EMBO/sd-character-level-ner huggingface dataset:
a–c) Retinal flat mounts of Pde6b−/− mutants at PW7 stained for LacZ to detect cones (see Methods and Supplementary Fig.figf8(a-c) Retinal flat mounts of Pde6b−/− mutants at PW7 stained for LacZ to detect cones (see Methods and Supplementary Fig. 9). An example of untreated control is shown in a, an example of a mouse injected with streptozotocin is shown in b and an example of a mouse injected daily with insulin is shown in c. (d) Quantification of cone survival after four weeks of treatment. Data represents an average of at least eight retinae. The y axis represents the percentage of the cone surface area versus the surface area of entire retina (see Supplementary Figs. 8 and 10). (e, f) Measurements of blood glucose levels (e) and body weight (f) performed weekly over the time span of the experiment. (g, h) Immunofluorescence microscopy staining on retinal flat mounts for HIF-1α (red) and PNA (green) in untreated control Pde6b−/− (g) and Pde6b−/− mice treated for 4 weeks with insulin (h). DAPI is shown in blue. Error bars in d-f represent s.d.
Comparison of simultaneous 5-AzadC + SAHA combined treatment with its corresponding sequential treatment in ex vivo cultures of CD8+-depleted PBMCs isolated from aviremic cART-treated HIV+ patients.Figure 7A Reactivation status of patient cell cultures presenting no viral reactivation in mock condition isolated from aviremic cART-treated HIV+ patients (shown in Appendix Table S2).B Reactivation status of patient cell cultures presenting no viral reactivation in mock condition isolated from aviremic cART-treated HIV+ patients (shown in Table 2).Data information: The results were reported as the actual HIV RNA copy numbers/ml or as an estimated value calculated as 50% of the smallest value when HIV RNA was not detected in order to assign a log value. Means are represented by a line.
Figure 2(A) Purified MAC (denoted as C5b6MAC) can be formed by mixing preassembled C5b6 complexes with C7, C8 and C9. (B) Lysis of human erythrocytes after exposure to a concentration range of preassembled C5b6 in the presence of 100 nM C7. After washing, erythrocytes were exposed to 20 nM C8 and 100 nM C9 for 30 minutes after which the OD405 nm of the supernatant was measured. (B-E) Data represent mean ± SD of 3 independent experiments. C) Bacterial viability of three Gram-negative strains after exposure to buffer, 10% human serum or C5b6MAC. Buffer and serum conditions are the same as Fig. 1E. (D) Permeabilization of the outer, but not inner membrane of perimCherry/cytoGFP E. coli cells exposed to C5b6MAC (different concentrations of C5b6 with fixed concentrations of C7-C9). (B-E) Data represent mean ± SD of 3 independent experiments. E) Inner membrane damage of three Gram-negative strains exposed to buffer, 10% serum or C5b6MAC. Buffer and serum conditions are the same as Fig. 1D.
Figure 7A The levels of ERp57 protein in the muscle were monitored by Western blot. ERp57WT (n = 4), ERp57Nes+/- (n = 5) and ERp57Nes-/- (n = 4) mice. HSP90 was determined as loading control. B Tibialis anterior muscles from ERp57WT (n = 5), ERp57Nes+/- (n = 5), and ERp57Nes-/- (n = 4) mice were dissected and cryosectioned (20 µm) for the analysis of several parameters of muscle physiology. Upper panel: hyaluronic acid (HA) staining was performed to assess the gross morphology of the muscle. Middle panel: Bismarck brown staining to monitor skeletal muscles fibrosis. Lower panel: Cryosections were then double stained with wheat germ agglutinin (WGA) to visualize plasmamembrane and nuclei. The absence of central nuclei in all genotypes suggests that ERp57 deficiency does not affect degeneration/regeneration cycles. Images are representative of each genotype and where acquired in the same anatomical region of the TA muscles. C Tibialis anterior cryosections (20 µm) from ERp57WT, ERp57Nes+/-, and ERp57Nes-/- mice were stained for NADH-TR activity to identify oxidative myofibers (upper panel). Representative micrographs of transversal cryosections show variable proportions of light, intermediate and dark positive staining in the different genotypes: (middle panel) NADH-TR positive (slow-twitch) and negative (fast-twitch) myofibers were quantified as percentage of total for every genotype (lower panel) The mean diameter of slow- and fast-twitch muscle fibers was evaluated at the light microscope using the ImageJ software. Data represent the average ± SEM of >300 fibers per animal.Scale bars represent 50 m in B and C.
Figure 3A In vitro Ub nucleophile discharge assays for U7BR/Ubc7 with indicated Hrd1 (left) and Doa10 (right) variants. E2 stimulation is shown as the ratio of rates derived from RING-catalyzed reactions and the "no E3" control (see Fig. EV2C-D). Values are reported as means ± standard deviation (n = 3). Significances for pairwise comparisons were determined by One-way ANOVA Test; * p < 0.05. For clarity only significances related to the "no E3" control are shown. In addition, reactions for Hrd1(400R) and Doa10(94R) are significantly faster than all other reactions and the reaction for Doa10(94H) is significantly faster than that for Doa10(94E). The wild-type E3s are identified by black frames for each set. B In vitro Ub chain formation assay by Ubc7 with indicated Hrd1 (left) and Doa10 (right) variants in the presence of indicated Cue1 variants. Rates for reactions of mono-Ub to di-Ub and di-Ub to tri-Ub with fluorescently-labeled Ub are shown on a logarithmic scale. Values are reported as means ± standard deviation (n = 3). Significances for pairwise comparisons were determined by One-way ANOVA Test; * p < 0.05. For clarity only significances related to the "no E3" control of given reaction set are shown. C Steps of ERAD protein degradation with associated rate constants; S = ER protein; S* = misfolded protein, i.e. ERAD substrate; kobs = observed rate of protein degradation in assays; kUb1 = rate of the priming reaction; kUb2 = rate of the first elongation step, i.e. mono-Ub to di-Ub; kUb3 - kUbn = rates of subsequent elongation steps; k0 - kj = rates for other steps of ERAD; the black box identifies steps of substrate poly-ubiquitylation with priming highlighted by the green box and chain elongation by the yellow box. The light yellow background highlights the mono-Ub to di-Ub reactions. D Protein degradation in indicated yeast strains monitored by pulse-chase experiments for the Hrd1 model substrate PrA*-3xHA (left) and by CHX decay assays for the Doa10 model substrate Deg1-eGFP2 (right). Values for each time point are reported as means ± standard deviation (n = 4 for PrA*-3xHA and n = 3 for Deg1-eGFP2).
Figure 6Patient-derived and control fibroblasts were used. (A) FLAG-tagged tandem ubiquitin binding entity (TUBE) assay was performed to pull down linear ubiquitin linkages upon stimulation with 500ng/ml TNF. (B) The TNFR1-SC was isolated using 500ng/ml TAP-TNF. (C) Cells were stimulated with 100ng/ml TNF for the indicated times and analyzed by western blot. One representative (A - C) of three independent experiments is shown. Patient-derived and control fibroblasts were used. (D) Cells were stimulated with TNF for 24 hours and IL-6 was determined by ELISA. Data are presented as mean ± SD of 3 individual experiments performed in two technical replicates; *, P = 0.014; **, P = 0.007; multiple t-test corrected for multiple comparisons using the Holm-Sidak method. (E) B cells were incubated with 50µg/ml cycloheximide (CHX) for the indicated times and subjected to analysis by western blot. One representative (left panel) out of 4 individual experiments analyzed by densitometry (right panel) is shown. Data are presented as mean ± SD. *, P = 0.02, unpaired t-test. (F) Patient-derived and control B cells were cultivated for 24 hours and TNF was determined by ELISA. Data are presented as mean ± SD of 3 individual experiments performed in two technical replicates; *, P = 0.028; unpaired t-test. (G) Fibroblasts were incubated with 50µg/ml cycloheximide (CHX) or DMSO and stimulated with TNF as indicated. Viability was measured after 24 hours. Data are presented as mean ± SD of 5 individual experiments performed in three technical replicates; ***, P = 0.0008, mixed-effects analysis with Tukey's multiple comparisons test. (H) Intracellular TNF in CD11b-positive cells from five healthy controls, patient (before anti-TNF treatment), mother and father was determined by FACS.
Figure 3A. Quantification of the amount of ciliated cells in primary embryonic fibroblasts from KV10.1 knockout mice. Actively growing cultures, cultures after 24 h serum starvation, and four hours after subsequent serum reintroduction were stained for the presence of cilia using anti-acetylated α-tubulin. An increase in frequency of cilia in the knockout cells was evident under all conditions, but especially marked in the ciliary resorption test (4h after serum reintroduction), indicating an influence of KV10.1 expression on deciliation. B. Exogenous expression of human KV10.1 reverted the phenotype in MEFs. Cells obtained from three animals were transfected with KV10.1 or empty vector (mock), serum starved for 48 h and cilia were revealed by immunocytochemistry using anti-acetylated α-tubulin in the indicated number of images. KV10.1 expression decreased the frequency of ciliated cells. C. MEFs from knockout mice did not show primary cilia when transfected with KV10.1. Cells were co-transfected with human KV10.1 and mVenus, serum starved for 48 hours and then fixed and stained for cilia (AcTub). Venus-positive cells did not show cilia. Scale bar: 25 µm. D. In wild type MEFs, transfection with KV10.1 reduced the presence of primary cilia. When a gain-of-function mutant (L352V) that originates developmental defects in human patients was transfected, the frequency of cells with cilia was further reduced. E. Sensitivity to SHH signaling was significantly increased in primary fibroblasts from KV10.1 knockout mice, in agreement with the higher abundance of primary cilia. Cells were serum starved and SHH was stimulated after 24 hours by addition of culture supernatant of SHH-expressing HEK293 cells. Expression of mRNA for Gli1 was used as reporter of SHH activation by qRT-PCR.
Control of Wnt4 expression A Epifluorescence stereo micrograph of inguinal mammary gland from a 5‐day‐old Wnt4::Cre; mT/mG female (n = 7).Figure 2A Epifluorescence stereo microscopy of inguinal mammary gland from a 5‐day‐old Wnt4::Cre; mT/mG female (n = 7). Scale bars: 0.5 mm and 0.1 mm (inset). B, C Histology sections of mammary glands from a 5‐day‐old (B; n = 7) and a 10‐day‐old (C; n = 5) Wnt4::Cre; mT/mG female stained by double immunofluorescence microscopy for EGFP (green) and PR (magenta, not detected), counterstained with DAPI (blue). Scale bar: 50 mm. D-F EGFP (green) and PR (magenta) co‐immunofluorescence microscopy counterstained with DAPI (blue) on histology sections from mT/mG; Wnt4::Cre mammary glands at different developmental stages. (D) TEB of a 4‐week‐old female (n = 4); scale bar: 30 μm. (E) Ducts of an 8‐week‐old female (n = 3); scale bar: 100 μm; inset, scale bar: 20 μm. (F) Duct of a female at day 10.5 of pregnancy (n = 3); scale bar: 150 μm; inset, scale bar: 30 μm. G-I EGFP (green), PR (magenta), and p63 (white) triple co‐immunofluorescence microscopy counterstained with DAPI (blue) on histological sections from mT/mG; Wnt4::Cre mammary glands at different developmental stages. (G) Ducts of 5‐day‐old female (n = 3). (H) TEB of a 4‐week‐old female (n = 3). (I) Duct of an 8‐week‐old female (n = 3). Scale bars: 30 μm. J-O Epifluorescence stereo microscopy of mammary glands harvested from 15‐day‐old Wnt4::GFP females either WT (n = 18) (J, M), ERα−/− (n = 4) (K, N), or PR−/− (n = 3) (L, O). dTomato expression (J-L); EGFP expression (M-O) is not abrogated in ERα−/− nor PR−/− epithelia. Arrowheads mark the main duct originating from the nipple. Scale bar: 50 μm. P Scheme of ex vivo hormone stimulation of mammary organoids. Q Bar plots showing relative PR and Wnt4 mRNA expression normalized to CK18 mRNA in mammary organoids from 5 pubertal (6 weeks old) and 3 adult (11 weeks old) mice exposed for 6 h to vehicle (C), 17β‐estradiol (20 nmol) (E2), or R5020 (20 nmol) (P). Bars represent the mean ± SD of 3 independent experiments. R-X Epifluorescence stereo microscopy of contralateral mammary glands that were engrafted with Wnt4::GFP epithelium from 8‐week‐old females, either PRWT (R, T, V, X) or PR−/− (S, U, W). dTomato expression (R, S); EGFP expression (T, U) double epifluorescence (V, W, X) on contralateral engrafted glands 3 weeks after surgery when recipients were 6 weeks old. Representative result from three independent experiments. Arrowheads point to TEBs (V, X) or to origin of growth (W). Scale bar (R-W): 5 mm, (X): 1 mm.
Figure 7(a-f). Pedigrees of PD families with PINK1 mutations and Sanger sequencing conformation. Pedigrees of three families with four PD patients (a, c, e) and corresponding sequencing chromatogram of four PD patients harboring PINK1 mutations (b, d, f) are shown. Red arrows indicate the mutations in each patient. (g, h). Reduction of Drp1S616 phosphorylation in dermal fibroblasts derived from PD patients with PINK1 mutations. Lysates of cells derived from three unrelated normal control individuals (Control, C) and four familial PD patients with PINK1 mutations (PINK1mutant) were immunoblotted with antibodies recognizing either phosphor-Drp1S616 (pS616) or Drp1 protein. β-actin was detected as a loading control (g). A quantitative analysis of phospho-Drp1S616 (pS616/Drp1) is shown (h). Student's test. **p<0.01, ns: no significance. Data was presented as mean ± SEM of three independent experiments. Drp1S616 phosphorylation in dermal fibroblasts derived from sporadic PD patients. Lysates of cells derived from five unrelated normal control individuals (Control, C) and seven unrelated sporadic PD patients (Sporadic PD, SP) were immunoblotted with antibodies recognizing either phospho-Drp1S616 (pS616) or Drp1 protein. β-actin was detected as a loading control (i). Drp1S616 phosphorylation in dermal fibroblasts derived from sporadic PD patients. A quantitative analysis of phospho-Drp1S616 (pS616/Drp1) in sporadic cases is shown (j). Student's test. *p<0.05. Data was presented as mean ± SEM of three independent experiments.
Figure 6A. FoxM1 or/and ICAT plasmids were co-transfected with Axin-2 promoter luciferase and TK-Renilla plasmid into U87 cells. Luciferase activity was measured with the Dural Luciferase reporter assay, and relative luciferase activity was normalized to that of the vector group. Values are mean ± SD for triplicate samples. B. FoxM1 siRNA#1, #2 or control siRNA were co-transfected to U87 cells with Axin-2 promoter reporter. Luciferase activity was measured with the Dural Luciferase reporter assay, and relative luciferase activity was normalized to that of the control siRNA group. Values are mean ± SD for triplicate samples. C. ICAT siRNA or control siRNA were co-transfected into LN229 cells with Axin-2 promoter reporter. Luciferase activity was measured as in (C). D. FoxM1 or/and ICAT plasmids were co-transfected with TOP-Flash reporter and TK-Renilla plasmids into U87 cells. Luciferase activity of TOP-Flash in the cells was measured as in A. Values are mean ± SD for triplicate samples. E. ICAT, FoxM1 or FoxM1-∆FB (FoxM1 mutant lacking the forkhead box domain) plasmid or their combinations were co-transfected with TOP-Flash reporter and TK-Renilla plasmids into LN229 cells. Luciferase activity of TOP-Flash in the cells was measured as in A. Values are mean ± SD for triplicate samples. F. FoxM1 siRNA and/or ICAT siRNA were co-transfected with TOP-Flash reporter and TK-Renilla plasmids into LN229 cells, and the cells were treated with Wnt3a (50 ng/ml) or control PBS. Luciferase activity of TOP-Flash in the cells was measured as in A. Values are mean ± SD for triplicate samples. G. Vector-, β-catenin-NLS-, sh-FoxM1-, FoxM1-shR- and/or ICAT plasmids were co-transfected with TOP-Flash reporter and TK-Renilla plasmids into U87 cells. Luciferase activity of TOP-Flash in the cells was measured as in (A). Values are mean ± SD triplicate samples. H. Levels of Axin-2 and cyclin D1 protein in LN229 cells transfected with FoxM1 siRNA#1 or #2 were determined by Western blotting. I. Levels of Axin-2 and cyclin D1 protein in LN229 cells transfected with ICAT siRNA were determined by Western blotting. J. Levels of Axin-2 and cyclin D1 protein in LN229 cells transfected with FoxM1 or/and ICAT plasmids were determined by Western blotting.
Fig.Figure 1Macrophages produce little to no inflammatory cytokines when incubated with either (a) whole iRBC for 24 h. One-way ANOVA with Tukey test for multiple comparisons was performed to determine statistical significance Each symbol represents the value obtained for cells from an independent donor in an independent experiment. (a) n=4; Asterisks indicate significance (*p<0.05, **p<0.01 and ***p<0.001) when values were compared with RBC Macrophages produce little to no inflammatory cytokines when incubated with either (b) fresh iRBCL for 24 h. One-way ANOVA with Tukey test for multiple comparisons was performed to determine statistical significance Each symbol represents the value obtained for cells from an independent donor in an independent experiment. (b) n=5 Asterisks indicate significance (*p<0.05, **p<0.01 and ***p<0.001) when values were compared with RBC (c) Macrophages were pre-incubated with IFNγ (1000U/ml) for 18 h before iRBCL stimulation. One-way ANOVA with Tukey test for multiple comparisons was performed to determine statistical significance Each symbol represents the value obtained for cells from an independent donor in an independent experiment. (c) n=3 Asterisks indicate significance (*p<0.05, **p<0.01 and ***p<0.001) when values were compared with RBC (d) Phase-contrast microscopy of macrophages 24 h after the addition of RBC, iRBC or LPS. Dark hemozoin crystals can be observed within macrophages. Bar is 30 µm. (e) Macrophages were incubated with iRBCL 24 h before addition of LPS. Paired t-test was performed to determine statistical significance. Each symbol represents the value obtained for cells from an independent donor in an independent experiment. (e) n=3. Asterisks indicate significance (*p<0.05, **p<0.01 and ***p<0.001) when values were compared RBCL (e).
Figure 4A. Deconvolved inverted fluorescent micrographs of BiFC signal of Chm7-VC with either Heh1-VN or Heh2-VN. Cell borders are outlined. Scale bar is 5 μm. B. Deconvolved inverted fluorescent micrographs of yeast strains expressing Heh1-VN and Snf7-VC with either Chm7-mCherry or Mps3-mCherry and merge of both channels. Scale bar is 5 μm. C. Deconvolved inverted fluorescent micrographs of BiFC signal of the indicated VN and VC fusions in the indicated strains. Cell borders outlined. Scale bar is 5 μm. D,E. Plots of the percentage of cells with BiFC signal of the indicated VN VC pairs in the indicated null backgrounds. Data are from three independent replicates where > 225 cells per strain per replicate were quantified. Error bars are SD from the mean of each replicate. p values from un-paired student's T-test where ns is p > 0.05. *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001. F. Deconvolved inverted fluorescence micrographs of Heh1-VN Snf7-VC BiFC signal in a chm7Δ strain with either an empty plasmid ("-"; pRS416) or one expressing CHM7 (pRS416-HA-CHM7). Cell borders outlined. Scale bar is 5 μm.G. Plot of the percentage of cells with BiFC signal from F. Error bars are SD from the mean of each replicate. p values from un-paired student's T-test where ns is p > 0.05. ***, p ≤ 0.001.
Figure 2A Histological analysis of RFP+ cells in Siglechdtr/dtr:Ccr2RFP/+ mice. Lumber spinal cord after EAE induction, and hippocampal CA1 2 or 4 days after PBS or DT injection were analyzed. Iba1 immunoreactivity (green) and RFP fluorescence (red) are shown. Images were acquired using the same laser power and sensitivity, and image processing was the same for all RFP signals (red). Scale bar, 50 µm. B Flow cytometric analysis of RFP+Ly6Chigh inflammatory monocytes in peripheral blood of Siglechdtr/dtr:Ccr2RFP/+ mice 2 days after PBS or DT administration. Representative data and a quantification graph (n = 5 animals per group, two-tailed unpaired Student's t-test) are shown. C Immunohistochemical localization of CD206+ perivascular macrophages (arrows) in hippocampal CA1 of Siglechdtr/dtr mice. Sections prepared 2 days after administration of PBS or DT were stained with anti-Iba1 (green), anti-CD206 (red) and anti-CD31 (blue) antibodies. Scale bar, 50 µm. D Number of perivascular macrophages in hippocampal CA1 of Siglechdtr/dtr mice 2 days after PBS or DT administration (n = 5 animals per group, two-tailed unpaired Student's t-test).
NeutrobodyPlex: multiplex competitive binding assay to monitor a neutralizing immune response in patients.Figure 5A Schematic illustration of the NeutrobodyPlex. The displacement of serum-derived neutralizing IgGs binding to SARS-CoV-2 antigens upon addition of bipNb is measured. In presence of neutralizing IgGs, the fluorescent signal from anti-human-IgG-PE is inversely proportional to the applied bipNb concentration. B For the NeutrobodyPlex, antigen-coated beads comprising RBD, S1 or spike were co-incubated with serum samples from 5 patients and a dilution series of NM1267 (1 µM to 6 pM) (n = 1). Mean fluorescent intensities (MFI) derived from antigen-bound IgGs in the presence of bipNb normalized to the MFI values of IgGs in the serum-only samples, illustrated as MFI (% control), are shown. C For two patient derived serum samples (#289, #265), differences in the NM1267-mediated displacement of IgGs binding the three spike-derived antigens (RBD, S1, spike) are shown.
a) 2,3,5-Triphenyltetrazolium chloride (TTC)-stained brain sections from WT and HDC−/− mice showing the infarct area in those receiving saline, the H3R antagonist thioperamide (THIO), the H1R antagonist pyrilamine (PYRI; 10 mg kg−1, i.f3(a) 2,3,5-Triphenyltetrazolium chloride (TTC)-stained brain sections from WT and HDC−/− mice showing the infarct area in those receiving saline, the H3R antagonist thioperamide (THIO), the H1R antagonist pyrilamine (PYRI; 10 mg kg−1, i.p., at reperfusion), the H2R antagonist cimetidine (CIME; 10 mg kg−1, i.p., at reperfusion) and α-FMH (50 mg kg−1, i.p., 2 h before ischemia) after 24 h reperfusion (n=6 per condition; scale bar, 5 mm). (b) Infarct volumes and (c) neurological deficit scores (*P0.05, **P0.01, ***P0.001 with analysis of variances followed by the Bonferroni/Dunn post hoc test). Data are presented as mean±s.e.m.