EMBO/sd-character-level-ner huggingface dataset:
Figure 3The pyramids correspond to gp106 VrlC and, possibly, gp107 proteins. Structure of the A511 baseplate in the pre-host attachment state (extended sheath). The above- and below-the-plane of the baseplate pyramids (colored plum and light green, respectively) are very similar (this color scheme is maintained in all panels). Three pyramids that do not interact with the sheath are disordered. The red arrow points to a density feature that is clearly different in the post-attachment conformation of the pyramid. Structure of the spontaneously- and urea-contracted A511 tail (respectively) with the baseplate in the post-host attachment state. The blue arrow points to a density feature that is missing in the urea-contracted structure.
Figure 6(A-C) BMDMs were transfected with scrambled miRNA mimic (40 nM), miR-2137 mimic (40 nM) or miR-205 mimic (40 nM) for 24 hours and cells were processed for lipidomics analysis as described in materials and methods section. The ratio of lipids from this analysis is shown for (A) PI(3,4,5) P3 to PIP2.; (B) PI(3,4,5) P3 to PI; (C) PI(3,4,5) P3 to PIP. Data from LC/MS/MS using a Waters Xevo TQ-S MS/MS in MRM mode represent mean values ± SEM of peak areas normalized to internal standards as described in Methods. (n=3 biological replicates) BMDMs were transfected with (D) scrambled miRNA mimic (40 nM) or miR-2137 mimic (40 nM) for 48 hours and protein lysates were analyzed by western blotting using specific antibodies against INPPL1, pAKTS473, AKT, p70S6KT389, p70S6K, pS6S235/236, S6, p4E-BP1S65, 4E-BP1, pmTORS2448, mTOR and β-actin. Protein quantifications are shown next to the western blots (n=3 biological replicates). BMDMs were transfected (E) with control antagomir (200 nM) or miR-2137 antagomir for 24 hours and protein lysates were analyzed by western blotting using specific antibodies against INPPL1, pAKTS473, AKT, p70S6KT389, p70S6K, pS6S235/236, S6, p4E-BP1S65, 4E-BP1, pmTORS2448, mTOR and β-actin. Protein quantifications are shown next to the western blots (n=3 biological replicates).
Figure 1(A) Immunostaining of ES cells (Oct4), NPCs (day4, Nestin), and neurons (day 8, Tuj1). Scale bar: 200µm. (B) Expression levels of candidate lncRNAs during three stages of differentiation, measured by RNA-seq. Gene models of lncRNAs appear in green, adjacent protein-coding genes appear in grey. Blue depicts transcripts from the '+' strand, orange represents transcripts from the '-' strand. All three RNA-seq tracks were scaled separately to the maximum coverage in each locus. (C) qRT-PCR of Reno1, Cox10as1, and lnc-Nr2f1 in ES cells (top) and following eight days of neuronal differentiation (bottom), following knockdown using two different shRNAs, normalized to sh-NT. Mean ± SEM is shown for 3-6 independent experiments, * P<0.05, ** P<0.01 (unpaired two sample t-test).
Overall structure of Dln1 dimer.Figure 1A. Cartoon representation of the Dln1 dimer (PDB 4ZNO). Subunit A is colored in gray. The lectin module of subunit B is colored in yellow, whereas the middle and C-terminal moieties of the aerolysin module are colored in hot pink and green, respectively. The pre-stem hairpin is colored in blue. The bound sucrose molecules are shown as light pink sticks.B. The dimeric interface. Residues involved in dimeric interactions are shown as sticks. C. Multiple-sequence alignment of the pre-stem hairpin (the putative transmembrane region) from different aerolysin members. The members include Dln1, the earthworm Eisenia foetida lysenin, the mushroom Laetiporus sulphureus lectin (LSL) and Aeromonas hydrophila aerolysin. The alignment is generated according to the alternating pattern of hydrophilic and hydrophobic residues by GeneDoc. The hydrophilic and hydrophobic residues are marked in hot pink and gray, respectively.
a, b) Stable Mito-DsRed (pseudo coloured; blue)-expressing HeLa cells were transfected with Flag–Parkin wild-type or pathogenic mutants.figf5(a, b) Stable Mito-DsRed (pseudo coloured; blue)-expressing HeLa cells were transfected with Flag-Parkin wild-type or pathogenic mutants. Cells were treated with CCCP for the indicated times before immunostaining with anti-Flag (green) and anti-p62 (red). (a) In contrast to the pathogenic mutants, wild-type Parkin colocalizes with p62 at the site of clustered mitochondria, after 6 h of CCCP treatment. (b) After 24 h of CCCP treatment, wild-type Parkin-transfected cells show complete loss of mitochondria, whereas K161N and R275W Parkin mutants colocalize with both p62 and mitochondria. In contrast, translocation-compromised mutants K211N, T240R and G430D, lacking the mitochondria-associated poly-ubiquitin signal (Fig. 4b), did not colocalize with p62. Representative images of four independent experiments are shown. Data from untreated control cells and quantification of colocalization of Parkin, mitochondria and p62 upon CCCP treatment are shown in Supplementary Information, Fig. 5a and b, c, respectively. (c, d) Control siRNA- or p62 siRNA-silenced HeLa cells were transfected with EGFP-Parkin wild-type. Cells were treated with CCCP for the indicated times and stained with anti-TOM20 (pseudo coloured; blue) to visualize mitochondrial morphology and anti-p62 (red) to monitor silencing efficiency. Parkin wild-type was visualized by EGFP epifluorescence (green). Parkin-positive cells with cleared mitochondria are surrounded by a line. ( (c-f) Knockdown of p62 has no influence on Parkin translocation to mitochondria after 6 h of CCCP treatment (c-e), but completely blocks final clearance after 24 h (c, d, f). (e, f) Relative parkin translocation (e) and mitochondrial clearance (f) after p62 knockdown and CCCP treatment (n = 4). Data represent the mean ± s.d., ***P ≤ 0.0005 compared with control siRNA-transfected cells. Representative images of four independent experiments are shown. Scale bars, 10 μm. Immunostainings of control cells are shown in Supplementary Information, Fig. S5d, e. (g) Western blot of siRNA-transfected HeLa cells used in c-f demonstrates the efficiency of p62 silencing. β-Actin probing served as a loading control. WT, wild-type.
Figure 2(A) Schematic representation of the mouse experiment. (B) Immunoblot analysis of Wap-Myc mammary tumor lysates for the indicated TGFβ signaling markers and hepsin (T# denotes individual tumors). GAPDH was used as the loading control. Lysates were derived from Wap-Myc mammary tumors from 6 mice with and 6 mice without DOX-induced hepsin overexpression (C) Gene set enrichment analysis (GSEA) enrichment map of pathways upregulated in hepsin overexpressing Wap-Myc tumors (DOX+) compared with control tumors (DOX-) (N = 5 tumors each). Node size correlates with the number of genes in the signature; node color red correlates with enrichment in hepsin overexpressing Wap-Myc tumors. (D) GSEA graph comparing the expression of the HALLMARK_TGF_BETA_SIGNALING gene set in hepsin overexpressing (DOX+) to control (DOX-) Wap-Myc tumors (FDRp - p-value; FWERq -false discovery rate; NES-Normalized Enrichment Score). (E) Heatmap showing changes in expression of all genes in the HALLMARK_TGF_BETA_SIGNALING gene set in hepsin overexpressing (DOX+) compared to control (DOX-) Wap-Myc tumors. Red color indicates upregulation, and blue color indicates the dowregulation of the indicated genes.
Figure 4Oxygen consumption rate (OCR) upon sequential injection of oligomycin (Oligo), FCCP and rotenone (Rot) + antimycin (AA), in untransfected, sorted "GFP-+" and "GFP++". The error bars represent the mean±SEM of 5-7 separate experiments Quantification of 4-6 independent experiments comparing the basal respiration, maximal respiration, spare respiratory capacity and ATP-linked respiration of the UNT versus "GFP-+", "GFP++" and WT cells. The cells were analyzed between 25-28 days after sorting Coupling efficiency, measured as the ratio of ATP-linked/Basal respiration x 100. Error bars represent the mean±SEM of 4-6 separate experiments Representative membrane of a mitochondrial protein synthesis experiment. The results were normalized to the gel loading Western-blot showing the levels of NDUFB8 and COXI in untransfected and sorted cells. The SDHA and tubulin levels were also analyzed. The panel also shows the quantification of these markers normalized by the stain-free loading of 6 independent sortings/experiments
Figure 3A) In vitro cell adhesion assays employing HUAECs plated on increasing concentrations of purified laminin 411 and 511, compared to the non-endothelial cell laminin 111 and fibronectin, showing high levels of HUAECs adhesion to laminin 511 and low binding to laminin 411. B) Enhanced fold change (Δ) in COX2 mRNA expression in HUAECs plated on laminin 511 versus laminin 111 in response to shear. mRNA data are means ± s.e.m of 4 independent experiments with triplicates/ experiment, paired t-test. B) Enhanced fold change (Δ) in COX2 protein levels in HUAECs plated on laminin 511 versus laminin 111 in response to shear. Protein quantification data are means ± s.e.m of 7 independent experiments, Mann-Whitney test. C) Angle histogram of HUAEC orientation after 120 min of 10dyn/cm2 shear stress showing the percent cells at 10-90° in relation to the direction of flow. Cells were plated on 25 nM laminin 511 and laminin 111. Data are means ± s.e.m of 6 independent experiments, t-test. D, E) Inhibition assays performed at 25nM laminin 511 (D) in the presence or absence of function blocking integrin antibodies. Data are means ± s.e.m of 4 experiments with triplicates/ experiment, unpaired t-test . *P<0.05, ** P <0.01, *** P < 0.001, **** P < 0.0001. D, E) Inhibition assays performed at 25nM laminin 111, laminin 411 or fibronectin (E) in the presence or absence of function blocking integrin antibodies. Data are means ± s.e.m of 4 experiments with triplicates/ experiment, unpaired t-test . *P<0.05, ** P <0.01, *** P < 0.001, **** P < 0.0001.
Figure 7(A) Representative pictures of colon tumors in AOM/DSS-induced Colitis-Associated Cancer (CAC) model (n = 6 biological replicates). (B and C) The tumor load (B) or tumor number (C) in the whole colon was measured (n = 12 biological replicates, mean ± s.e.m., *P = 0.0431, **P = 0.003, two-tailed unpaired Student's t-test). (D) Representative H&E (Hematoxylin-eosin) staining pictures of colon tumors (The scale bars represent 1000 μm). (E) Flow cytometric analysis of TFRC expression in intestinal epithelial cells (IECs) from wild-type (WT) and Otud1─/─ mice treated with AOM/DSS. MFI, mean fluorescence intensity. (n = 4 biological replicates, mean ± s.e.m., **P = 0.0062, two-tailed unpaired Student's t-test). (F) GSEA of genes expressed in colon tissues from wild-type (WT) and Otud1─/─ mice treated with AOM/DSS (n = 3 biological replicates). ES, enrichment score; NES, normalized enrichment score. (G) Representative immunohistochemistry staining pictures of CD3+ T cells in colon tumors (The scale bars represent 400 μm ). (H) Flow cytometric analysis of various T cell subsets in lamina propria mononuclear cells (LPMC) isolated from wild-type (WT) and Otud1─/─ mice treated with AOM/DSS (n = 5 biological replicates, mean ± s.e.m., **P = 0.0025, ***P = 0.0005, two-tailed unpaired Student's t-test). (I) Intracellular ROS levels in colon tissues from wild-type (WT) or Otud1─/─ mice treated with AOM/DSS (n = 4 biological replicates, mean ± s.e.m., **P = 0.0072, two-tailed unpaired Student's t-test) were detected by DCFDA staining. MFI, mean fluorescence intensity. (J) ATP level in tumor interstitial fluid (TIF) of mice treated with AOM/DSS (n = 4 biological replicates, mean ± s.e.m., *P = 0.0292, two-tailed unpaired Student's t-test). (K) Model for the role of OTUD1 in iron absorption and tumor suppression. OTUD1 facilitates TFRC-mediated iron absorption through stabilization of IREB2 under steady condition. However, during colorectal cancer development, downregulation of OTUD1 restricts iron absorption and promotes resistance to ferroptosis, thereby subverting host immune surveillance against cancer.
Transferrin and EGF trafficking is inhibited by COPI depletion.Figure 9.Transferrin and EGF trafficking is inhibited by COPI depletion. 293/GFP-LC3 cells were treated with β′-COP siRNA or control siRNA. After 48 h cells were incubated with Alexa 555 transferrin (A) for 2 min, (B) 30 min, or (C) 30 min followed by a 30-min chase, then washed and fixed and analyzed by confocal microscopy. (D and E) 125I-EGF was added to HeLa cells after 48 h of siRNA depletion of β′-COP, or control siRNA for 10 min. The cells were then washed as described in the Materials and methods, and chased for 30, 60, 120, and 180 min (including 10-min incubation). (D) The amount of 125I-EGF internalized was shown as a percentage of the total 125I-EGF taken up in 10 min. (E) The amount of degraded 125I-EGF was determined as a percentage of the total radioactivity present after TCA precipitation over the total 125I-EGF internalized. The experiment was performed three times in duplicate and the data shown is the mean ± SEM. The significance was determined by Student's t test. *, P ≤ 0.05.
Figure 1A Microarray analysis of microRNA expression changes in newborn mouse lungs exposed to 21% O2 vs. 85% O2, at post-natal day (P) 5 and P14. B Quantitative RT-PCR detection of microRNA-34a/b/c-5p family members in the lung over the course of normal (21% O2) and aberrant (85% O2) alveolarization. C Quantitative RT-PCR detection of microRNA-34a/b/c-3p family members in the lung over the course of normal (21% O2) and aberrant (85% O2) alveolarization.
Levels of PPCA associated with the lysosomal membrane correlate with rates of CMA.Figure 2Levels of PPCA associated with the lysosomal membrane correlate with rates of CMA. Membranes of lysosomes incubated with increasing concentrations of GAPDH (A) or MgCl2 (B, top panel) as labeled, were analyzed by immunoblot for all forms of lamp2 (upper), lamp2a (middle) or PPCA (lower). The arrowhead indicates a previously identified truncated form of lamp2 lacking the cytosolic/transmembrane region ( Cuervo and Dice, 2000b). (B, bottom panel) Chart showing the effect of increasing concentrations of MgCl2, CaCl2 or EDTA on the degradation of [14C]GAPDH by intact ratliver lysosomes. Values are means ± SE of six different experiments. Differences from the control value were significant at P 0.001 (**) or P 0.01 (*). (C) Immunoblot analysis for PPCA (upper) and cathepsin L (lower) of rat liver lysosomes (100 μg of protein) with high (H+) and low (H−) activity for CMA and of the lysosomal membranes (L.Memb) and matrices (L.Mtx) from both groups of lysosomes (200 μg of protein). (D) Immunoblot analysis for lamp2a, PPCA and cathepsin D, as labeled, of lysosomes (100 μg of protein) from fed or 48 h starved rats and the membranes (L. Memb) and matrices (L. Mtx) of both goups of lysosomes (200 μg of protein). Only mature forms of the cathepsins are shown (E) Correlation between CMA activity, measured as in (B, bottom panel) and levels of PPCA at the lysosomal membrane determined by denstometric quantification of immunoblots similar to those shown in Figures 1 and 2 in lysosomes from fed or 20 h starved rats (Starv 20h), highly active (H+) or less active (H−) lysosomes, or lysosomes incubated with 1 mM CaCl2, MgCl2, EDTA, or increasing amounts (2, 5, 10 μg) of GAPDH. Values are expressed as times the value in fed rats (control) and are means ± SE of 3-6 different experiments.
Characterisation of stroma IMCs.Figure 2Giemsa staining of isolated stroma IMCs. These cells show round or oval-shaped nuclei that lack indentations, and have an abundant basophilic cytoplasm without showing prolonged cytoplasmic processes, suggestive of immature myeloid-lineage cells that are morphologically distinct from monocytes/DCs. Scale bar, 25 μm. Cell surface marker analysis of IMCs showing lack of common hematopoietic marker expression except for CD11b, which is expressed at low levels (overlaid with isotype control histograms shown in grey). Cell surface marker analysis (dot plots) of IMCs demonstrating CD11c+ IMCs do not express macrophage (F4/80), DC (CD24) or M1-polarisation (CD86) markers, but weakly express the M2 marker CD206. Insets show isotype control staining. Morphological evaluation of IMCs cultured for 2 weeks without additional cytokines. The cells grown on culture plates were imaged by phase-contrast microscopy (left). Then, the cells were trypsinised and smeared on glass slides for Giemsa staining (right). These cells are intermediate to large sized, round or amoeboid cells with cytoplasmic vacuolation, consistent with a macrophage morphology. Scale bars, 50 μm. Cell surface marker analysis of cultured IMCs showing differentiation into F4/80+ macrophages with CD11bhighCD11c+CD86+CD206low surface phenotype (overlaid with isotype control histograms shown in light grey). PCR detection of Braf recombination in purified IMCs, AT2 cells and tumour cell aggregates.
Figure 1(A) UBQLN4 associates with polyubiquitinated proteasomal substrates. Flag-tagged UBQLN4 protein was affinity-purified from extracts of HeLa cells that were treated with (+) or without (-) 20 μM MG-132 for 4 h before harvesting, and the Flag-precipitates (IP:Flag) were blotted with anti-polyubiquitin (FK2), anti-BAG6, and anti-Flag antibodies. Mock indicates empty vector transfection. Note that UBQLN4-BAG6 co-precipitation can be observed exclusively in the presence of polyubiquitinated substrates. (B) Polyubiquitinated proteins associated with UBQLN4 are sensitive to the translation inhibitor cycloheximide (CHX). HeLa cells expressing Flag-tagged UBQLN4 were treated with 5 μM MG-132 as well as CHX (either 10 or 25 μg/mL) for 4 h as indicated, and then subjected to immunoprecipitation with an anti-Flag antibody as in (A). (C) Schematic representation of MG-132 and CHX chase experiments in (D). During the course of MG-132 (5 µM) treatment for 4 h, CHX (10 µg/mL) was added at the indicated time points before harvesting. (D) HeLa cells were treated as in (C), and Flag-precipitates were blotted as in (B).
Figure 3Representative images of st30 medaka embryos injected with hccs-MO (B) alone or co-injected with miR-181a/b-MOs Co-injection of miR-181a/b-MOs rescues microphthalmia and microcephaly in both hccs-MO embryos. hccs-MO/miR-181a/b-MO- b-MO-injected embryos were treated with Baf-A1, PD98059 or HA14-1. Baf-A1 treatment counteracts the protective effect of miR-181a/b downregulation in hccs-MO/miR-181a/b-MO embryos PD98059 treatment does not interfere with the modulation of the MLS phenotype mediated by miR-181a/b downregulation HA14-1 treatment counteracts the effect of miR-181a/b downregulation in the hccs-MO/miR-181a/b-MO model Scale bars are 100μm. Representative images of st30 medaka embryos injected with cox7b-MO (H) alone or co-injected with miR-181a/b-MOs Co-injection of miR-181a/b-MOs rescues microphthalmia and microcephaly in cox7b-MO embryos. cox7b-MO/miR-181a/b-MO-injected embryos were treated with Baf-A1, PD98059 or HA14-1. Baf-A1 treatment counteracts the protective effect of miR-181a/b downregulation in cox7b-MO/miR-181a/b-MO embryos PD98059 treatment does not interfere with the modulation of the MLS phenotype mediated by miR-181a/b downregulation Scale bars are 100μm. Percentage of embryos with or without MLS phenotype in the different conditions in hccs-MO embryos. N≥300 embryos/conditions. Percentage of embryos with or without MLS phenotype in the different conditions in cox7b-MO (N) embryos. N≥300 embryos/conditions.
Figure 6A Phenotype and activation of newborn microglia in presence of CVF, CLys and CSN (10µg/ml). B RT-PCR for pro- and anti-inflammatory genes from microglia and culture supernatant levels of PGE2. Confocal microscopy immunofluorescence of Iba-1 staining (left panel) and 3D microglia morphology and shape in presence of CVF, CLys and CSN. Scale bar (50 µm) applies to all images. D Red-fluorescent latex bead uptake by CVF-, CLys- and CSN-stimulated microglia by confocal microscopy and total number of bead uptake per microglia. Arrowheads show bead uptake by single microglia. Scale bar (100 µm) applies to all images. E Treg induction following naïve CD4+CD62L+ T cells incubation with CVF-, CLys-, CSN-microglia conditioned media.
Figure 1A, B, and C. Immunostaining of Tom20 (mitochondria) in cells positive for β3-tubulin in DIV6 Ifnb+/+ and Ifnb-/- CNs with or without rIFN-β for 24 h. A. Projection of 3D image of Tom20. Scale bars equal 20 microns. B. Quantification of total mitochondrial mass per neuron. C. Mean individual mitochondria volume in Ifnb+/+ and Ifnb-/- CNs. D, E, F, and G. 3D electron microscopy of thalamus in 12-month-old Ifnb+/+ and Ifnb-/- mice. D. Volume rendering of mitochondria (left panel) and corresponding mitochondria in 2D electron microscopy image (right panel) in cell bodies. Each individual mitochondrion is colored differently. Scale bars equal 2 microns. Voxel size equals 0.00878908x0.00878908x0.03 microns for the Ifnb+/+ neuron and 0.00439453x0.00438453x0.04 microns for Ifnb-/-. Nuclei are in grey and marked with 'N.' E. Reconstituted axonal mitochondria. Images extracted from Movies EV1 and 2. F. Size of individual mitochondria in cell bodies and non-myelinated and myelinated axons. N>30; error bars are SD. G. Number of branches per mitochondrion. N>30; error bars are SD.