 6Volcano plot showing differentially expressed miRNAs from unstimulated COVID-19 patient-derived macrophages (SC-conv) compared to those from naïve individuals (SC-naïve). Negative log10 adjusted p-values are plotted against the log2 fold-change. Genes of interest are indicated. For miRNA analyses data with a padj value of smaller 0.05 and log2-fold change of ≤-0.85 (blue) and ≥ 0.85 (red) were included. Average profile plot showing distribution of histone modifications identified in CUT&RUN experiments displayed as normalized read counts per million around known transcription start site (TSS) of known genes. Color code for H3K4me3 (SARS-CoV-2 naïve/green; COVID-19/red) and for H3K27ac (SARS-CoV-2 naïve/yellow; COVID-19/purple). GO biological process analysis performed on differentially enriched loci of H3K27ac peaks (enhancer) showing associated pathways from SC-conv monocytes compared to SC-naïve monocytes. KEGG enrichment analysis performed on differentially enriched loci of H3K27ac peaks (enhancer) showing involved pathways from SC-conv monocytes compared to SC-naïve monocytes. Adjusted p-values are indicated (color code) and size of the circles corresponds to the number of detected genes associated with each pathway (count). CNET-plot (derived from analysis shown in D) illustrating genes involved in tuberculosis and phagosome KEGG pathways. Representative gene loci (TLR2 (top) and S100A7A/8/9/12 (bottom) H3K27ac peaks) comparing SC-naïve (blue) and SC-conv (red) monocytes. Red peaks reach higher values than blue peaks (representative examples of four samples for each condition). Quantification of IL-1β concentrations (pg/ml) in the supernatant of primary macrophage cultures from patients with active/untreated tuberculosis (n = 6; red/black bars) or healthy/SARS-CoV-2 naïve individuals (n = 3; blue bars) or the same patients after 6 months of anti-tuberculous treatment; n = 6; white/red bars) stimulated with LPS or S-protein (0.1 µg/ml) for 4h and subsequent incubation with nigericin for 2h. For statistical analysis, two-way ANOVA with tukey post hoc test was used.
a) Immunoblot with the antibody that recognizes PS–CFP2 of homogenates (Hom), cytosol (Cyt) and lysosomal membranes (MB) and matrices (Mtx) isolated from mouse fibroblasts stably expressing PS–CFP2 or KFERQ–PS–CFP2 proteins 16 h after serum removal.f5(a) Immunoblot with the antibody that recognizes PS-CFP2 of homogenates (Hom), cytosol (Cyt) and lysosomal membranes (MB) and matrices (Mtx) isolated from mouse fibroblasts stably expressing PS-CFP2 or KFERQ-PS-CFP2 proteins 16 h after serum removal. (b) Immunoblot for the indicated proteins of homogenates (Hom), mitochondria (Mit) and lysosomal membranes and matrices isolated from cells stably expressing KFERQ-PS-CFP2 maintained for 16 h in media supplemented (+) or not (−) with serum. (c) Quantification of the amount of KFERQ-PS-CFP2 associated to lysosomes (matrix and membrane) performed in blots as the one shown in (b). Values are expressed as percentage of the total KFERQ-PS-CFP2 in the cell and are mean+s.e. of three different experiments. Difference in the lysosomal associated KFERQ-PS-CFP2 between serum+ and serum− condition (*) were significant for P=0.033 (t-test). (d) Changes in the intracellular content of KFERQ-PS-CFP2 on serum removal. Values are expressed as percentage of KFERQ-PS-CFP2 present in serum-supplemented cells and area mean+s.e. of three different experiments. Values are mean+s.e. of three different experiments and differences are significant (*) for P=0.007 (t-test). (e-g) Effect of inhibition of macroautophagy by treatment with 3-methyladenine (3-MA) on the lysosomal content of KFERQ-PS-CFP2 in cells maintained in serum-free media for 16 h. (e) Immunoblot for the indicated proteins of the same fractions as described in (b). The amount of KFERQ-PS-CFP2 associated to lysosomes (f) and the changes in the intracellular content of KFERQ-PS-CFP2 were calculated as in (c, d). Values are mean+s.e. of three different experiments and differences are significant (*) for P=0.032 (t-test).
SH-BC-893 preserves mitochondrial function in the WAT of mice with HFD-induced obesity.Figure 4Body weight of mice fed the SD or HFD for 16 weeks; n=5. Mice are grouped to reflect their randomization to vehicle or 120 mg/kg SH-BC-893 groups, weight measured prior to treatment. NAD(P) H autofluorescence was evaluated in epididymal fat (eWAT) freshly resected from the mice in (A) 4 h after treatment with vehicle or 120 mg/kg SH-BC-893 by gavage at ZT8.5; n=5. NAD(P) H intensity from (B) calculated on a per cell basis; 16 - 25 cells from each of the 5 mice per group were quantified (87 - 109 total cells per treatment group). Body weight of mice fed the SD or HFD for 16 weeks; n=5. Mice are grouped to reflect their randomization to vehicle or 120 mg/kg SH-BC-893 groups, weight measured prior to treatment. TMRE (100 nM) and MitoTracker Green staining (200 nM) in eWAT resected from mice in (D) treated as in (B,C); n=5. TMRE (F) or MitoTracker (G) intensity for (E) calculated on a per cell basis; 15 - 24 cells from each of the 5 mice in each group were quantified (99 - 104 total cells per treatment group). MitoSOX (5 μM) and MitoTracker Green staining in eWAT resected from the mice in (D) treated as in (B and E); n=5. MitoSOX (I) or MitoTracker (J) intensity calculated as in (F and G); 10 - 13 cells from each of the 5 mice in each group were quantified (57 - 58 total cells per treatment group). Chop, Xbp1s, Atf6, and Grp78 mRNA measured in eWAT resected from mice maintained on a HFD for 13 weeks and gavaged with vehicle or 120 mg/kg SH-BC-893 on Monday, Wednesday, and Friday for the final 3 weeks; n=7-9.
Figure 2A Representative photographs of fly eyes expressing (CGG)90-EGFP under a GMR-GAL4 driver, with various belle disruptions. B Quantitation of GMR-GAL4, (CGG)90-EGFP eye phenotypes with belle disruptions (Mann-Whitney U test with Bonferonni corrections for multiple comparisons; n=35-77/genotype). Data Information: For all panels, * P≤0.05, ** P≤0.01, *** P≤0.001, **** P≤0.0001 for the specified statistical test. All data in all panels are presented as mean ± SD (compiled from ≥3 replicates). Longevity assays of (CGG)90-EGFP; Tub5-GS (Log-rank Mantel-Cox test with Bonferroni corrections for multiple comparisons; n=110-219/genotype) flies with belle knockdown. Data Information: For all panels, * P≤0.05, ** P≤0.01, *** P≤0.001, **** P≤0.0001 for the specified statistical test. All data in all panels are presented as mean ± SD (compiled from ≥3 replicates). Longevity assays of (CGG)90-EGFP; ElaV-GS (n=147-299/genotype) flies with belle knockdown. Data Information: For all panels, * P≤0.05, ** P≤0.01, *** P≤0.001, **** P≤0.0001 for the specified statistical test. All data in all panels are presented as mean ± SD (compiled from ≥3 replicates). Longevity assays of (CGG)90-EGFP; Tub5-GS (Log-rank Mantel-Cox test with Bonferroni corrections for multiple comparisons; n=110-219/genotype) flies with belle knockdown. Data Information: For all panels, * P≤0.05, ** P≤0.01, *** P≤0.001, **** P≤0.0001 for the specified statistical test. All data in all panels are presented as mean ± SD (compiled from ≥3 replicates). F Quantitation of FMRpolyG-EGFP band density, normalized to β tubulin band density, from blots in E (Student's t test; n=4-5/genotype). Data Information: For all panels, * P≤0.05, ** P≤0.01, *** P≤0.001, **** P≤0.0001 for the specified statistical test. All data in all panels are presented as mean ± SD (compiled from ≥3 replicates). G Abundance of (CGG)90-EGFP mRNA normalized to RPL32 mRNA, following belle knockdown, determined by qRT-PCR (n=8/genotype). H Western blot of AUG-driven EGFP in EGFP; Tub5-GS flies with and without belle knockdown. I Quantitation of EGFP band density, normalized to β tubulin band density, from blot in H (n=4/genotype).
Figure 1(A) Genetic strategy to selectively knock out Nrp1 in GnRH-expressing cells, using Nrp1loxP/loxP mice crossed with Gnrh::cre mice. These mice were also crossed with Gnrh::gfp mice expressing green fluorescent protein under the control of the GnRH promoter in order to generate triple transgenic mice. (B) Isolation of GFP-positive cells by FACS: schematic diagram showing the selection of the GFP-positive population (green). (C) Relative mRNA expression from real-time PCR analysis of the GnRH transcript in GFP-positive cells, in comparison with GFP-negative cells. The GnRH transcript appears to be selectively expressed in GFP-positive cells. Mann-Whitney U test, n=4 mice from 2 litters. (D) Relative mRNA expression from real-time PCR analysis of Neuropilin-1 (Nrp1), Nrp2, Plexin A1 (PlxnA1), and PlxnA4 transcripts in FACS-isolated GFP-positive GnRH neurons from control (Nrp1loxP/loxP; grey) and mutant (Gnrh::cre; Nrp1loxP/loxP; red) P0 mice. Unpaired t-test, n=5 to 6 mice per group from 3 litters. (E) Representative immunofluorescence images showing GnRH neurons migrating along Nrp1-immunoreactive vomeronasal/terminal nerve fibers (empty arrowheads) at the nose-brain junction in sagittal slices from E14.5 control and mutant embryos. GnRH neurons (green) themselves express Nrp1 (red, white arrows) in control Nrp1loxP/loxP mice whereas Nrp1 is undetectable in GnRH neurons from mutant Gnrh::cre; Nrp1loxP/loxP littermates (black arrows). Scale bar: 50μm.
Figure 4(A) Schematic representation of yeast growth assay. The model proteins consisted of an UbL domain and S. cerevisiae imidazoleglycerol-phosphate dehydratase (His3), followed by stop codon (no tail) or a 51 amino acid tail derived from subunit 9 of the Fo component of the Neurospora crassa ATPase (Su9) at the C-terminus. In his3 mutant cells, the absence of His3 protein causes a growth defect when grown on selective media. Stable His3 proteins escape proteasome degradation and rescue the his3 mutant cells from the growth defect. (B) UbL domains mediated degradation of His3 fusion proteins with a Su9 tail, and affected yeast growth under selective condition (+3-AT, -his). Replacing UbL domains with DHFR domains rescued the growth defect. Cells expressing His3 fusion proteins in late log phase were serially diluted and stamped on selective plates. Plates were incubated at 30 ˚C for 3 daysfor imaging.
a) Brain sections from HdhHD/+ Dnchc1+/+ or HdhHD/+ Dnchc1Loa/+ mice were stained with antibody to huntingtin (EM48; green).figf6(a) Brain sections from HdhHD/+ Dnchc1+/+ or HdhHD/+ Dnchc1Loa/+ mice were stained with antibody to huntingtin (EM48; green). No EM48-stained aggregates were detected in brains of Hdh+/+ Dnchc1Loa/+ mice. Arrows indicate cells with aggregates. (b) Western-blot analysis of mouse brain lysates from wild-type (WT) or Loa mutant mice using antibodies to LC3 and to actin.