EMBO/sd-character-level-ner huggingface dataset:
Figure 1A. One-color fluorescence autocorrelation spectroscopy (FCS) and photon counting histogram (PCH) are methods based on fluorescence fluctuation analysis of fluorescence intensity detected at a sub-femtoliter volume. B. FCS analysis yields a fluorescence autocorrelation function (G(τ)), which reports the average number of fluorescence molecule, N (N is inversely proportional to the correlation function amplitude G(0)) , and the mobility parameter of the molecules diffusing through the diffraction-limited volume (τD is the translational diffusion time, and D is the diffusion coefficient). Faster mobility correlates with smaller τD. C. PCH analysis is a reporter for the oligomerization state of fluorescence proteins. It reports the measured photon counts per molecule and the average number of molecules within the observation volume. Diagram illustrates potential oligomerization state of monomer (black), dimer (red), and trimer (blue) from PCH analysis.
Reduction of Nurr1 and Foxa2 in mDA neurons during aging and degeneration.Figure 2A-C Nurr1 and Foxa2 protein levels in mDA neurons decrease in the midbrains of old mice in vivo (A) Nurr1 and Foxa2 protein levels were compared in individual mDA neurons of the midbrains of young (10 weeks) and old mice (18 months) of the same mouse strain (C57BL/6, male). All the midbrain sections were immunofluorescently co-stained with Nurr1/TH (upper) and Foxa2/TH (lower) under identical conditions, and levels of Nurr1 and Foxa2 proteins were determined in individual TH+ mDA neurons by measuring mean fluorescence intensities (MFI) using LAS image analysis (Leica). Dots in the graphs represent the Nurr1 and Foxa2 MFI values of individual TH+ DA neurons in the SN of each animal. The average MFI values (indicated by horizontal lines) of three animals from each group were compared (***P = 5.25E-88 for Nurr1 intensity, 5.57E-40 for Foxa2 intensity, one-way ANOVA followed by Bonferroni post hoc test). A-C Nurr1 and Foxa2protein levels in mDA neurons decrease in vitro after long-term culture (B and C).Nurr1 and Foxa2protein levels were also quantified in cultured mDA neurons over 6-34 days in vitro by Western blotting (B) A-C Nurr1 and Foxa2protein levels in mDA neurons decrease in vitro after long-term culture (B and C).Nurr1 and Foxa2protein levels were also quantified in cultured mDA neurons over 6-34 days in vitro immunocytochemical analysis (C). Significantly lower MFI values on day 24 of culture (D24) compared to D12 at *P = 0.027 (Nurr1), *P = 0.012 (Foxa2), n = 60-70 TH+ cells from two cultures in each group, unpaired Student's t-test. D, E Loss of Nurr1 and Foxa2 expression in mDA neurons after treatment with the neurotoxin MPTP (or MPP+). Mice (10 weeks old) were treated with MPTP for 5 days as described in Materials and Methods. Three days after the last MPTP injection, Nurr1 and Foxa2protein levels in the TH+mDA neurons of the MPTP-treated SN were compared with in the mDA neurons of untreated mice (D) (***P = 5.47E-103 for Nurr1 intensity, 1.53E-111 for Foxa2 intensity, one-way ANOVA followed by Bonferroni post hoc test.). The effects of neurotoxin treatment were also determined in mDA neuron cultures treated with MPP+ (250 μM, 8 h, E). *P = 0.015 (% Nurr1+/TH+ cells), *P = 0.018 (% Foxa2+/TH+ cells), unpaired Student's t-test.
Figure 8A, Timeline of therapeutic treatments undergone by mice intracranially injected with BT463NS. B left, Representative images of ex vivo epi-fluorescent GFP signal of intracranial tumors, generated by injection of BT463NS, and explanted 62 days after IR in the absence (2 Gy × 3 days) or in the presence (combo) of the MET inhibitor JNJ38877605 (50 mg/kg). B right, Quantification of epi-fluorescent GFP signal from tumors represented in (B, left) (n = 4/condition). Vehicle: non-irradiated vehicle-treated tumors. *: one-way ANOVA, P=5.5×10-5. C, Growth curves of tumors, generated by subcutaneous injection of BT308NS, irradiated in the absence (2 Gy × 3 days) or in the presence (combo) of JNJ38877605, which was administered for 15 days as indicated (n = 7/condition). Vehicle: non-irradiated vehicle-treated tumors. *: one-way ANOVA, P=0.0006. D, Survival analysis of mice bearing tumors generated and treated as in C. Black dot: censored mouse. Log-rank (Mantel-Cox) test (combo) vs. (2 Gy × 3 days), P=0.0019. E, Experimental design to measure GSC frequency in tumors treated as in C (JNJ was administered for 10 days). Explanted tumors were dissociated and viable cells were tested by in vitro sphere forming LDA or in vivo single-passage (p1) LDA. F, In vitro LDA (sphere-forming) measuring GSC frequencies as described in E (n = 3/condition). *: 2 test, p<10-5. G, Tumor incidence in the in vivo LDA performed on tumors generated and treated as described in E. H, Histogram showing GSC frequencies measured by in vivo LDA, as described in E. *: 2 test, P=5.47×10-6. I, Table showing GSC frequencies measured by in vivo LDA, represented in H. Data are represented as mean ± SEM or ± CI in F,H,I.
Increased oxidative stress in PD.Figure 1A, B. Oxidative stress biochemical markers (lipid peroxidation, ROS production) (A) and p-ERK expression (B) in tissues from 3-months old PD mice (KO) (n=3) and from wild-type (WT) mice. In all instances indicators of stress were increased in PD, compared to the respective control samples. Data information: In each experiment at least biological triplicates were analyzed for each cell line or tissue sample; each assay was performed at least in duplicate. Data are presented as mean ± SD. Student's t-test was applied. Statistically significant comparison p-values are indicated. C, D. Oxidative stress biochemical markers (lipid peroxidation, ROS production, GSH levels) (C) and p-ERK expression (D) in PD and control (CNTR) fibroblasts (n=6). E, F. Oxidative stress biochemical markers (lipid peroxidation, ROS production, GSH levels) (E) and p-ERK expression (F) in PD and control (CNTR) myoblasts (n=4). In all instances indicators of stress were increased in PD, compared to the respective control samples. Data information: In each experiment at least biological triplicates were analyzed for each cell line or tissue sample; each assay was performed at least in duplicate. Data are presented as mean ± SD. Student's t-test was applied. Statistically significant comparison p-values are indicated. Fi
Figure 4(A) Coomassie-stained protein gels with cell lysates and concentrated culture supernatants of MCF10A-pIND20-HPN cells, with (+DOX) or without (-DOX) hepsin overexpression. M indicates the media only control. R and NR above the gels indicate reducing and non-reducing conditions, respectively. Numbered arrowheads indicate areas of the gel that were analyzed by mass spectrometry; corresponding proteins differently expressed in hepsin overexpressing cells are listed on the left. The image with two lanes on the right is a copy of the indicated area with supernatant under reducing conditions, highlighting the part from which fibronectin was identified. Red boxes indicate the lanes that were analyzed by mass specrtometry (B) Immunoblot analyses of fibronectin expression in concentrated culture supernatant from MCF10A-pIND20-HPN cells with (DOX+) or without (DOX-) hepsin overexpression. Ponceau staining of the western blot is shown as the loading control. (C) Immunoblot analysis of fibronectin, hepsin, and β-tubulin (loading control) in cell lysates from MCF10A-pIND20-HPN cells with (DOX+) or without (DOX-) hepsin overexpression. The graph shows the quantification of fibronectin levels. (D) Silver-stained protein gel with samples from an in vitro protease activity assay with recombinant hepsin and either full length purified plasma fibronectin (upper panel) or the 70 kDa most N-terminal fragment of fibronectin (lower panel). Full-length fibronectin (1μg) or the 70 kDa fibronectin fragment (1μg) were incubated with increasing concentrations of recombinant hepsin. Arrowheads indicate full-length fibronectin, the 70kDa fragment, the 50 kDa cleavage fragment generated by hepsin, and hepsin. The schematic figure shows the full-length fibronectin protein and the 70KDa N-terminal fragment. The red arrow indicates the location of the putative hepsin cleavage site. RGD indicated the RGD-binding domain in fibronectin.
Figure 6A Prolactin signaling pathway plotted in IPA. Red and green colors indicate up- and down-regulated DEGs, respectively, relative to thresholds (|Log2FC |>0.58, Padj<0.1). Orange arrows indicate activation of signaling. B Gene Ontology (GO) molecular function clusters are schematically shown on a two-dimensional scaling plot. Cluster groups 1-11 are marked by colors and explained on panel 6C. Clusters of potentially pro-nociceptive molecular functions are circled. C Molecular function clusters are summarized on the best-match average (BMA) distance heatmap. Heatmap colors correspond to a number of GO terms in each cluster.
Relative and absolute cell numbers of B‐cell populations in induced mb1‐CreERT2;Sykfl/fl mice A, B Flow cytometric analysis of B cells from (A) the BM and (B) the SP of mb1‐CreERT2 control (left) and mb1‐CreERT2;Sykfl/fl mice (right) treated with Tam as described in the Materials and Methods section.Figure 2A, B Flow cytometric analysis of B cells from (A) the BM and (B) the SP of mb1‐CreERT2 control (left) and mb1‐CreERT2;Sykfl/flmice (right) treated with Tam as described in the Materials and Methods section. The BM cells were stained with anti‐IgM and anti‐B220, and the SP cells with anti‐CD19 and anti‐CD93 or anti‐CD23 and anti‐CD21. The gated regions in the dot blots correspond to individual B‐cell populations: (A) Bone marrow: gate P (B220+IgM−) pro‐/pre‐B cells, gate I (B220loIgM+) immature B cells, gate M (B220hiIgM+) mature B cells; (B) spleen: gate T (CD19+ CD93+) transitional B cells (T), gate M (CD19+ CD93−) mature B cells; in CD93−splenic B cells, gate M (CD23hiCD21int CD93−) mature follicular B cells and gate MZ (CD23lo CD21hi CD93−) marginal zone B cells. The numbers in the dot plots indicate the mean relative frequency of cells in the gate.C-E Statistical analysis of absolute cell numbers per Tam‐treated mouse: filled circles indicating cells obtained from mb1‐CreERT2 control mice and open circles from mb1‐CreERT2;Sykfl/flmice. (C) Statistical analysis of the absolute cell numbers of pro‐/pre‐, immature and mature recirculating B cells in the BM. (D) Absolute cell numbers of transitional, M and MZ B cells found in the SP. (E) Statistical analysis of the absolute cells numbers of peritoneal B cell subsets B1‐a and B1‐b. An asterisk (*) marks statistically significant differences (P 0.05), two asterisks (**) indicate P 0.01, P‐values were obtained using two‐tailed Student's t‐test. Cell numbers of four to five mice per group are shown, with each dot representing an individual animal.
Distribution of CEP250, CETN3 and SYCP3 in control and Plk1(Δ/Δ) spermatocytes.Figure 9(A) Triple immunolabelling of CEP250 (green), CETN3 (magenta) and SYCP3 (grey) in (a-b) late diplotene and (c-d) metaphase I in control and Plk1(Δ/Δ) spermatocytes. (B) Double immunolabelling of KIF11 (grey/green), and SYCP3 (red) in (a) control spindle, (b) Plk1(Δ/Δ) monopolar spindle, (c) BI2536 monopolar spindle and (d) MLN8237 monopolar spindle. Chromatin has been stained with DAPI (bue). Data is presented for 100µM BI2536 and 10µM MLN8237 for 8h treatments on organotypic cultures of seminiferous tubules.
Increasing GABA levels endogenously also inhibits pexophagy and mitophagy, and these defects are suppressed by rapamycinWT cells expressing OM45-GFP, along with the uga2? strain over-expressing the GAD1 gene and expressing OM45-GFP were grown in YPL medium to mid-log-phase.Figure 3A WT cells expressing OM45‐GFP, along with the uga2∆ strain over‐expressing the GAD1 gene and expressing OM45‐GFP were grown in YPL medium to mid‐log‐phase. To monitor mitophagy, strains were transferred to SD‐N starvation medium (with or without rapamycin). B WT strain along with the uga2∆ strain over‐expressing the GAD1 gene was grown in oleate medium and pexophagy was monitored as described in Fig , with or without rapamycin. Samples were taken at the indicated time points, and Pot1 degradation was analyzed by immunoblotting (45 kD). C To monitor autophagy, WT cells expressing GFP‐Atg8 along with the uga2∆ strain over‐expressing the GAD1 gene and expressing GFP‐Atg8 were grown in SD medium and transferred to SD‐N.
Inhibition of IRE1α reduces skin fibrosis.Figure 5A The effect of the IRE1α inhibitor 4μ8C on TGFβ-induced skin fibrosis in C57BL/6 mice as measured by the percentage of sirius red areas in the hypodermis. Ctrl (n = 6); Ctrl + 4μ8C (n = 5); TGFβ (n = 8); TGFβ + 4μ8C (n = 8). * P = 0,046.B Representative skin tissue sections stained with sirius red for collagen (top) and αSMA (bottom). Scale bars = 50 μm.C Percentage αSMA-positive cells in the hypodermis of mice from the experimental setup in A. * P = 0,024. D Representative image of the area selected for laser capture microdissection (scale bars = 100 μm).E Expression levels of αSMA mRNA in samples of hypodermis obtained by laser capture microdissection. Ctrl (n = 5), Ctrl + 4μ8C (n = 5), TGFβ (n = 5), TGFβ + 4μ8C (n = 5). * P = 0,044.F miR-150 expression levels in the same samples as shown in E. G Immunohistochemistry staining of αSMA in primary skinfibroblasts isolated from healthy mice after treatment with TGFβ ± 4μ8C. Scale bars = 20 μm. H αSMA mRNA expression in primary mouse skin fibroblasts after treatment with TGFβ ± 4μ8C. * P = 0,049, n = 4. I Agarose gel showing levels of spliced and unspliced XBP-1 in primary mouse skin fibroblasts treated as indicated with TGFβ and 4μ8C. J BiP mRNA expression in primary mouse skin fibroblasts after treatment with TGFβ in the absence and presence of 4μ8C. * P = 0,00007 for TGFβ vs. Ctrl and P = 0,00002 for TGFβ vs. TGFβ + 4μ8C. n=4. K Western blot showing CHOP protein expression in primary mouse skin fibroblasts treated as indicated with TGFβ and 4μ8C. L miR-150 mRNA levels in primary mouse skin fibroblasts treated as indicated with TGFβ and 4μ8C. * P = 0,00031 compared to untreated control, n = 3.
a, Autophagosome formation in the heart indicated by GFP–LC3.figf1a, Autophagosome formation in the heart indicated by GFP-LC3. Hearts were isolated from GFP-LC3 transgenic mice at multiple stages, including 18.5 day embryos and 0.5 h, 3 h, 6 h, 24 h and 2.5 day neonates, immediately fixed, cryosectioned, and analysed by fluorescence microscopy. Scale bar, 10 µm. b, Quantification of GFP-LC3 dots in neonatal tissues. Cryosections were prepared from tissues isolated at the indicated times. The ratio of the total area of GFP-LC3 dots to the total cellular area is shown as a percentage. Values represent mean ± s.d. of three mice. Diaph., diaphragm; Panc., pancreas; Muscle; gastrocnemius muscle. c, Electron microscopic analysis of the heart from wild-type neonates. Typical autophagosomes (arrows) and autolysosomes (arrowhead) were observed in the heart at 10 h after birth. Scale bar, 1 µm. d, Conversion of LC3-I to LC3-II in neonatal hearts. Heart homogenates from five embryos/neonates were pooled and analysed by immunoblotting using an anti-LC3 antibody. The bands representing LC3-I and LC3-II are marked with arrows.
Figure 3A. The levels of histone H3K122 succinylation, histone H3K122 acetylation, histone H3K27 acetylation, histone H3, HAT1, and β-actin were examined by Western blot analysis in HepG2, HAT1 knockout (KO) Clone 1 HepG2 cells, and in HAT1 KO cells reconstituted with either HAT1 or mutant HAT1 (T188A). B. HAT1-catalyzed histone H3K122 succinylation was analyzed by mixing purified HAT1, histone H3 or histone H3 mutant (K122R), and succinyl-CoA in the in vitro succinylation assays. Heat-inactivated HAT1 was used as a negative control. Western blot analysis was performed with indicated antibodies. C. HAT1-catalyzed histone H3K122 succinylation was analyzed by mixing purified HAT1, histone H3, and succinyl-CoA (2 μM) with or without the addition of the indicated concentrations of CoA. Western blot analysis was performed with indicated antibodies. D. The effect of HAT1 on histone H3K122 succinylation and histone H3K122 acetylation at the indicated gene promoters was determined by ChIP assays with anti-H3K122 succinylation antibody and anti-H3K122 acetylation antibody, and followed by quantitative PCR with primers of the promoter regions of CREBBP, BPTF and RPTOR in WT HepG2 cells, HAT1 KO HepG2 cells and HAT1 KO HepG2 cells reconstituted with either wildtype HAT1 or mutant HAT1 (T188A). N = 3 biological replicates. Data were presented as mean ± SD. Student's t-test, ***P < 0.001; ns, no significance. E. The mRNA levels of CREBBP, BPTF, and RPTOR were measured by RT-qPCR in HepG2 cells, HAT1 KO HepG2 cells, and HAT1 KO HepG2 cells reconstituted with either wildtype HAT1 or mutant HAT1 (T188A). N = 3 biological replicates. Data are presented as mean ± SD. Student's t-test, **P < 0.01. F. A model illustrating how HAT1-catalyzed histone H3K122 succinylation modulates epigenetic regulation and gene expression.
Comparison of immunostainings for mitochondrial proteins after fixation with either PFA or glyoxal.Figure 12 Figure 12 Comparison of immunostainings for mitochondrial proteins after fixation with either PFA or glyoxal. Cells were stained with MitoTracker Orange prior to fixation with the respective fixative and immunostained for the mitochondrial proteins ATP5B, COA6, NDUFA9 and TIM23. Quantification of the staining intensity shows a significant increase of fluorescence (signal over background) for 2 markers (ATP5B and Mitotracker) after fixation with glyoxal, whereas for the remaining 3 proteins immunostainings seem to be more efficient after fixation with PFA, albeit the differences are small. N = 18 - 128 cells per condition. Scale bar = 5 µm. *** p < 0.001
pat1 mutants display EDS1‐dependent, constitutive defense responsesPR gene expression is elevated in mpk4‐2 and pat1‐1 mutants.Figure 4PR gene expression is elevated in mpk4‐2 and pat1‐1 mutants. Four‐week‐old plants were used for RNA extraction, followed by qRT‐PCR. Fold‐change in PR1 (gray bars) and PR2 (black) expression is relative to Col‐0, normalized to UBQ10. Standard error of the mean is indicated by errors bars (n = 3). Statistical significance between the mean values was determined by ANOVA followed by Fisher's LSD test, P‐values are only shown for mean values significantly different from Col‐0. pat1 is more resistant to colonization by P. syringae pv. tomato DC3000. Bacteria were syringe‐infiltrated and samples taken 3 days post‐infiltration. Data are shown as log10‐transformed colony‐forming units/cm2 leaf tissue (cfu/cm2). Standard error of the mean is indicated by errors bars (n = 4). Statistical significance between the mean values was determined by ANOVA followed by Fisher′s LSD test.
Figure 6(A) CD11c-GFP+ DC cluster formation near venous vessel with (B) protrusions attracting 2d2.tdRFP Th17 cells. (C) Low-velocity motility of 2d2.tdRFPT cells in DC cluster compared to outside of the cluster. SHG in upper panel on the left (arrow). In addition, intercellular matrix fibers (arrow) are visualized that are only found in inflamed CNSparenchyma and are due to second harmonic generation (SHG) at 1110 nm wavelength excitation and detection at 535 ± 50nm. Tracking of 2d2.tdRFPTh17 in right panel (color-coded tracks, low velocity: red, high velocity: yellow). (D) Quantitative analysis of T cell tracking: higher mean velocity of 2d2.tdRFPTh17 cells in the onset of the disease with differential distribution at the peak of the disease. Cluster associated tracks have significantly lower mean velocities than Th17 cells outside the CD11c-GFP clusters. (E) Analogously for track straightness; Kruskal-Wallis Test and Dunn's Multiple Comparison Test (*** p < 0.0001) were performed; pooled data from at least three independent experiments per group.
GFP-LC3–positive AVs colocalize TGN46 and early endocytotic markers after COPI depletion.Figure 8.GFP-LC3-positive AVs colocalize TGN46 and early endocytotic markers after COPI depletion. (A) 293/GFP-LC3 cells were incubated with siRNA for β′-COP or control siRNA (Cont) for 48 h, processed for immunofluorescence, and labeled with antibodies for TGN46. (B) siRNA-treated cells were fixed and labeled, or starved for 2 h (St), and labeled with antibodies to EEA1. ( (C) Control-starved HEK293 cells were labeled for endogenous LC3 and EEA1 by double labeling after methanol fixation. (D) After siRNA treatment, rhodamine-labeled dextran was internalized for 20 min, followed by a 40-min chase after which the 293/GFP-LC3 cells were fixed and analyzed by confocal microscopy.
PR, RANKL, and Wnt4 and their role in the regenerative capacity of the mammary gland A Experimental scheme.Figure 1A Experimental scheme. Mammary tissue fragments dissected from wild‐type (WT) or mutant (MT) donor mice were engrafted to contralateral mammary fat pads of RAG1−/− recipient mice surgically divested of the endogenous epithelium. Between 8 and 12 weeks later, the engrafted glands were assessed by fluorescent stereomicroscopy and new fragments were dissected for serial engraftment. B, C Serial transplantations of PR+/+ and PR−/− mammary epithelia. (B) Fluorescence stereo microscopy of third‐generation mammary outgrowths derived from 8‐week‐old PR+/+; EGFP and PR−/−; EGFP donor mice. (C) Table summarizing 3 independent serial transplant experiments with PR+/+; EGFP and PR−/−; EGFP. Each engrafted gland is represented by a micrograph; black sectors represent area of fat pad filled by engrafted epithelium. Scale bar: 200 μm. D, E Serial transplantation of RANKL+/+ and RANKL−/− mammary epithelia. (D) Fluorescence stereo microscopy of third‐generation mammary outgrowths derived from 5‐week‐old RANKL+/+; EGFP and RANKL−/−; EGFP donor mice. Insets: higher magnification showing side branches present in the WT control (arrowheads) absent from RANKL−/−; EGFP epithelium. (E) Table summarizing three independent serial transplant experiments with RANKL+/+; EGFP and RANKL−/−; EGFP donor mice. Scale bar: 200 μm. F, G Serial transplantation of Wnt4+/+ and Wnt4−/− mammary epithelia. (F) Fluorescence stereo microscopy of third‐generation mammary outgrowths derived from mammary buds of E12.5 and E13.5 Wnt4+/+; EGFP and Wnt4−/−; EGFP embryos. Scale bar: 200 μm. (G) Table summarizing three independent serial transplant experiments with Wnt4+/+; EGFP donor mice. Scale bar: 200 μm. H Box plot showing the difference between percentage of reconstitution between WT and MT contralateral grafts in each transplant generation. P values were determined by Mann-Whitney U‐test.