EMBO/sd-character-level-ner huggingface dataset:
Fig.Figure 7A, B. HEK293T HOM1 cells were plated on a planar lipid bilayer containing (NIP)1-H12-Hylight647 (A) or His-Activin A-Atto647N (B) and an automated time-lapse was started, where 2-4 positions within a well were imaged every 40 s during a 27-min time period; 3 time points from one position are shown. TIRF, upper panels, brightfield, lower panels. The cluster intensities from all positions were quantified at 6 time points indicated (right panel) and plotted as individual cluster values. The horizontal gray bar denotes the mean. Cells + bilayer denotes bilayer areas under the cells, as identified from the brightfield images, and bilayer denotes areas where no cells were present. A representative experiment from 2 biological replicates quantified is shown. Scale bar 10 μm. In B, the region under cell 2 is shown enlarged in the top right-hand corner. C. Mobile His-Activin A-Atto647N on a planar lipid bilayer was imaged for a 20 sec period; 3 time points are shown. Scale bar 10 μm. D. Mean cluster intensity per cell over time. Quantifications are the mean cluster intensities at 5 time points of 35 cells (NIP + cells), 99 cells (Activin A + cells), and 28 bilayer areas ± SEM, from a representative experiment imaged as in A and B. The p values are from F test. ns, not significant; **, p<0.01.
Naïve Hdac3-/- mESCs differentiate into PrE.Figure 1Nanog>GFP intensity of WT and Hdac3-/- TNG-A cells in 2iL and at d2 and d5 of SL exposure. Pairwise Pearson correlations of in vitro TNG-A and in vivo embryo (Boroviak et al, 2015) RNAseq samples. k-means clustering of gene expression changes relative to naïve WT TNG-A mESCs. Immunofluorescence and reporter expression in spheroids derived from single WT and Hdac3-/- naïve mESCs. DNA counterstain is Hoechst. Dashed white lines indicate the embryonic Gata6::mCherry negative part. Scale bar: 10 μm.
Figure 4A The position of ura4+ reporter alleles in centromere 1 is shown in the top panel. Strains containing the imr::ura4+ allele were grown to log phase in YE5S medium, subjected to 5-fold serial dilutions and spotted onto YE5S agar or YE5S agar supplemented with 5-FOA (1 mg/ml). Data are representative of three independent biological repeats. B Strains carrying the otr::ura4+ were analysed as described for A. C RNA was purified from the indicated strains and the level of centromeric transcripts determined by qRT-PCR. Data is the mean of three independent repeats and error bars indicate ± SEM. P-value was calculated using a two-tailed unpaired t-test. D RNA was purified from the indicated strains and subjected to qRT-PCR for the subtelomeric gene, tlh1+. Data is the mean of three biological repeats and error bars indicate ± SEM. P-value was calculated using a two-tailed unpaired Student's t-test. E The position of insertion of ade6+ reporter allele in the silent mat locus is shown in the top panel. Log phase cells containing the mat3-M::ade6+ allele were subjected to five-fold serial dilution and spotted onto YE5S agar plates lacking adenine (Low Ade) were incubated for 4 days at 30°C. Data are representative of at least three independent biological repeats. Note, microarray analysis indicates that deletion of abo1+ does not influence the expression of ade6+ (or ura4+) when these genes are present at their normal genomic loci.
Fig.Figure 5Effect of MB on proliferation of human CD8+ T cells. CFSE labeled human peripheral blood mononuclear cells (PBMC) were pre-incubated with DMSO, MB, 25 μg/ml of pembrolizumab or 20 μg/ml of Nivolumab for 1 h and then seeded in the 96-well plate precoated with 10 μg/ml of aCD3/aCD28 in the presence of 10 μg/ml human PD-L1 Protein. Cell proliferation was analyzed through FACS analysis of dilution of CFSE. Data information: Data are representative of three independent experiments, and were analyzed by unpaired t-test. Error bars denote s.e.m. **P < 0.01; ****P < 0.0001. Cytokine and cytolytic granule production in CD8+ T cells. Human peripheral blood mononuclear cells from healthy donators were stimulated with 5 μg/ml of phytohaemagglutinin for 2 days and then rested for 1 day. Cells were pretreated with DMSO, MB, 25 μg/ml of pembrolizumab (Pem) or 20 μg/ml of nivolumab (Niv) for 1 h and then seeded in a 96-well precoated with 10 μg/ml aCD3/aCD28, with media supplemented with 10 μg/ml of human PD-L1 Protein. Cytokine and cytolytic granule production were determined by intracellular flow cytometry by gating on CD8+ populations. Data information: Data are representative of three independent experiments, and were analyzed by unpaired t-test. Error bars denote s.e.m. **P < 0.01; ****P < 0.0001. Comparison of the efficacy of MB with PD1 inhibitors currently used in clinic to activate T cell. JP-luc cells were stimulated with Raji-L1 or parental Raji cells preloaded with 1 μg/ml of superantigen (SEE) in the presence of MB at indicated concentration or 25μg/ml of pembrolizumab (Pem) or 20μg/ml of nivolumab (Niv) for 6 hours. Luciferase activity was measured by luminometer. Data are representative of three independent experiments, and were analyzed by unpaired t-test. JP-luc: Jurkat cell harboring NFAT-luciferase transgene and overexpressing PD-1; Raji-L1: Raji overexpressing PD-L1. Data information: Data are representative of three independent experiments, and were analyzed by unpaired t-test. Error bars denote s.e.m. **P < 0.01; ****P < 0.0001.
Diet and NOD2 influence the gut microbiome.Figure 5A Unweighted UniFrac principal coordinates analysis (PCoA) plot illustrating the relative degree that genotype (NOD2−/− versus WT, PCoA1: 28%) and diet (chow versus HFD, PCoA2: 16%) defined the diversity of the microbiota in the cecum. The most abundant bacterial genus are labeled and superimposed on the same PCoA plot. Each small dot is a mouse, and the size of the sphere representing a taxon is proportional to the mean relative abundance of the taxon across all samples. B Bar graph showing the relative abundance of phyla from each mouse cecum, where each bar is a separate mouse. C Alpha diversity represented by the Shannon index in chow-fed (n = 6) and HFD-fed (n = 5) WT and NOD2−/− mice. Values are mean ± SEM. D Impact of diet on the Firmicutes/Bacteroidetes ratio in the cecum of chow-fed (n = 12) and HFD-fed (n = 10) mice. *Significantly different from chow-fed mice, P = 0.03. An unpaired t-test was used. Values are mean ± SEM.
Figure 3(A) Retroviral transduction workflow for γδ thymocytes. (B) Gating strategy for identification of the GFP+ versus GFP- cells. (C) RT-PCR analysis of the expression of miR-181a(-5p and -2-3p) (normalized to the values obtained with the empty virus) in transduced (GFP+) and untransduced (GFP-) γδ thymocytes, cultured with IL-7 plus IL-2 for 11 days (n=4 independent biological samples). (D) FACS analysis of the expression of Annexin V in miR-181a versus empty transduced (GFP+) and untransduced (GFP-) γδ thymocytes, cultured with IL-7 plus IL-2 for 11 days (n=5 independent biological samples). (E) RT-PCR analysis of the expression of Bcl2 and Bcl-xL (normalized to the values obtained with the empty virus) in transduced (GFP+) γδ thymocytes, cultured with IL-7 plus IL-2 for 11 days (n=5 independent biological samples). (F) FACS-derived expression of indicated surface and intracellular markers in miR-181a versus empty transduced (GFP+) and untransduced (GFP-) γδ T thymocytes, cultured with IL-7 plus IL-2 for 11 days (n=7-11 independent biological samples).
TIP30 overexpression inhibits cardiac hypertrophy.Figure 2(A) Western blot for TIP30 and GAPDH in neonatal rat cardiomyocytes (NRCM) after adenoviral transduction with Ad.Control (Ad.Con) or Ad.TIP30. Quantification of cardiomyocyte area (B), N=6 to 8 samples/group in NRCM transduced with Ad.Con or Ad.TIP30 and stimulated as indicated. ET-1: endothelin-1, FBS: fetal bovine serum, PE: phenylephrine. Quantification of protein/DNA ratio (C), N=9 samples/group in NRCM transduced with Ad.Con or Ad.TIP30 and stimulated as indicated. PE: phenylephrine. Quantification of Acta1 mRNA transcript abundance (D), N=3 samples/group in NRCM transduced with Ad.Con or Ad.TIP30 and stimulated as indicated. ET-1: endothelin-1 PE: phenylephrine. Quantification of cell death with a 7-AAD assay (E), N=7 samples/group in NRCM transduced with Ad.Con or Ad.TIP30 and stimulated as indicated. PE: phenylephrine. (F) Schematic representation of AAV-MLC-TIP30 study design. (G) Western blot for TIP30 and Actin in mouse hearts with AAV9 mediated overexpression of TIP30 (AAV-TIP30) or from mice treated with a control AAV9 construct (AAV-Con) followed by 2 weeks of TAC surgery. Quantification of HW/TL ratio (H), N=8 to 13 mice/group 2 weeks after sham or TAC surgery in AAV-Con or AAV-TIP30 treated C57BL/6 WT mice. * Quantification of cardiomyocyte area (I), N=4 to 5 mice/group 2 weeks after sham or TAC surgery in AAV-Con or AAV-TIP30 treated C57BL/6 WT mice. (J) Serial echocardiography with quantification of echocardiographic fractional area change 2, 4 and 6 weeks after sham or TAC surgery in AAV-Con or AAV-TIP30 treated C57BL/6 WT mice (N=10 to 14 mice/group and time point).
Quantification of autophagic vacuoles induced by ABT737.Figure 4Quantification of autophagic vacuoles induced by ABT737. HeLa cells transfected with the indicated siRNAs (specific for Emerin or for Beclin‐1 at 0 h) were re‐transfected with LC3‐GFP finally cultured in nutrient‐free (NF) conditions (60-72 h), in the presence or absence 1 μM ABT737 and then subjected to electron microscopy detection of immature (AV1) or mature (AV2) autophagic vacuoles. Representative pictures are shown in (A). The number of AV1 and AV2 was determined for a minimum of 50 cells (means±s.e.m.) (B).
Figure 3, Dependence of clockwise rotation on the level of FliMN-CheY~Ac. The red and purple curves are two experimental days in which the clockwise production by fliM∆N ∆cheA cells expressing FliMN-CheY was measured as a function of FliMN-CheY concentration (strain EW696; Concentrations are estimates based on the calibration curves or which a similar CheY expression system was used. ach data point is a mean ± SEM of the data or the second and third experimental days of this strain (in the presence of acetate). B, Response of the strain to addition and removal of varying acetate concentrations (pH 7.0). FliMN-CheY concentration was estimated to be ~300 μM. Lines and shaded regions are the mean time spent in clockwise rotation ± SEM. The arrows indicate the estimated time points at which acetate entered or left the flow chamber. Black lines mark the linear part of the biphasic response to acetate removal. N is the number of cells. C, Distribution of clockwise interval lengths at different FliMN-CheY~Ac concentrations in the strain FliMN-CheY was acetylated by acetate (10 mM, pH 7.0). The black lines illustrate the slopes of the short- and long-clockwise interval distributions. The color bar indicates the estimated FliMN-CheY~Ac concentration. Distribution of clockwise interval lengths with respect to the average clockwise levels of the cells. E, Average clockwise interval length, calculated as the inverse of the fast (blue) and slow (red) rate constants
Mfa1+Pg up-regulate the expression of DC-SIGN in human MoDCs.Fig 2A) DC-SIGN mRNA expression in P. gingivalis-infected MoDCs at 0.1, 1 and 10 MOIs. The figure shows the gene expression after 12 hours of Pg381 and mutant strains infections. The target gene (DC-SIGN) was normalized using the endogenous control GAPDH (ΔCt) and fold regulations were calculated using 2-(ΔΔCt) method. The statistical analysis was performed using the t-test, which accounts for the clustering of infected and un-infected controls within 3 different experiments (* p<0.001). B) Immuno-electron microscopy of un-infected MoDCs (Cont.) (upper panel), MoDCs infected with Pg381 (middle panel) and Mfa1+Pg mutants (lower panel). Gold particles (marked with red rings) for positive DC-SIGN were detected in the cell membrane and cytoplasm of cells infected with Mfa1+Pg strains. Minimal positive staining for DC-SIGN was detected in the membranes of MoDCs infected with Pg381, while no cytoplasmic gold labeling was detected in these cells. C) Flow cytometry analysis of surface DC-SIGN in human MoDCs after infection with Pg381, Mfa1+Pg and FimA+Pg. The analysis of the intensity used Kruskal-Wallis test analysis of different groups and Dunn's test for multiple comparisons 3 different experiments (* p<0.01).
Figure 6Quantification of oxygen consumption rate (OCR) of reprogramming intermediates treated with small molecules (A, C) and the indicated OCR parameters (B, D). n = 9. Fluorescence images of DCFH-DA staining for ROS levels in cells treated with DMSO, HA, NAC, and R406 on d12 (E) and the quantification of the ROS levels (F). n = 4. Scale bar, 100 μm. MDA quantification in cells treated with DMSO, HA, NAC, and R406 on d12. n = 9. A summary diagram of this study.
Knockdown of p19ink4d and p27kip1 in SCC25 cells.Figure 4(A) Short interfering RNAs (siRNA) knockdown of p19ink4d messenger RNA and protein in SCC25 cells. SiRNAs were directed against p19ink4d, scrambled p19ink4d (Scr.) or cyclophilin (cyc.) controls. (B) Combined knockdown of p27KIP1 and p19INK4D in SCC25 cells analysed by western blotting for p27KIP1 (top) and p19INK4D (bottom). (C,D) Fluorescence‐activated cell sorting analysis of cell‐cycle distribution of control SCC25 cells, or cells transfected with scrambled (Scr), p19ink4d‐ or p27kip1‐directed siRNAs, individually or together (Dbl). (C) Cells were treated with vehicle or 1,25‐dihydroxyvitamin D3 (1,25D3), as indicated. (D) Cells were treated with DMSO vehicle, retinoic acid (RA) or 1,25D3, as indicated. (E) Loss of p19 enhances lysosomal β‐galactosidase activity in SCC25 cells. (Left) Histogram of numbers of cells staining for β‐galactosidase activity in the absence (−) or presence of 1,25D3 in control cells (−) or in cells transfected with scrambled (Scr) or p19 siRNAs. (Right) Bright field images of untransfected cells (−/−) or p19INK4D‐depleted cells treated with 1,25D3 (p19/D3). (F) Loss of p19INK4D induces lysosomal fusion. Analysis of lysosomal fusion (autophagosomes) in SCC25 cells treated as in (E), stained for incorporation of lysotracker red. Nuclei were counterstained with Hoechst (blue).
Identification of proteins differentially secreted by CAFs upon SOM230 treatment and involved in CAF-CM-induced chemoresistance on pancreatic cancer cells.Figure 6A Membrane antibody array assay using CM from SOM230-treated or not CAFs (representative of n = 3). Controls are circled in blue dashed line and IL-6 in red square. B, C Anti-IL-6 ELISA assay (mean ± SD) using CM or protein extracts from SOM230-treated or not CAF (from left to right: §§§P = 0.0002, §§§P = 0.0002) (B), or CM from siCTR- or si4E-BP1-transfected CAFs treated or not with SOM230 (from left to right: ***P = 0.0003, $$P = 0.004) (C) (n = 3). B, C Anti-IL-6 ELISA assay (mean ± SD) using CM or protein extracts from SOM230-treated or not CAF (from left to right: §§§P = 0.0002, §§§P = 0.0002) (B), or CM from siCTR- or si4E-BP1-transfected CAFs treated or not with SOM230 (from left to right: ***P = 0.0003, $$P = 0.004) (C) (n = 3). D Panc-1 cell viability was assessed by MTT. Results (mean ± SD) are presented for each treatment as the percentage of the respective untreated cells (= 100%) (n = 3; from left to right: **P = 0.004, ##P = 0.001, §P = 0.023, §P = 0.019). E, F Immunoblotting using an anti-cleaved caspase-3, anti-PARP or anti-GAPDH (loading control) antibody of protein extracts from Panc-1 cells incubated with CAF-CM pre-incubated or not with an anti-IL-6 neutralizing antibody (E), or with human recombinant IL-6 (representative of n = 3) (F). G, H Immunoblotting using an anti-survivin or anti-GAPDH (loading control) antibody of protein extracts from Panc-1 cells stimulated or not with CAF-CM pre-incubated or not with anti-IL-6 neutralizing antibody (G), or with human recombinant IL-6 (representative of n = 3) (H). I Immunoblotting using an anti-P-Akt, anti-P-S6 or anti-β-actin (loading control) antibody of protein extracts from SOM230-treated or SOM230-untreated CAF incubated or not with IL-6 neutralizing antibody (representative of n = 3).
Compound C inhibits the effects of AICAR on cytokine-induced atrophy.Figure 5
Figure 6Restoration of glucoseuptake or Akt signaling suppresses the thermogenic defect in AdRiKO mice. (A) HKII mRNA expression level in BAT of AdRiKO and control mice infected with either AAV9-HKII or AAV9-empty (n=8/group). (B) Cold-induced 2-Deoxyglucose-6-phosphate (2DG6P) accumulation in BAT of AdRiKO and control mice infected with either AAV9-HKII or AAV9-empty housed at 4°C for 4h (n=8/group). (C) Body temperature of AdRiKO and control mice infected with either AAV9-HKII or AAV9-empty housed at 22°C (n=8/group). (D) Body temperature upon cold exposure of AdRiKO and control mice infected with either AAV9-HKII or AAV9-empty. The left panel represents body temperature after each hour of cold exposure while the right panel represents body temperature as a bar graph for the 3h cold exposure time point (n=8/group). a=significant difference between AdRiKO and control mice infected with AAV9-empty, b= significant difference between AdRiKO and control mice infected with AAV9-HKII, d= significant difference between AdRiKO mice infected with AAV9-empty and AAV9-HKII. (E) Immunoblot analysis of BAT from AdRiKO and control mice infected with either AAV8-Akt2S474D or AAV8-empty (n=6/group, each lane represents a mix of 3 mice). (F) Body temperature of AdRiKO and control mice infected with either AAV8-Akt2S474D or AAV8-empty housed at 22°C (n=11/group). (G) Body temperature upon cold exposure of AdRiKO and control mice infected with either AAV8-Akt2S474D or AAV8-empty. The left panel represents body temperature after each hour of cold exposure while the right panel represents body temperature as a bar graph for the 3h cold exposure time point (n=11/group). a=significant difference between AdRiKO and control mice infected with AAV8-empty, b= significant difference between AdRiKO and control mice infected with AAV8-Akt2S474D, d= significant difference between AdRiKO mice infected with AAV8-empty and AAV8-Akt2S474D. (H) Cold-induced 2-Deoxyglucose-6-phosphate (2DG6P) accumulation in BAT of AdRiKO and control mice infected with either AAV8-Akt2S474D or AAV8-empty housed at 4°C for 4h (n=7 (control AAV8-null), n=6 (AdRiKO AAV8-null), n=6 (control AAV8-AktS474D), n=6 (AdRiKO AAV8-AktS474D)). Data represent mean ± SEM. Statistically significant differences between AdRiKO and control mice were determined with unpaired Student's t-test and are indicated with asterisks (*=p<0.05; **=p<0.01, ***=p<0.001). Statistically significant differences between viruses were determined with unpaired Student's t-test and are indicated with a number sign (#=p<0.05; ##=p<0.01; ###=p<0.001). The exact p-value for each significant difference can be found in Appendix Table S2.
Localization and Characterization of ZDHHC5 in HeLa Cells.Figure 4(A) Primary screen anti-CD8 antibody-uptake images for control cells (top) and ZDHHC5 KD cells (bottom). (B) Cells stably expressing ZDHHC5-Myc show localization of ZDHHC5 to the plasma membrane and to intracellular tubules and vesicles. Some colocalization between ZDHHC5 and retromer VPS35 is observed (arrowheads in inset). Following nocodazole treatment endosomes are dispersed and some are labeled with ZDHHC5 and VPS35 (arrowheads in inset). (C) Control and ZDHHC5 siRNA-treated SFT2D2-Myc cells were mixed and stained for ZDHHC5, Myc, and VPS35. KD cells are marked by an asterisk. (D) Control and ZDHHC5 siRNA-treated HeLa cells were mixed and stained for ZDHHC5, α5-integrin, and VPS35. KD cells are indicated with an asterisk. (E) Control (top) and ZDHHC5 KD (bottom) HeLa cells were fixed and stained for TGN46 and β1-integrin.
Implanted constructs of Mabs in PF on the surface of TA.Figure 2A Gross morphology of the PF implant containing Mabs-nLacZ/PlGF immediately after implantation on the surface of underlying TA (arrow).B, C Artificial muscles (arrows) developed within 8 weeks over the host TA surface (arrowheads), encapsulated by the host perimysium (B) and released after tendon resection of the TA (C).D The isolated artificial muscle from the TA surface (arrow) is comparable in size to the underlying host TA (arrowhead). E Stereomicroscopy images revealing blood vessel formation in the artificial muscle. Scale bar: 100 μm. F X-Gal and H&amp;amp;E staining on artificial muscle section revealing vessel connection with host vasculature by Indian ink, injected into the femoral artery immediately before sacrifice. Scale bar: 20 μm. G Western blot analysis of protein extracts from artificial muscles (PF) and host TA (Ctrl) reveals remarkable expression of muscle- and vessel-specific proteins (n = 5). I-L X-Gal and H&amp;amp;E staining of artificial muscle sections at 4 (I, K) and 8 weeks (J, L) after implantation, showing donor origin of muscle cells (nLacZ+ nuclei) and correlation between implant duration and myofibre maturation (I, J images are the results of the collage of several photos). Scale bar: (I, J) 200 μm, (K, L) 100 μm. M-P Immunofluorescence analysis on adjacent sections revealing laminin organization (green) and myosin heavy chain (MyHC) expression (red) at 4 weeks (M) and 8 weeks (N) after grafting (M, N displayed items are obtained from a multi-photo collage); DAPI staining (blue) shows centrally located nuclei in developing fibres (O) and peripheral nuclei in mature myofibres (P). Scale bar: (M, N) 200 μm, (O, P) 50 μm. Q Confocal isosurface image of neurofilament (green) and bungarotoxin (red) immunofluorescence in the artificial muscle section shows a mature neuromuscular plaque. Scale bar: 10 μm. R Immunofluorescence against smooth muscle actin (blue), laminin (green) and VE-Cad (red) superimposed on phase contrast image shows blood vessels (arrows) adjacent to the myofibres in the artificial muscle section. Scale bar: 20 μm. S X-Gal staining on mature artificial muscle (arrowhead) and the underneath host TA (arrow) revealing no tendon development at the extremity of the artificial tissue; implanted LacZ-positive Mabs are present within the host TA. Scale bar: 200 μm.