EMBO/sd-character-level-ner huggingface dataset:
DOT1L-deficient B cells fail to mount an efficient immune response A.Figure 4A. Representative flow cytometry plots showing gating strategy to identify splenic GC B cells 14 days after LCMV Armstrong infection in WT and Dot1L-KO mice (left panel) and statistical analysis of their relative number. Results represent the data from two experiments and numbers represent biological replicates for each group (WT; n = 7, KO; n = 8). B. Representative flow cytometry plots showing gating strategy to identify plasma cells from the spleen 14 days after LCMV Armstrong infection (left panel) and statistical analysis of their relative number (right panel). Results represent the data from one experiment and numbers represent biological replicates for each group (WT; n = 3, KO; n = 4). C. Statistical analysis of serum titers of LCMV-specific IgM and IgG of WT and KO mice before (D0) and 14 days after (D14) LCMV Armstrong infection. Results represent the data from two experiments and numbers represent biological replicates for each group (WT; n = 7, KO; n = 8). D. Statistical analysis of serum titers of NP-specific IgG1 and IgM quantified by ELISA from WT and KO mice at the indicated days following immune challenge. Adjusted p-value were calculated using two-way ANOVA. Error bars indicate mean ± standard error of mean (SEM). Results represent the data from one experiment and numbers represent biological replicates for each group (WT; n = 6, KO; n = 6).*** denotes p- value < 0.0001 and ** denotes p- value < 0.001.
Figure 5Representative images of E11.5 lung explants after treatment for 48 h with 100 μM Scra or mo142-3p reveals reduced size and branching morphogenesis after miR142-3p knockdown. Organoid diameter (n=30-50 per group) in µm before addition of 4 μM Scra or mo142-3p at day 6 (control) and 5 days after treatment (at day 11 BALO culture) in n=3 biological replicates Representative images of β-galactosidase staining in TOPGAL epithelium (C) BALO cultures before (day 6, control) or 5 days after treatment with either 4 μM Scra or mo142-3p (day 11 of co-culture). BASC and rMC were isolated form the lung homogenate of TOPGAL mice. β-galactosidase+ rMC at day 11 of culture are indicated with arrows. Representative images of β-galactosidase staining in TOPGAL rMC (D) BALO cultures before (day 6, control) or 5 days after treatment with either 4 μM Scra or mo142-3p (day 11 of co-culture). BASC and rMC were isolated form the lung homogenate of TOPGAL mice. β-galactosidase+ rMC at day 11 of culture are indicated with arrows. Representative transmission and confocal images after LysoTracker staining indicating branching and number of branching points in n=4 BALO 15 days after treatment with 4 μM Scra or mo142-3p (day 21 of co-culture) in n=3 biological replicates. mRNA levels of epithelial and mesenchymal miR142-3p expression in Scra and mo142-3p-treated organoids 5 days after treatment (n=3-4 biological replicates with pooled cells from 4 cultures per replicate).
a) Confocal analysis of HeLa cells subjected to nutrient deprivation for 2 h.f3(a) Confocal analysis of HeLa cells subjected to nutrient deprivation for 2 h. Cells were transfected with HA-PLD1 and GFP-ATG14L (upper panels) or with HA-PLD1 alone (lower panels). Upper panels: HA-PLD1 (red), GFP-ATG14L (green), endogenous LC3 (blue). Lower panels: HA-PLD1 (green), endogenous ATG16L (red) and LC3 (blue). Insets show high magnification of the selected areas. The triple insets on the right indicate high magnification of the selected areas for two channels in various combinations. Scale bar, 5 μm. (b) Confocal analysis showing lack of co-localization of HA-PLD1 (green) with the isolation membrane marker ATG16L (blue) in HeLa cells expressing the strawberry-ATG4-C74A mutant (red) and subjected to 1 h of nutrient deprivation. Insets denote high magnification of the selected area. Scale bar, 5 μm. (c) EM analysis showing that GFP-PLD1 does not localize to the isolation membrane (arrows) in cells expressing the strawberry-ATG4-C74A mutant. Immuno-gold EM micrographs of HeLa cells transfected with GFP-PLD1 and pStrawberry-ATG4-C74A and subjected to 1 h of nutrient deprivation. Inset denotes high magnification of the selected area. Scale bar, 250 nm.
Co-localization of R-LPS and the essential porin Omp2b.Figure 3Co-localization of Omp2b (red) and R-LPS (green) in exponential phase bacteria. Scale bars: 2 μm. Co-detection of Omp2b (red) and R-LPS by AFM. R-LPS was detected using AFM tips functionalized with mAb against R-LPS. AFM images of whole bacteria and of the separated areas (red squares in first images, 0.4 x 0.4 μm2) are shown. The percentages of adhesion Padh are indicated below each area. Scale bars: 1 μm. Adhesion ratio between the Omp2b positive area compared to the Omp2b negative area. Grey dashed line shows ratio = 1 corresponding to homogeneity in surface structure. nWT = 13 bacteria, nΔgmd = 13 bacteria. Statistical analysis with Mann-Whitney U test showed **P < 0.01.
CatSper inhibitors abolish alkaline‐evoked Ca2+ influx in A.Figure 2A Alkaline‐evoked Ca2+ signals in sperm mixed with NH4Cl; sperm were loaded with the Ca2+ indicator Fluo‐4. ΔF/F (%) indicates the change in Fluo‐4 fluorescence (ΔF) with respect to the basal fluorescence (F, mean of the first 3-5 data points).B Ca2+ signals evoked by mixing of sperm simultaneously with NH4Cl (30 mM) and the CatSper inhibitor MDL12330A.C Dose-response relation of inhibition of the Ca2+ signals shown in panel (B), Ki = 20 μM, amplitudes were determined at t = 4 s.
Figure 7Figure 7. Proposed models predicting effector loading mechanisms. Model I: the VgrG-PAAR-effector-adaptor complex forms first before binding to the membrane-associated baseplate complex. Model II: VgrG binds to baseplates first prior to be loaded with the effector-adaptor complex. In both, the formation of an effector-loaded VgrG-baseplate complex is critical for initiating the polymerization of the Hcp and TssBC sheath and ejecting the effector complex outward. In the absence of either cargo effectors or adaptors, VgrG may not bind to the baseplate efficiently or the VgrG-baseplate complex is incompetent for triggering T6SS assembly. The effector shown in this model represents a PAAR/DUF4150-fused effector (such as Tde2 of C58, V6 of 1D1108) but also applicable to those where the effector and PAAR are encoded in separate genes (such as Tde1 of C58, V3 of 12D1, V6 of 15955), based on the evidence of VgrG-PAAR-Tde1-Tap-1 complex formation shown previously [16]. The adaptor protein is depicted as being released from the associated effector but the step which happens remain to be determined. The proposed models do not consider the role of Tae that may differentially regulate the outcome of effector loading in T6SS assembly.
Rab25 regulates bioenergetics in ovarian cancer cell through the AKT pathway.Figure 3A. Rab25 expression increases phospho but not total AKT and GSK3 levels in A2780 cells leading to decreased phosphorylation of GS (pGS). B. WB analysis of AKT and GSK3 phosphorylation confirming the inhibitory effect of PI3K pathways inhibitors PI103, GPi and synthase kinase 3 inhibitor (GSK3i) in HEY cells. C. Inhibition of GSK3 activity increases total cellular glycogen levels. pcDNA transfected and Rab25 expressing ovarian cancer cells were cultured in complete media in the presence of 10 µM GSK3i for the indicated times. Total cellular glycogen content was measured and normalized with total protein content. (a) p < 0.001 versus no GSK3i treated pcDNA cells. D-E. Effect of PI3K inhibitor PI103 on cellular glycogen content and ATP production. Ovarian cancer cells were pretreated with PI103 for at least 2 h in complete media. Cells were then subjected to 2 hglucose and FBS withdrawal to deplete endogenous glycogen followed by culturing cells in RPMI-glucose media in the presence of PI103 for 2 h (2 hSF). After 2 h of nutrient stress, complete media containing PI103 was added to the cells for 30 min (5% FBS) for recovery. A second glucose and FBS withdrawal (15 min SF) followed immediately to examine the effect on glycogen and ATP levels. a, p < 0.001 5% FBS versus 2 hSF, b, p < 0.01 15 min SF versus 5% FBS.D. There is an increase in glycogen content during recovery phase (5% FBS) and a decrease in glycogen when cells are subject to a second nutrient stress (15 min SF). D-E. Effect of PI3K inhibitor PI103 on cellular glycogen content and ATP production. Ovarian cancer cells were pretreated with PI103 for at least 2 h in complete media. Cells were then subjected to 2 hglucose and FBS withdrawal to deplete endogenous glycogen followed by culturing cells in RPMI-glucose media in the presence of PI103 for 2 h (2 hSF). After 2 h of nutrient stress, complete media containing PI103 was added to the cells for 30 min (5% FBS) for recovery. A second glucose and FBS withdrawal (15 min SF) followed immediately to examine the effect on glycogen and ATP levels. a, p < 0.001 5% FBS versus 2 hSF, b, p < 0.01 15 min SF versus 5% FBS.E. Utilization of glycogen to produce ATP. F. Addition of AKT inhibitor MK2206 (AKTi) abolishes Rab25-dependent glycogen storage. (a) p < 0.05 versus shRNA control and (b) p < 0.01 SF + AKTi versus SF. G. Inhibiting AKT pathway and glucose metabolism decreases cell viability. Cells were pretreated with 10 µM of PI103 or AKTi, 100 µM LND or 3BrPy for 2 h before switching to SF media in the presence of inhibitors for an addition16 h before cell titre blue viability assay. *p < 0.001 shRNA Rab25 versus shRNA control.
Knockout of TMEM41B impairs autophagosome biogenesis.Figure 2 A H4 Cas9 cells were infected with sgRNAs targeting ATG7, TMEM41B or NT control, treated with 500 nM AZD8055 or vehicle control for 24 h, and analyzed by immunoblotting. B Ratio of LC3-II to LC3-I band intensities are depicted as mean ± SD (n=6 independent experiments) with Wilcoxon test values  C H4 Cas9 TMEM41B and NT control cells were probed by LC3B immunostaining and imaged with an automated CV7000 confocal microscope. Scale bar: 20 μm. D The number of LC3 puncta per cell was quantified using Yokogawa Analysis Software (YAS) and depicted as mean ± SD (n=3 technical replicates)  E LC3 and WIPI2 were co-stained and imaged using an LSM700 confocal microscope. Arrowheads point at co-localized LC3 and WIPI2 puncta. Scale bar: 5 µm  F The number of WIPI2 puncta positive for LC3 was quantified using ImageJ and depicted as mean ± SD (n=5 technical replicates). G The total number of WIPI2 puncta per cell was quantified using the Harmony software on images acquired with an automated Operetta microscope. Data are depicted as mean ± SD (n=3 technical replicates)  H H4 Cas9 TMEM41B and NT control cells were infected with a RFP-DFCP1 expression construct for 72h, fixed, stained with LC3 antibodies and imaged with an automated CV7000 confocal microscope. Arrowheads point at co-localized LC3 and RFP-DFCP1 puncta. Scale bar: 5 µm. I The number of RFP-DFCP1 puncta positive for LC3 was quantified using ImageJ and depicted as mean ± SD (n=10 technical replicates)   J mCherry-GFP-LC3 was expressed in H4 Cas9 TMEM41B KO and NT control cells which were treated with 50nM BafA1 or vehicle control for 24 h, fixed and imaged. Scale bar: 20 μm. The number of mCherry- and GFP-positive puncta per cell was quantified using YAS and are depicted as mean ± SD (n=3 technical replicates)
A conserved sRNA motif is enriched in laboratory selection experiments A) Experimental strategy of the laboratory selection experiments: an sRNA library was generated using the rybB scaffold with nine randomized nucleotides at the 5' end, cloned into a broad-range plasmid backbone, and transferred into V.Figure 5Experimental strategy of the laboratory selection experiments: an sRNA library was generated using the rybB scaffold with nine randomized nucleotides at the 5' end, cloned into a broad-range plasmid backbone, and transferred into V. cholerae ΔrpoE cells. These colonies were pooled, grown to OD600 of 0.2 and treated with ethanol (3.5% final conc.) for 6 hours. Surviving cells were recovered on agar plates, pooled and subjected to another round of selection (3 selections total). After each selection, the plasmids of surviving cells were analyzed using high-throughput sequencing. V. cholerae wild-type and ΔrpoE strains carrying an empty vector control (pCtr), pRybB, or the sRNA library after consecutive selection experiments (Sel1, Sel2, Sel3) were grown in LB to OD600 of 0.2. Cells were treated with ethanol (3.5% final conc.) for 6 hours. Serial dilutions were prepared and spotted onto agar plates. R1 and R2 indicate two independent biological replicates. Plasmid contents of the strains carrying the sRNA libraries before selection (input) and after consecutive ethanol treatments (Sel1, Sel2, Sel3) were analyzed using high-throughput sequencing. Relative library complexity (left y-axis) was determined by counting sequence variants present in the normalized samples. To test for the enrichment of possible sequence motifs, the sequence variants present in each sample were counted and normalized for sequencing depth. The resulting data were analyzed for the enrichment of the conserved CRCUGCUUUU motif (right y-axis).
We used 20-month-old heterozygous littermates surviving grossly tumor free.f6We used 20-month-old heterozygous littermates surviving grossly tumor free. (a,b) The sequential state III oxygen consumption rate of heart isolated mitochondria (300 μg) was monitored with a fibre-optic system. Substrates and inhibitors were added sequentially at the final concentrations indicated as follow. FCCP, 1 μM carbonylcyanide-p-trifluoromethoxyphenylhydrazone; M/P, 5 mM malate+5 mM pyruvate; Rot, 100 nM rotenone; Suc, 5 mM succinate; AA, 50 nM antimycin A; TMPD/Asc, 0.4 mM N,N,N′,N′-tetramethyl-p-phenylenediamine +1 mM ascorbate; KCN, 5 mM potassium cyanide. Representative results are shown (a) and oxygen consumption by each complex was calculated (n=5-8 per group, b). (c,d) Mitochondrial particle size (c) and membrane potential (d) were determined using flow cytometry for TMRE in cardiac mitochondria preparations. Results are shown from 4-8 hearts analysed in duplicate. (e) H2O2 production with 0.5 mM NADH, complex I-linked substrate, was determined in the absence or presence of 2 μM complex I inhibitor rotenone using Amplex Red (n=5-8 per group). (f) Myocardial high-energy phosphate metabolism under 8γ dobutamine loading was measured biochemically with HPLC (n=5 per group). (g) Maximum dP/dt was examined using cardiac catheterization with graded a dobutamine infusion (n=5 per group). (h) Photographs showing heart sections after SA-β-gal staining; original magnification, × 200; scale bar, 40 μm. SA-β-gal-positive cells were quantified (n=4 per group). Five images per mouse were taken on brightfield and DAPI staining was used to count the total cell number per section. HT indicates heterozygous; DHT double heterozygous. Data are shown as the means±s.d. *P0.05; **P0.01; ***P0.001 (two-tailed unpaired Student's t-test)
Bob1 and Oct2 synergistically transactivate the Btla and Bcl6 promoter.Figure 9(A) NIH/3T3 cells were transfected with luciferase reporter constructs of the Btla (left panel) or Ifng (right panel) promoter along with expression vectors for Bob1 and/or Oct2. (B) Analysis of the Bcl6 enhancer/promoter construct in untreated or TPA/Ionomycin-stimulated EL-4 cells. In addition, we have analyzed mutant Bcl6 enhancer/promoter constructs in which either the motif M3 or M6 had been functionally inactivated by site directed mutagenesis. Data are expressed as fold-induction above the activity of the wild-type promoter without transfection of Bob1 or Oct2 (n=3 for NIH3T3 cells; n=5 for wild-type Bcl6 promoter analysis in EL-4 cells and n=3 for mutant Bcl6 promoter analysis in EL-4 cells). Data are shown as mean ± SEM.
Figure 4(A) qRT-PCR expression of endogenous repeats (left) and ISGs (right) following shRNA-mediated KAP1 depletion in HeLa cells (day 6 post transduction), normalized to B2M and GAPDH respectively. n=2 and two-tailed unpaired t test p-values are: HERVK14C: 0.025, 0.003; SVA VNTR: 0.00027, 0.01; ISG56: 0.002; CCL5: 0.007, 0.00003; CXCL10: 0.0008; Mxa: 0.011; IKba: 0.008, 0.01. See also Fig S2AB. (B) qRT-PCR expression of endogenous repeats (left) and ISGs (right) following 5-AZA treatment of HeLa cells and PBMCs (day 6 post transduction). A representative experiment of two is shown in each case. Two-tailed unpaired t tests were performed. HeLa cell p-values: ISG56: 0.005; CCL5: 0.0006; CXCL10: 0.0007; Mxa: 0.0001; IFNb: 0.02. For repeat expression (left panel), p values not available as N=2. PBMC p-values: HERVK14C: 0.1; SVA D VNTR: 0.003; ISG56: 0.001; CCL5: 0.012; CXCL10: 0.012; Mxa: 0.02; IKba: 0.08. Results were normalized to GAPDH and B2M (GAPDH results shown). See also Fig S2C for results in 293T cells. All error bars show SD. (C) DNA methylation at endogenous SVAs was measured over 18 CpGs in the stated treatment groups (day 5 post transductions, or day 2 post 5-AZA treatment). Each point represents the average methylation state of one sequence with at least 10 sequences analyzed per group. Mann-Whitney tests were performed to compare shControl to shKAP1 (p=0.6593) and DMSO to 5-AZA (0.0027). 5-AZA was added to all experiments at 7uM. All numbers above bars depict fold changes compared to control cells (to one decimal place).
Prions induce lysosomal dysregulation via TFEB.Figure 5A: Time course of TFEB phosphorylation in prion-infected C57BL/6 mice. Total TFEB was unchanged but reduced TFEB phosphorylation was evident at ≥90 days. Quantification: Each dot represents an individual biological replicate. Statistics: Unpaired t-test. *: p0.05; **: p<0.01. Error bars represent s.e.m. B: TFEB-controlled transcripts in brains of terminally scrapie-sick C57BL/6 mice were measured by qPCR. Prion infection led to upregulation of these genes, but not of STAT3. Control: NBH-inoculated mice. ANOVA on independent triplicates. ***: p<0.001. Error bars represent s.e.m. C: Prion-infected Gt1 cells (75dpi) showed reduced TFEB phosphorylation. Total levels of TFEB remain unchanged. Quantification: Each dot represents an individual biological replicate. Statistics: Unpaired t-test. **: p<0.01. Error bars represent s.e.m. D: COCS prepared from tga20 mice were infected with RML prions and cultured for 5 weeks. Cell lysates were prepared at 4 wpi followed by Western blot analysis using anti-phospho-TFEB antibody. Activated (dephosphorylated) TFEB was increased in RML infected COCS. Total levels of TFEB remain unchanged. Quantification: Each dot represents an individual biological replicate. Statistics: Unpaired t-test. Error bars represent s.e.m. E: Gt1 cells (as in D) were fixed and stained with anti-TFEB antibody. Prion-infected cells showed nuclear translocation of TFEB. DAPI: Blue. Quantification: Each dot represents an individual biological replicate (30 images were quantified per replicate). Statistics: Unpaired t-test. **: p<0.01. Error bars represent s.e.m. F: TFEB responsive genes were upregulated in Gt1 cells chronically infected with RML prions (75 dpi). Panels depict independent triplicates. Statistics: ANOVA. *: p0.05. Error bars represent s.e.m. G: TFEB responsive genes were upregulated in cells transfected with shPIKfyve for 5 days compared to shCtrl (shRNA against luciferase). Panels depict independent triplicates. Statistics: ANOVA. *: p0.05. Error bars represent s.e.m.
Figure 1 A) Treadmill analysis (n=8/group). Black: untreated WT; grey: rapamycin-treated WT; red: untreated Cox15sm/sm; blue: rapamycin-treated Cox15sm/sm. Error bars represent SEM. The asterisks represent the significance levels calculated by unpaired, two-tailed Student's t test ***p=0.005; ***p=0.0007 (T3) and ****p<0.0001 (T4). Only p values for rapamycin-treated vs. untreated Cox15sm/sm mice are shown  B) Histological and histochemical characterization of skeletal muscle in rapamycin-treated and untreated Cox15sm/sm and WT mice. H&E: hematoxylin and eosin; PAS: periodic acid Schiff reaction; COX: cytochrome c oxidase; SDH: succinate dehydrogenase. White bars correspond to 50µm  C) Analysis of the cross-sectional area of muscle fibers in the different genotypes (n=3/group). Black: untreated WT; grey: rapamycin-treated WT; red: untreated Cox15sm/sm; blue: rapamycin-treated Cox15sm/sm. Error bars represent SEM. The asterisks represent the significance levels calculated by one-way ANOVA with Tukey post-hoc multiple comparison test: ***p=0.001; ***p=0.006, ****p<0.0001  D) Analysis of the number of centralized nuclei in muscle fibers (n=3/group). Black: untreated WT; grey: rapamycin-treated WT; red: untreated Cox15sm/sm; blue: rapamycin-treated Cox15sm/sm. Error bars represent SEM. The asterisks represent the significance levels calculated by one-way ANOVA with Tukey post-hoc multiple comparison test **p=0.0018, **p=0.0052  E) Analysis of PAS-reaction intensity in muscle fibers (n=4/group). Black: untreated WT; grey: rapamycin-treated WT; red: untreated Cox15sm/sm; blue: rapamycin-treated Cox15sm/sm. Error bars represent SEM. The asterisks represent the significance levels calculated by one-way ANOVA with Tukey post-hoc multiple comparison test *p=0.0293 (WT vs. Cox15sm/sm) and *p=0.0150 (Cox15sm/sm vs. Cox15sm/sm Rapa)  F) Analysis of COX-reaction intensity in muscle fibers (n=3). Black: untreated WT; grey: rapamycin-treated WT; red: untreated Cox15sm/sm; blue: rapamycin-treated Cox15sm/sm. Error bars represent SEM. The asterisks represent the significance levels calculated by one-way ANOVA with Tukey post-hoc multiple comparison test ****p<0.0001; **p=0.0031  G) Analysis of SDH-reaction intensity in muscle fibers (n=3). Black: untreated WT; grey: rapamycin-treated WT; red: untreated Cox15sm/sm; blue: rapamycin-treated Cox15sm/sm. Error bars represent SEM. The asterisks represent the significance levels calculated by one-way ANOVA with Tukey post-hoc multiple comparison test *p=0.0199, **p=0.0091
Figure 1NE activates mTORC2 in vitro via cAMP, PI3K and Epac1. (A) Immunoblot analysis of BAT cells stimulated with norepinephrine (NE) for the indicated proteins. (B) Immunoblot analysis of BAT cells stimulated with NE for 5 minutes in the presence of Rapamycin (Rapa), Torin or Wortmannin (Wrtm) for the indicated proteins. (C) Immunoblot analysis of BAT cells stimulated with 8-Br-cAMP for 5 minutes in the presence of Rapa, Torin or Wrtm for the indicated proteins. (D) Immunoblot analysis of BAT cells stimulated with NE for 5 minutes in the presence of Wrtm, H89 or ESI-09 for the indicated proteins. (E) Immunoblot analysis of BAT cells stimulated with 8-Br-cAMP for 5 minutes in the presence of Wrtm, H89 or ESI-09 for the indicated proteins. All experiments were performed in triplicates, a representative replicate is presented.
Figure 3A, B Core histones were prepared from NSPCs treated with various concentrations of crotonate (A) and 1 μM MS-275 (B) for 24 h and subjected to western blotting analysis using anti-Kcr antibody, and the experiments were repeated three times. C NSPCs were treated with 20 μM C646, 10 mM acetate (Ac), 20 μM C646 and 10 mM acetate (Ac+C646), 10 mM crotonate (Cr), and 20 μM C646 and 10 mM crotonate (Cr+C646) for 24 h, then core histones were extracted and subjected to western blotting analysis using anti-Kcr antibody, the experiments were repeated three times. D ChIP-seq density heatmaps in NSPCs for histone Kcr at ± 1 kb from the center of peak regions with differential histone Kcr after crotonate (left panel) and MS-275 (right panel) treatment. There were 3308 and 836 peak regions with increased histone Kcr after crotonate and MS-275 treatment, respectively, and 1075 and 2942 peak regions with reduced histone Kcr after crotonate and MS-275 treatment, respectively. E Heatmaps displaying overlap enrichment for various genomic regions (Up and Down: peak regions with increased and reduced histone Kcr in NSPCs after crotonate or MS-275 treatment). A darker color in blue, red, or purple corresponds to greater fold enrichment for a column-specific coloring scale. F GO analysis (biological process) of 2442 genes with increased histone Kcr (left panel) and 726 genes with reduced histone Kcr (right panel) after crotonate treatment in NSPCs.