EMBO/sd-character-level-ner huggingface dataset:
Figure 2A, B. H3122 and H2228 cells were stained for anti-ALK (green), anti-GRB2 (red), anti-SOS1 (red), anti-pC-KITY721 (red), anti-PI3K p85β or anti-pPLCγ2Y759 and DAPI (blue). Scale bars, 10 μm; magnified views of a selected area are shown. C. HEK293 cells transfected with YFP-EM4L-ALK V1 WT or V3 WT for 48 hrs and treated with 5% or 10% 1,6-hexanediol. Representative still images after 5 min (taken from time-lapse movies) are shown of cells treated with 10% 1,6-Hexanediol or DMSO. Scale bars, 10 μm; the arrowheads indicate cytoplasmic foci. D. Box plot showing the number of cytoplasmic foci in DMSO and 5% or 10% Hexanediol (5 min). Data represent 5-10 counts from 3 independent experiments (n=3). Error bar represents SD of three biological replicates. ****P< 0.0001 in comparison to YFP-EML4-ALK V3 WT DMSO by one-way ANOVA analysis. E. Representative western blots of HEK293 transfected EML4-ALK V1 WT and V3 WT for 48 hrs. Cells were treated with either 5% or 10% 1,6-Hexanediol for 5 minutes. DMSO was used a control. Lysates were analysed for the phosphorylation and expression of the indicated antibodies. GAPDH was used as a loading control. Data representative of n=2 experiments.
Generation of CA VII-deficient mice.Figure 1(A) Targeting strategy for the Car7 locus. Endogenous Car7 locus (top). Targeting construct (middle). Targeted locus after Cre-mediated recombination (bottom). Exons are indicated as grey boxes and loxP sites as arrowheads. Multiple tissue (eye, brain, adrenal gland, submandibular gland, oesophagus, lung, heart, liver, kidney, spleen, pancreas, stomach, duodenum, jejunum, ileum, caecum, colon, spinal cord, dorsal root ganglia, skeletal muscle, testis, epididymis, female gonad, skull) northern blot for CAVII. In the P56 mouse, CAVII is prominently expressed in the central nervous system including brain and spinal cord. Gapdh served as the loading control. (C) Developmental profile of CAVII protein expression. Western blot analysis of protein lysates of whole hippocampus shows increasing CAVII levels from P3 to P30. The band of the appropriate size (∼30 kDa) was absent in protein lysates from Car7 KO tissue.beta Actin served as the loading control in C and D. (D) In western blots from protein lysates of cultured cells, CAVII was detected in mixed glial/neuron cultures but not in glia cell cultures. The presence of astrocytes in all cultures was confirmed by detection of GFAP. Actin served as the loading control in C and D.
Figure 4A. WB analysis of nuclear and cytosolic fractions demonstrated that the duration of p65 in the nucleus is longer in the VI10 PDFs than in CFs. B. ChIP assay shows that p65 is associated more strongly with inflammatory cytokine gene promoters in LPS-stimulated PDFs than in LPS-stimulated-CFs. The binding activity at the IL1B promoter was set as 1. Differences between knockdown and control with p value= 0.00436 (Student's t test) are indicated with an asterisk. C. NF-kB inhibitor BOT-64 blocked the inducible mRNA expression of the inflammatory cytokines. Differences between knockdown and control with p values (Student's t test) are indicated with an asterisks and exact values.
Attenuation of mitochondrial apoptosis by HBV.Figure 5(A-C) Parkin silencing accelerates HBV-induced mitochondrial apoptotic signaling. HepG2 and HepAD38 cells grown in the absence or presence of tetracycline were transfected with non-targeting (NT) and Parkin siRNA, respectively, for 72 h. Cells were used for Western blot analysis using antibodies specific to the indicated proteins (A), caspase-3/7 activity assay (B), and TUNEL assay (C) (D) A model elucidating HBV-induced aberrant mitochondrial dynamics in mitochondrial apoptosis. HBV induces immoderate stimulation in mitochondrial fission and mitophagy through mitochondrial translocation of Drp1 and Parkin, respectively, eventually causing disruption in mitochondrial dynamics. Inhibition of mitophagy by Parkin silencing can lead to apoptotic cell death in HBV-infected host cells via accumulation of apoptotic signal (red).
Figure 2Blood vessels were observed in MOs (control, cep41 AUG (2.5 ng), or cep41 SB (2 ng))-injected or cep41-mutated Tg(kdrl:eGFP) zebrafish at 40 hours post-fertilization (hpf) by fluorescent microscopy. Asterisks and arrowheads indicate impaired ISVs and DLAVs, respectively. The representative images for analysis of ISV lumen diameter are indicated by dotted rectangles. A, anterior; P, posterior; DLAV, dorsal longitudinal anastomotic vessel; ISV, intersegmental vessel. Scale bars, 100 μm. Quantification of ISV lumen diameter in (B), the numbers of defective ISVs in (C), the numbers of embryos with aberrant DLAVs in (D), and the numbers of ruptured DLAVs in (E) from data observed in equivalent fields of view (within eight somites). The severity of blood vessel defects in cep41-deficient zebrafish: B (narrowed ISVs) < C (shorten, fused, and missing ISVs) < D and E (ruptured DLAVs). Data are shown as mean ± SD of three independent experiments with ≥ 20 embryos per condition. CV protrusions (arrowheads) and vascular loops (asterisks) were observed in zebrafish at 35 hpf. The images within the dotted rectangles are magnified in the right panels. A, anterior; P, posterior; CA, caudal artery; CV, caudal vein. Scale bars, 40 μm. Quantification of the length of the CV protrusions in (G) presented graphically by measuring them in equivalent fields of view (within five somites).
Figure 4U87 cells were treated with 20 µM LXR623 for 7h and 24h. Thereafter, cells were stained with annexin V/propidium iodide and analyzed by multi-parametric flow cytometry. The lower left quadrant indicates the percentage of viable cells. U87 cells were treated with LXR623 as indicated for 7h and analyzed for total ATP levels by a luminescence assay. To account for dead cells, assays were normalized to DNA-content (cell number). U87 cells were treated with LXR623 as indicated for 24h. Cell lysates were prepared and subjected to LC/MS for the determination of ATP levels. U87 cells were treated with increasing concentrations of LXR623 for 7h and 24h. Thereafter, whole cell protein lysates were collected and analyzed by capillary electrophoresis for the expression of phosphorylated AMPK (threonine 172) and total AMPK. U87 cells were treated with increasing concentrations of LXR623 in the presence or absence of exogenous ATP for 7h. Thereafter, whole cell protein lysates were collected and analyzed by capillary electrophoresis for the expression of ATF4. U87 cells were treated with increasing concentrations of LXR623 for 24h in the presence or absence of cholesterol. Thereafter, whole cell protein lysates were collected and analyzed by capillary electrophoresis for the expression of phosphorylated AMPK (threonine 172) and total AMPK. Cells were treated as in F and whole cell protein lysates were collected and analyzed by capillary electrophoresis for the expression of ATF4 and Vinculin (G) and by the standard western blot for the expression of Noxa and Actin. U87 cells were treated with increasing concentrations of LXR623. Thereafter, whole cell protein lysates were prepared and analyzed for classical ER-stress markers, GRP78, ATF4 and Noxa by capillary electrophoresis. U87 cells were transfected with non-targeting siRNA (n.t.-siRNA), ATF3-siRNA, ATF4 siRNA or the combination of both ATF4 and ATF3 siRNAs. After 48h, cells were treated with LXR623, whole cell protein lysates were harvested and analyzed by capillary electrophoresis for the expression of ATF3, ATF4, Noxa and Vinculin.
Figure 6(A) Schematic diagram of histone H3 indicating the position of Y41. The pY41-peptide (amino acids 37-46, red) was used for immunizing mice and the subsequent generation of the hybridoma cell line 8B2-C1 The longer phosphorylated (pY41) and non-phosphorylated peptides (amino acids 29-53, black) were used for ELISA and immunoblot analysis. (B) Specific detection of the pY41-peptide by the antibody produced by the hybridoma cell line 8B2-C1. The plate-bound pY41-peptide, in contrast to the corresponding non-phosphorylated peptide, is specifically detected by antibodies in the serially diluted supernatant of the hybridoma cells by ELISA assays, as measured by absorption at the wavelength of 450 nm. Mean values with SEM are shown for two independent experiments. (C) Specificity of the anti-H3Y41ph antibody, as shown by immunoblot analysis. One phosphorylated (pY41) or non-phosphorylated peptide was coupled to ubiquitin (Ubi, 8.5 kDa), followed by separation of the protein conjugates on SDS-PAGE and immunoblot detection with the anti-H3Y41ph antibody present in the 8B2-C1 cell supernatant. Following treatment with calf intestinal alkaline phosphatase (CIP), the dephosphorylated Ubi-pY41 peptide conjugate could no longer be detected with the anti-H3Y41ph antibody. (D) Immunoblot analysis of whole-cell extracts prepared from Pax5Jak2/+ and control Pax5Etv6/+ Cdkn2ab+/- B-ALL cells as well as from the human HEL, TMD8 and K1106 cell lines. Phosphorylated (p) STAT5 was detected with an anti-STAT5 (pY694) antibody and the pY41-peptide with the purified anti H3Y41ph antibody One to five pY41-peptides were coupled to ubiquitin, which was added in the range of 50 ng per well. The Gapdh and histone H3 proteins were analyzed as loading control. An unspecific protein is denoted by an asterisk. One representative of 5 immunoblot experiments is shown. (E) No evidence for an epigenetic function of Pax5-Jak2 in early B cell development. The genome-wide distribution of the histone marks H3K4me3, H3K27ac and H3K27me3 in in vitro cultured Pax5Jak2/- and Pax5Prd/- progenitor cells was determined by ChIP-seq analysis The average density of the three histone marks in these progenitor cells was determined for a region from -2.5 kb to +2.5 kb from the summit of the Pax5-Jak2 peaks identified in Pax5Jak2/+ B-ALL cells The results of two different ChIP-seq experiments per cell type are shown.
Figure 1A) Designed F-domain scaffold structures. Red and Purple dots indicate N and C terminal sites of F-domain conjugation, respectively. The letter N is the maximum number of conjugated F-domains and the dashed lines is the distance between F-domain conjugation sites. Top two rows: Cyclic homo-oligomers. The trimeric nanocage subunits Icos1-A, Tet1-A and Tet1-A.2allow precise testing of the effect of valency on signaling independent of geometry as they can be tested as trimers alone or as nanocages upon addition of the other component of these two component nanoparticles. Tetrameric scaffolds C4 and AkC4 bridges the gap between 3 and 6 copies of F-domain. H3, H6, and H8 are helical bundle scaffolds with nearly identical geometry but different valency. Third row: Tetrahedral nanoparticle scaffolds Tet1 and Tet2. Fourth row: Icosahedral nanoparticles. Icos1 and Icos2 present sixty copies of the F-domain on the trimer and pentamer subunits, respectively. B) Table showing the average percentage for F-domain conjugations and the maximum number of F-domains for each scaffold. C) Example of F-domain conjugated scaffold. Negative stain electron micrograph of Icos1 scaffold with 60 conjugation sites for F-domains. Scale bar is 1000 nm. D) Serum starved HUVECs were stimulated with 18 nM of angiopoietins or PBS (vehicle) for 15 minutes and phosphorylation levels were analyzed by immunoblotting for pAKT(S473), pERK1/2 (T202/Y204), pFAK(Y397), and β-Actin.
APOE inhibits subretinal MP clearance.Figure 4Representative image of a RPE flatmount 12 h after the subretinal injection (red marking) of 4 μl PBS with 12,000 CFSE-stained thioglycollate-elicited peritoneal cells that contain 70% macrophages (inset close-up view).Quantifications of CFSE+F4/80+ macrophages at different time points after subretinal injections of C57BL/6J and Cx3cr1GFP/GFP CFSE+ macrophages (n = 5/per group (12 h) and n = 6/per group thereafter; Mann-Whitney U-test, C57BL/6J versus Cx3cr1GFP/GFP: 1 day n = 20/group *P < 0.0001; 2 day n = 6/group *P = 0.0317). Representative image of TUNEL/Hoechst double-staining 12 h after subretinal injection of C57BL/6J CFSE+ macrophages (experiment repeated three times, inset close-up view). Representative cytometry images of SSC-A/CFSE and CD11b/F4/80 gated analysis of eye cell suspensions prepared 24 h after the injection of Cx3cr1GFP/GFP CFSE+ macrophages and cytometric quantification of eye cell suspensions at 24 h after the injection of C57BL/6J and Cx3cr1GFP/GFP macrophages into C57BL/6J (n = 16-20/group; Mann-Whitney U-test, *P = 0.0024). Quantification of subretinal CFSE+ cells on RPE and retinal flatmounts 24 h after subretinal injections of CFSE+ magnetic-bead-sorted bone marrow-derived monocytes (Mo) from C57BL/6J and Cx3cr1GFP/GFPmice into C57BL/6Jmice (n = 8-12/group; Mann-Whitney U-test, *P = 0.0006).Quantification of subretinal CFSE+ cells on RPE and retinal flatmounts 24 h after subretinal injections of CFSE+CD11bFACS-sorted brainmicroglial cells from C57BL/6J and Cx3cr1GFP/GFPmice into C57BL/6Jmice (n = 9-12/group; Mann-Whitney U-test, *P = 0.0087). Quantification of subretinal CFSE+F4/80+macrophages on RPE and retinal flatmounts 24 h after subretinal injections of CFSE+macrophages from C57BL/6J, Cx3cr1GFP/GFP, Cx3cr1GFP/GFPApoE−/−, and ApoE−/−mice into C57BL/6Jmice (n = 8-12/group; one-way ANOVA/Dunnett test of Cx3cr1GFP/GFP versus any other group *P ≤ 0.0001; Mann-Whitney U-test, Cx3cr1GFP/GFP versus Cx3cr1GFP/GFPApoE−/− *P = 0.0006). Quantification of subretinal CFSE+F4/80+ macrophages on RPE and retinal flatmounts 24 h after subretinal injections of C57BL/6J CFSE+ macrophages into C57BL/6J and with exogenously added APOE3 at 1, 10, or 100 μg/ml calculated intraocular concentrations (n = 6-7/group; one-way ANOVA/Dunnett test: C57BL/6J versus 10 μg *P = 0.0488; C57BL/6J versus 100 μg ‡P = 0.006. Mann-Whitney U-test: C57BL/6J versus 10 μg *P = 0.0012; C57BL/6J versus 100 μg ‡P = 0.0013).
Figure 1(A) Srcap expression in murine different tissues was examined by Northern blot. A 277 nt probe of Srcap (81-358nt) was labeled for Northern blot analysis. 18S RNA was used as a loading control. (B) Two-week-old SRCAP-C-HA tag mice were sacrificed for longitudinal sections followed by immunofluorescence staining. A global look of the section was shown. Scale bar: 500 μm. Green: EpCAM, Red: HA tag, Nuclei were counterstained by DAPI. (C) Murine intestine tissues were fixed and stained according to iDISCO staining protocol. SRCAP was highly expressed in vast majority of crypts in mouse intestine. Scale bar: 50 μm. Green: EpCAM, Red: SRCAP, Nuclei were counterstained by DAPI. (D) Duodenum region of intestinal tissues were obtained from Lgr5GFP-CreERT2 (Lgr5GFP) mice for immunofluorescence staining by indicated antibodies with OpalTM 7-color fIHC kit. Nuclei were counterstained with DAPI. Scale bar: 50 μm. (E) ISCs from Lgr5GFP mice were collected for organoid formation. Organoids were fixed for immunofluorescence staining with indicated antibodies. Scale bar: 50 μm. Data information: Green: EpCAM, Red: SRCAP, Purple: PCNA, Nuclei were counterstained by DAPI.
a) Colocalization of Rubicon–EGFP-associated structures with the late endosome/lysosome marker Lamp1 (arrows) in HeLa cells transfected with Rubicon–EGFP.figf4(a) Colocalization of Rubicon-EGFP-associated structures with the late endosome/lysosome marker Lamp1 (arrows) in HeLa cells transfected with Rubicon-EGFP. Note that some of the Rubicon-EGFP-associated structures show a 'ring' shape (yellow arrows). Scale bar, 10 μm. (b) Partial colocalization of Rubicon-EGFP-associated structures with the MVB marker LBPA (arrows) in HeLa cells transfected with Rubicon-EGFP. Scale bar, 10 μm. (c) Representative ultrastructural images show aberrant expansion of late endosomal/lysosomal structures in HEK 293T cells overexpressing Rubicon-EGFP. These abnormal organelles are large in size, with high (orange arrows) or low (black arrows) electron density. Some enclose small vesicles (purple arrows) and some resemble the MVB (blue arrows). Scale bars, 500 nm. (d) Representative ultrastructural images show late endosome/lysosome-like structures that are labelled with anti-GFP gold particles (panels 3, 4) in HEK 293T cells transiently transfected with Rubicon-EGFP. These structures are enwrapped by double membranes (panel 4 inset) and co-labelled by anti-GFP (developed by DAB) and anti-Lamp1 (gold enhanced) (panels 5-7) antibodies. Note that mitochondria are mostly negative for Rubicon-EGFP (panel 4). The negative controls are without antibody (panels 1-2). M, mitochondria. Scale bars, 200 nm.
CHOP deletion accentuates muscle atrophy in AR113Q mice.Figure 2A. Muscle fiber size (100 fibers/mouse) was quantified from proximal hind limb muscle of AR113Q (black) or AR113Q, CHOP −/− mice (white) at 12 wks. Left panel shows fiber size distribution, middle panel shows cumulative percent of fibers as a function of fiber area, and right panel shows relative fiber cross sectional area (mean +/− SEM). Left, middle panels, p<0.0001 by Mann-Whitney test. Right panel, p<0.001 by Student's t test. B. Representative image of muscle fibers following NADH stain. Bar = 20 µM. C. Distribution of proximal hind limbmuscle fiber size from wt (black) and CHOP −/− (white) mice at 12 wks. Difference not significant by Mann-Whitney test.
Figure 3(A) Domain architecture and boundaries for WAVE1 fragments used in this study. (B) Scheme of domain architecture of surface-immobilized WAVE1 variants (top panel), time averages (middle panels, 7 min total) and representative kymographs (lower panel) from multi-color TIRF imaging of actin polymerization on WAVE1-coated coverslips. Conditions are 1uM actin, 5nM Alexa488-UTRNN. (C) Average barbed end growth velocities of actin polymerization on WAVE1-immobilized surfaces. At least 40 filaments were analyzed per condition. (D) Scheme of actin polymerization from NPF-actin complexes in solution. (E) TIRF microscopy of actin polymerization from 2uM actin in the absence (upper panel) or presence of near-saturating amounts of WAVE1 WCA. (F) Barbed end polymerization rates as a function of actin and WAVE1-actin ratios. The red line is a fit to a minimal kinetic model assuming two polymerization competent actin species that associate to filament ends with different on-rates as indicated (see Methods).
Scalp psoriasis patient skin shows increased expression of TSLP in hair follicles and epidermis A.Figure 7Representative composite immune-fluorescent images of whole hair follicle units by confocal from non-lesional and lesional scalp psoriasis patients. c-JUN (purple), K-15 (green), TSLP (red) and DAPI (blue). TSLP intensity measurement in hair follicle units and epidermis by ImageJ from healthy scalp, non-lesional and lesional scalp psoriasis patients. Representation of fold change relative to control healthy scalp. n= 3 samples. Representative composite immune-fluorescent images of whole hair follicle units by confocal from non-lesional and lesional scalp psoriasis patients (D) and magnification images of specific regions from the HFs (E). CD200 (green), c-JUN (white), TSLP (red), DAPI (blue).
A potential unique drug target pocket in SARS-CoV-2 N-NTDFigure legend: A.Figure 4:A potential unique drug target pocket in SARS-CoV-2 N-NTDFigure legend: A. Detailed view of ribonucleotide binding pocket in superimposition structures between SARS-CoV-2 N-NTD with HCoV-OC43 N-NTD AMP complex. AMP, interacting residues and equivalents are highlighted with stick representation; B. Electrostatic surface of the potential ribonucleotide binding pocket on SARS-CoV-2 N-NTD; C. Electrostatic surface of the ribonucleotide binding pocket on HCoV-OC43 N-NTD; D. Detailed view of phosphate group binding site in superimposition structures between SARS-CoV-2 N-NTD with HCoV-OC43 N-NTD AMP complex; E. Dot representation of SARS-CoV-2 residues Thr 55 and Ala 56, which indicates potential steric clashes with the ribonucleotide phosphate group; F. Dot representation of HCoV-OC43 N-NTD residues Ser 67 and Gly 68; G. Detailed view of nitrogenous base binding site in superimposition structures between SARS-CoV-2 N-NTD with HCoV-OC43 N-NTD AMP complex; H. Electrostatic surface of the potential ribonucleotide nitrogenous base binding pocket on SARS-CoV-2 N-NTD; I. Electrostatic surface of the ribonucleotide nitrogenous base binding pocket on HCoV-OC43 N-NTD. In electrostatic surface potential panels, blue denotes positive charge potential, while red indicates negative charge potential. The potential distribution was calculated by Pymol. The values range from −5 kT (red) to 0 (white) and to +5 kT, where k is the Boltzmann constant and T is the temperature.
Figure 4DNA sequences used in tandem target experiments. Complementary sequences to crRNA are marked in pink boxes and PAMs are marked in green boxes. Distances between the protospacers are indicated above the sequences. An illustration of Cas9 binding to each target site resulting in either high FRET (target H) or low FRET (target L) is shown on the right. Example fluorescence traces showing Cas9 transitioning between H and L targets with increasing target distances (right). FRET histograms constructed from events that show fluctuations showing two FRET peaks corresponding to binding to either target. High FRET peak stays the same in each histogram, but lower FRET peak moves to lower FRET values as the distance between targets increases. A scatter plot showing the probability of Cas9 transitioning between targets before dissociation. The values are averages from five measurements and error bars represent standard error of the mean. The transition probability (p = T / (T+D)) was calculated by summing up all the number of FRET transitions (T) and dividing T by the sum of T and the total number of dissociation events (D). A histogram showing all dwelltimes from single-target controls and tandem-target experiments. τ1 is similar for single-target control and tandem target experiments. τ2 only appears in tandem target experiments and is over 20-fold higher than τ1 or τst. The values represent the mean dwelltimes that were obtained by fitting dwelltime distributions by either single or double exponential decay function from five measurements. Error bars represent standard error of the mean.
MiR-342-3p associates with TB susceptibility (A) Expression changes of pri-, pre- and mature miR-342 transcripts were analyzed by semiquantitative PCR in C3H or B6 BMDMs after Mtb infection.Figure 1(A) Expression changes of pri-, pre- and mature miR-342 transcripts were analyzed by semiquantitative PCR in C3H or B6 BMDMs after Mtb infection. Representative blots from n = 3 biological replicates are shown. (B, C) The relative expressions of miR-342-3p and miR-342-5p were analyzed by northern blotting (B, representative blots from n = 3 biological replicates are shown) or quantitative real-time PCR (C, data are shown as the mean ± s.e.m. of n = 3 biological replicates) in Mtb-stimulated C3H and B6 BMDMs. (D-G) Cell death mechanisms of C3H BMDMs transfected with miR-342-3p mimic (D), or B6 BMDMs transfected with miR-342-3p inhibitor (F), followed by Mtb infection for 36 hours. Cell viabilities of C3H BMDMs transfected with miR-342-3p mimic (E), or B6 BMDMs transfected with miR-342-3p inhibitor (G), followed by stimulation with Mtb or z-VAD (20 μM) for 24 hours. Data are shown as the mean ± s.e.m. of n = 3 biological replicates. (H, I) Mtb growth rates of C3H BMDMs transfected with miR-342-3p mimic (H), or B6 BMDMs transfected with miR-342-3p inhibitor (I) after Mtb infection. Data are shown as the mean ± s.e.m. of n = 3 biological replicates. (J) Expression levels of miR-342-3p in PBMCs from healthy controls (n = 27) or TB patients (n = 34) were detected by qRT-PCR. Data are shown as the medians ± interquartile ranges.