EMBO/sd-character-level-ner huggingface dataset:
Src activity is required for targeting of Ret to puncta in both the presence and absence of FAK.Figure 4(A) FAK+/+ cells were treated with dasatinib, then lysates were immunoblotted with anti‐FAK, anti‐FAK Y397, anti‐FAK Y925, anti‐PY416 Src, anti‐Src and anti‐actin. FAK was immunoprecipitated and then immunoblotting performed using anti‐Src, anti‐FAK or anti‐Ret. (B) Cells were treated with dasatinib, then fixed and stained with anti‐Ret (green), anti‐paxillin (red) and DAPI (blue). Solid arrows indicate co‐localization in adhesions. Quantification of percentage of cells that contained Ret in intracellular puncta is shown. Data are presented as mean±s.d. and the significance calculated using a Student's t‐test (n=3). (C) FAK−/− cells treated with dasatinib were fixed and stained with anti‐Ret (green), anti‐paxillin (red) and DAPI (blue). Solid arrows indicate co‐localization in adhesions, while broken arrows show lack of co‐localization in puncta. Scale bars, 20 μM. Quantification of percentage of cells that contained Ret in intracellular puncta is shown. Data are presented as mean±s.d. and the significance calculated using a Student's t‐test (n=3). (D) Lysates from untreated and dasatinib‐treated FAK−/− cells were immunoblotted with anti‐Ret, anti‐PY416 Src, anti‐Src and anti‐actin. Ret was also immunoprecipitated from cells and then immunoblotted using anti‐Src and anti‐Ret. DAPI, 4,6‐diamidino‐2‐phenylindole; FAK, focal adhesion kinase; Ret, rearranged during transfection.
Figure 4A. Quantitative qRT-PCR of transcription factors during MΦ development. Data are normalized to β-actin and GAPDH and expressed as ratio of the mRNA expression compared to embryonic microglia and show mean ± s.e.m. Two to three mice per group were analyzed. B. Irf8-VENUS expression on A1 and A2 progenitor cells isolated from the E 9.0 yolk sac of Irf8-Venus PAC reporter mice. Representative blots obtained from 17 individual embryos are shown. Mean fluorescence intensity (MFI) of Irf8-VENUS signal in A1 and A2 progenitor cells. Each symbol represents one mouse. Data are from one representative experiment out of two and represented as mean ± s.e.m. C. Flow cytometry measurement of the Irf8-VENUS signal in brain, liver and kidneyMΦ from E 14.0, postnatal (P1) and adult (P42) Irf8-VENUS PAC reporter mice. Representative density blots are shown (left) and quantification thereof (right). Each symbol represents one mouse. Data are from one representative experiment and displayed as mean ± s.e.m. D. Representative immunofluorescence microscopy images of Venus (green) and Iba-1+ (red) MΦ from brain, liver and kidney of adult Irf8-VENUS PAC reporter mice. Stars indicate the cell shown in the insert. Scale bar = 10 µm.
TGF-β1 and Sema3A are downregulated in vivo by increasing VEGF doses.Figure 2Muscles were harvested 7 days after implantation of V Low, V Med, and V High clones or control cells (Ctrl).Gene expression of Pdgfb,Ang1,Ang2,Tgfb1, and Sema3a was quantified by qRT-PCR and expressed as fold-change versus control muscles. Data represent individual values, with mean ± SEM (n = 3 or 4, as indicated); *P < 0.05 and **P < 0.01 by one-way ANOVA with Bonferroni multiple comparisons test, after data normalization by logarithmic transformation. TGF-β1: V Low versus V Med P = 0.0047; V Low versus V High P = 0.0379; Sema3A: V Low versus V High P = 0.0042; V Med versus V High P = 0.0179. Muscles were harvested 7 days after implantation of V Low, V Med, and V High clones or control cells (Ctrl). Immunohistochemistry for Sema3A on frozen muscle sections confirmed a decreasing protein expression in the areas implanted with myoblasts producing increasing VEGF doses (n = 4). The right panels represent higher-magnification views of the left panels, which encompass the majority of the implantation sites. Scale bar = 50 μm. Muscles were harvested 7 days after implantation of V Low, V Med, and V High clones or control cells (Ctrl).In situ hybridization for Sema3A (green) and CD31 (red) mRNA, with nuclei staining by DAPI, on frozen muscle sections. The three lower panels represent a higher-magnification of the area in the white square in the top panel, as a merged image and as individual channels: arrows indicate cells expressing both transcripts (yellow), only Sema3A (green), or only CD31 (red). Scale bar = 30 μm.Quantification of the percentage of endothelial cells (CD31+) that express Sema3A (left group) and of Sema3A-expressing cells that are endothelial (CD31+, right group). Data represent the mean ± SEM of 12 individual fields of view from three independent muscles (15-94 nuclei/image, 642 total nuclei). No statistics was applied, as data represent complementary sets.
Figure 3A, Cumulative probability plots of mEPSCs amplitude (500 events per cell) and representative examples of mEPSCs (left panel) recorded from cultured cortical control neurons after 48 hours of 1 µM TTX or Veh exposure (Ctrl/Veh n=16 and Ctrl/TTX n=17). B, A linear fit of Ctrl/TTX and Ctrl/Veh amplitudes. C, The degrees of overlap between Ctrl/TTX and Ctrl/Veh data were assessed using various scaling factors. The largest nonsignificant p-value was obtained with 1.47 scaling factor. D, Cumulative probability plots of the mEPSCs amplitude of Ctrl/Veh, Ctrl/TTX and Ctrl/TTX divided by scaling factor 1.47, which yielded the maximum overlap with Ctrl/Veh data. Cumulative probability plots of mEPSCs amplitude (500 events per cell) and representative examples of mEPSCs (left panel) recorded from cultured cortical neurons after 48 hours of 1 µM TTX or Veh exposure (E) Fxr1overexpressing neurons (Fxr1/Veh n=10 and Fxr1/TTX n=8) Cumulative probability plots of mEPSCs amplitude (500 events per cell) and representative examples of mEPSCs (left panel) recorded from cultured cortical neurons after 48 hours of 1 µM TTX or Veh exposure (F) Gsk3 KO neurons (Gsk3KO/Veh n=10 and Gsk3KO/TTX n=11) Cumulative probability plots of mEPSCs amplitude (500 events per cell) and representative examples of mEPSCs (left panel) recorded from cultured cortical neurons after 48 hours of 1 µM TTX or Veh exposure (G) Fxr1 KO neurons (Fxr1KO/Veh n=16 and Fxr1KO/TTX n=8). H, A linear fit of Fxr1 KO/Veh and Ctrl/Veh amplitudes. I, The degrees of overlap between Fxr1 KO/Veh and Ctrl/Veh data were assessed using various scaling factors. The largest non-significant p-value was obtained with 1.27 scaling factor. J, Cumulative probability plots of the mEPSCs amplitude of Ctrl/Veh, Fxr1 KO/Veh and Fxr1 KO/Veh divided by scaling factor 1.27, which yielded the maximum overlap with Ctrl/Veh data. K, Cumulative probability plots of mEPSCs amplitude (500 events per cell) and representative examples of mEPSCs (left panel) recorded from cultured cortical Gsk3 and Fxr1 KO neurons after 48 hours of 1 µM TTX or Veh exposure (Gsk3/Fxr1KO/Veh n=8 and Gsk3/Fxr1KO/TTX n=11). L, A linear fit of Gsk3/Fxr1 KO/Veh and Ctrl/Veh amplitudes. M, The degrees of overlap between Gsk3/Fxr1 KO/Veh and Ctrl/Veh data were assessed using various scaling factors. The largest non-significant p-value was obtained with 1.68 scaling factor. N, Cumulative probability plots of the mEPSCs amplitude of Ctrl/Veh, Gsk3/Fxr1 KO/Veh and Gsk3/Fxr1 KO/Veh divided by scaling factor 1.68, which yielded the maximum overlap with Ctrl/Veh data. O, mEPSC mean amplitude of cultured cortical neurons after 48 hours of 1 µM TTX or Veh exposure. One way Anova with Bonferroni's Multiple Comparison Test *p<0.05, **p<0.01, ***p<0.001. P, mEPSC frequency of cultured cortical neurons after 48 hours of 1 µM TTX or Veh exposure.
Fig.Figure 5Space filling and cartoon representation of heterodimer structures in the Ca2+-free (A) and Ca2+-bound states (B). Both structures are built based on the MICU1 homodimer structure (PDB code: 4NSC) in the apo state. Ca2+-free and Ca2+-bound MICU1 were colored in wheat and gray, respectively. Ca2+-free and Ca2+-bound MICU2 were colored in purple and yellow, respectively. Calcium ions are shown as blue and red spheres in MICU1 and MICU2, respectively. Comparison of MICU1-MICU2 structures in the apo (wheat and purple) and Ca2+-bound states (gray and yellow). The conformational differences are highlighted by boxes (black dotted lines). The black arrows show the direction of the conformational change from apo to Ca2+-bound state. Proposed model for mitochondrial Ca2+ uptake regulated by MICU1-MICU2. In the apo state, MICU1-MICU2 form a loose complex which inhibits the uniporter. Upon Ca2+ binding, the conformational changes of the EF-hands promote a transition to a more compact state, which activates Ca2+ uptake.
Hypothetical model for the regulation of CMA by PPCA.Figure 8Hypothetical model for the regulation of CMA by PPCA. Under normal nutritional conditions (FED, left), binding of PPCA to the lysosomal membrane might trigger cleavage of lamp2a by an unidentified metalloprotease (at the transmembrane region) (1) and by PPCA (at the intersection between the transmembrane and luminal regions) (see inset). That cleavage releases a truncated lamp2a into the matrix (2), where it is completely degraded by the lysosomal proteases (3). During starvation (right), PPCA dissociates from the lysosomal membrane (1) so that lamp2a is not longer degraded and remains in the membrane, accessible for substrate binding (2) and uptake into the lysosomes (3).
Influence of bortezomib on cell viability and proliferation of chondrosarcoma cells.Fig 1A,C) After 24, 48, and 72 h incubation bortezomib inhibited cell growth in a concentration dependent manner. Untreated cells were measured as controls (n = 12, mean ± S.D.). B,D) Dynamic proliferation curves for Cal-78 and SW-1353 cells in the presence of 0 (red), 2.5 nM (green), 5 nM (blue), and 10 nM (purple) bortezomib. Data shown are representatives from three independent experiments (n = 3, measured in biological quadruplicates).
Figure 7A) Middle. Graphic reconstruction of an anteroposterior sequence of coronal sections of mouse striatum, reporting the coordinates relative to Bregma. HSV-LacZ (left, grey) and HSV-RGS9-2 (right, red) were injected into the left and right striatum, respectively, and mice sacrificed after 3-5 days. Left. Serial confocal images showing the extent of viral infection, as indicated by the robust dark β-gal staining. Right. The RGS9-2 immunolabelling (red) is particularly intense near the injection site, indicating enhanced protein expression with respect to the endogenous level. Bar = 200 µm B) A representative WB shows correlated viral-mediated increases in RGS9-2 and DRD2 protein levels in Tor1a+/- dorsal striata (lanes 4 and 5) . while in a sample where RGS9-2 level was not increased by viral infection (lane 6) DRD2 level remained low as in the sham-injected striatum (lane 7). C,D) The summary plots report mean ± SEM values of the ratio of protein vs. loading control intensity level measured from the injected (HSV/LV) or sham contralateral Tor1a+/- (+/-) striata, normalized to the Tor1a+/+ (+/+) samples of the same experiment. C) RGS9-2: Tor1a+/+ (N=10); Tor1a+/- injected (N=7) vs. contralateral (N=7): paired t test **P=0.0011; D) DRD2: Tor1a+/+ (N=9); Tor1a+/- injected (N=6) vs. contralateral (N=6): paired t test *P=0.0380
Figure 6Flow cytometric assay of the SARS-CoV-2 RBD and its mutants (K417R, Q498H and K417R-Q498H) binding to human, mouse, rat and European hedgehog ACE2s expressed on the cell surface of BHK21 cells. The SARS-CoV-2 NTD was used as negative control. The mFc-tagged ACE2s from human, mouse, rat and European hedgehog were captured by anti-mIgG Fc antibodies immobilized on the CM5 chip, and sequentially tested the binding with serially diluted SARS-CoV-2 RBD and its mutants (K417R, Q498H and K417R-Q498H). The binding affinities of the four ACE2s to the SARS-CoV-2 RBD and its mutants are shown. Mean ± SD represents the mean and standard deviation of three independent experiments.
Figure 4A. WISH for runx1/c-mybin 36 hpf evi1 morphant (left) and evi1 morphant transgenic Tg(5xUAS-E1b:6xMYC-notch1a;-1.5hsp70l:Gal4) embryos (right) with global heat-shock (HS) induction at 20 hpf.B. WISH for runx1/c-myb in 36 hpf evi1 morphant (left) and evi1 morphant transgenic Tg(5xUAS-E1b:6xMYC-notch1a;cdh5:gal4ff) embryos with endothelial-specific NICD induction (right).C. WISH for runx1/c-myb in 36 hpf evi1 morphant (left) and evi1 morphant transgenic Tg(Hsp70l:vegfaa;myl7:eGFP) embryos (right) with global heat-shock induction at 20 hpf.D. WISH for runx1/c-mybin 32 hpf evi1 MO injected embryos (left) and embryos injected with both evi1 MO and capped gata2 mRNA (right).E. Quantitation of all rescue experiments. Shown are the percentages of embryos displaying normal or changed runx1/c-myb expression. A Fisher´s exact test was applied to calculate statistical significance. (n.s., not significant; p < 0.001 (***); p<0.01 (**)).
PAT1‐GFP is present in P‐bodies after PAMP treatmentConfocal microscopy with root elongation zones.Figure 5Confocalmicroscopy with root elongation zones. Five‐day‐old Col‐0/PAT1‐GFP seedlings were treated with 1 μM flg22 on glass slides for 20 min (second panel) followed by treatment with 100 μg/ml cycloheximide for 60 min (bottom panel). The scale bar corresponds to 10 μm. Confocalmicroscopy with 5‐day‐old VCS‐GFP seedlings treated as in (A). The scale bar corresponds to 10 μm. PAT1 protein is induced by PAMP treatment. Immunoblot detection of equal amounts of protein from Col‐0 seedlings at times in minutes as indicated following vacuum infiltration with 1 μM flg22 or water (180 min after infiltration). Immunoblots were probed with anti‐PAT1 antibodies. Negative control pat1‐1 was loaded for comparison. Coomassie brilliant blue protein loading control is indicated by CBB. Anti‐GFP immunoblotting of proteins extracted from Col‐0/PAT1‐GFP seedlings treated over a time course with flg22 (top panel) and Coomassie brilliant blue (CBB)‐stained PVDF loading control is shown (bottom panel). No PAT1‐GFP was detected in negative control Col‐0.
Figure 4A. WT, and two independent clones each of 53bp1−/−, Shld2−/−/−, and Shld3−/− CH12 cells were stimulated with CIT and analyzed by flow cytometry for IgM and IgA expression. The Iglo population were reduced after 7 days in culture. Values are mean ± SD from 3 biological replicates; * P ≤ 0.05, ** P ≤ 0.01, **** P ≤ 0.0001, two-way ANOVA with post hoc Dunnett's test. B. WT, and two independent clones each of 53bp1−/−, Shld2−/−/−, and Shld3−/− CH12 clones were stimulated with CIT for 3 days. The IgM+, IgA+, and Iglo populations were sorted and reanalyzed for expression of IgM and IgA 5 days post sort as well as 12 days post sort (see Figure EV4B). Shown on bar graphs are sorted IgM+, IgA+, and Iglo populations (each column, 1 technical replicate) from WT and mutant CH12 clones, and the percent of cells expressing IgM, IgA, or low for both isotypes (Iglo) after 5 days of culture post-sort. A representative flow plot of 53bp1−/− CH12 3 days post CIT treatment is shown.
Figure 2(A) Immunostaining using anti-TUJ1 antibody of ES-cell-derived neurons following infection with either non-targeting shRNA (sh-NT), or two different shRNAs targeting Reno1. Scale bar: 200µm. (B) Quantification of cell numbers and neurite lengths of ten images of non-overlapping fields for each shRNA. Mean ± SEM is shown, ** P<0.01 (unpaired two sample t-test). (C) Mean expression levels of genes which were down- or upregulated following knockdown (KD) of Reno1 or lnc-Nr2f1 during an eight time point neuronal differentiation and maturation of mouse ES cells: Neurons1 corresponds to day in vitro (DIV) 1; Neurons2 - DIV7; Neurons3 - DIV16; Neurons4 - DIV21; and Neurons5 - DIV28 (Hubbard et al., 2013). (D) Transcriptional response of cell cycle and neuron-related genes. Heatmaps show log2FC of cells infected with shRNAs targeting Reno1 or lnc-Nr2f1 compared to cells infected with sh-NT, following eight days of neuronal differentiation, and of day eight of differentiation compared to ES cells during differentiation of WT cells.
Figure 6(A) UMUC3-PLKO and UMUC3-shcircFNTA cells were used to establish a subcutaneous nude mouse xenograft model, and cisplatin (1 mg/kg) was intraperitoneally administered. Images of tumors are shown after mice were sacrificed (N= 6). (B) Compared with the vector (PLKO) group, the tumor growth rate was significantly inhibited in shcircFNTA nude mice. (C) Tumor weights also decreased in the shcircFNTA group. (D-E) The qRT-PCR results using mouse tumor tissues revealed that circFNTA expression was significantly reduced (D), and the expression of its host gene FNTA was correspondently decreased in the shcircFNTA group as compared to the PLKO group (E). (F) Representative immunohistochemistry (IHC) images detecting FNTA, p-ERK1/2 and p-MEK1/2 in PLKO tumurs as compared to shcircFNTA xenografted tumors. Scale bars: 100 μm (upper panels) and 25 μm (lower panels). (G) Quantification of relative IHC staining intensity revealed that targeting circFNTA led to decreased expression of FNTA, p-ERK1/2 and p-MEK1/2. (H) The tumor tail vein injection model was established to observe lung metastasis (N=6). Representative images of lung metastasis foci in gross and microscopic HE examination. Scale bars: 400 μm (upper panels) and 100 μm (lower panels). (I) Knocking down circFNTA significantly decreased metastatic foci numbers as compared to the PLKO group. (J) Targeting circFNTA significantly reduced lung metastatic foci as compared to the PLKO group.
Increasing antigen dose does not improve antigen-specific CD8+ T cell responses to an EEF vacuolar membrane protein.Figure 5(A) Schematic overview of experimental design. C57BL/6 mice (n=4 per group) received an intravenous dose of 8,000 γ-radiation attenuated CSPSIINFEKL or UIS4SIINFEKL sporozoites or 64,000 γ-radiation attenuated UIS4SIINFEKL sporozoites. Spleens and livers were harvested at day 12 and IFN-γ-secreting lymphocytes following restimulation ex vivo with SIINFEKL peptide were quantified. (B) Flow cytometry plots show representative CD8+ T cells co-stained with IFN-γ and CD11a. (C) The upper panel of bar charts show the percentage of CD11ahi IFN-γ+ CD8+ T cells and the lower panel the absolute cell counts. Bar charts show mean values (±SEM) from representative experiments (***, p<0.001; one-way ANOVA with Tukey's multiple comparison test). See Appendix Table S4 for exact p-values.
Validation of GluK2/3 reduction on the surface of SEZ6KO neurons by immunoblot.Figure 2A) SEZ6KO and WT neurons were biotinylated with Sulfo-NHS-Biotin and surface proteins were enriched by streptavidin bead pull-down. The GluK2/3 antibody cannot discriminate the subunits 2 and 3 (Lerma & Marques, 2013), therefore the band is commonly indicated with the labeling GluK2/3. GluK2/3, GluA2 and GluN2B surface levels were quantified, normalized to SEZ6L2 surface levels (negative control) in the same sample (GluK2/3 / SEZ6L2) and divided by the WT levels, with the ratio for WT being set to 1.0 (plot shows mean ± S.E.M., at least 10 replicates in 4 independent biological experiments, Mann-Whitney ****p-value<0.005). B) mRNA levels of GluA1, GluA2, GluK2, GluK3 and GluN2B were quantified in SEZ6KO neurons, normalized to GAPDH and β-actin mRNA in the same sample and compared to the mRNA levels in WT neurons. No difference in GluK2/3 mRNA was detected in SEZ6KO neurons compared to WT (plot shows mean ± SD, 6 replicates in 2 independent biological experiments, multiple t-test was used). C) Editing of GluK2 mRNA was tested in SEZ6KO and WT neurons. The editing value was calculated as (intensity of 376 [edited] / intensity of [376 (edited) + 269 (unedited)]) × 100 and normalized to the band at 76 bp. No difference was detected in SEZ6KO neurons compared to WT (plot shows mean ± S.E.M., 6 replicates in 2 independent biological experiments, Mann-Whitney test was used).
C-circles interact with SAGA/TREX2.Figure 4A Senescence curves of two est2∆ and two est2∆ ADA2-Myc clones used to generate type II survivors for Co-IP-RCA. This experiment is representative out of the 4 independent experiments performed during this study. B Telomere length and recombination were analyzed by TG1-3-probed Southern blot of XhoI-digested DNA prepared from samples of the replicative senescence. C Detection of the C-circles co-immunoprecipitated with Ada2-Myc in type II survivors. The DNA co-immunoprecipitated with Ada2-Myc was amplified by RCA, loaded on a dot blot and detected with a telomeric TG1-3 probe. The numbers above the blot refer to clones in (A) and correspond to type II survivors produced in independent cultures. The lower panel shows the efficiency of Ada2-Myc IP analyzed by western blot with anti-Myc antibody. D Quantification of the dot blot signals in Ada2-Myc co-IP. Dot signals were quantified as in Fig 3D. The graph shows means and s.e.m. from n= 3 independent co-IP experiments each performed with 3-4 independent clones per genotype. The p-value is obtained for the Mann-Whitney two-tailed test (***p= 0. 00000005).
Hsp70 modulation of human monocytes is dependent on the SIGLEC14 genotypeTNFα secretion from SIGLEC14+/+ monocytes was measured after exposure to LPS or Hsp70 in the presence or absence of endotoxin‐neutralizing compound polymyxin B.Figure 7TNFα secretion from SIGLEC14+/+ monocytes was measured after exposure to LPS or Hsp70 in the presence or absence of endotoxin‐neutralizing compound polymyxin B. Hsp70 was able to induce secretion of TNFα even in the presence of polymyxin B, suggesting a mechanism independent of endotoxin contaminants. DnaK, the E. coli homolog of human Hsp70, did not induce significant secretion of TNFα above background levels. TNFα secretion from SIGLEC14+/+ monocytes by Hsp70 was reduced when the cells were exposed to an anti‐Siglec‐5/Siglec‐14 blocking antibody. Human monocytes isolated from SIGLEC14+/+, SIGLEC14+/−, and SIGLEC14−/− individuals were exposed to varying concentrations of Hsp70, and TNFα secretion was evaluated. The amount of TNFα secreted was normalized to the respective cells' response to 1 ng/ml LPS to minimize the impact of other possible polymorphisms and factors that contribute to variation between individuals. TNFα secretion from SIGLEC14−/− monocytes stimulated with 15 ng/ml LPS and various concentrations of Hsp70. TNFα secretion was reduced with increasing concentrations of Hsp70.