EMBO/sd-character-level-ner huggingface dataset:
SPIN90 binds to the actin filament binding interface on Arp2/3 complex and blocks filament binding.Figure 6A. Ribbon diagram of the co-complex structure showing where an actin filament (grey semi-transparent surface model) binds to Arp2/3 complex. To model the bound filament, ARPC2 and ARPC4 from the EM reconstruction of Arp2/3 complex at a branch junction were overlaid onto ARPC2 and ARPC4 from the co-complex structur B. SDS-PAGE gel showing results of actin filament copelleting assay. Actin filaments at 3.5 μM were used to co-pellet 0.6 μM Bos taurus Arp2/3 complex in the presence or absence of the indicated concentrations of SPIN90(269-722) (S: Soluble fraction, P: Pellet) C. Plot of co-pelleting data as shown in (B), run in quadruplicate with error bars showing the standard deviation D. Comparison of the contacts made by SPIN90 (left panel) and actin filament (right panel) with the αD helix in ARPC4 (ARPC4 is blue, with the αD helix highlighted in yellow). SPIN90 is colored according to the helix number within each repeat (helix 1 - cyan, helix 2 - yellow, helix 3 - red). The actin subunit from the filament that contacts ARPC4 is colored according to secondary structure (helix - red, strand - yellow). Select elements of secondary structure are labeled, as is the DNaseI binding loop of actin (DBL) E. Superposition of subdomains 1 and 2 of Arp3 from the inactive GMF-bound structur (4JD2, orange) with Arp3 from the branch junction mode (grey) and Arp3 from the SPIN90-bound structure (cyan - shown only in right panel). Regions disordered in the structure are shown as dashed lines. Cyan arrow indicates the direction of motion in the transition from the twisted to flattened conformation F. Plot of the nucleotide cleft width versus the twisting dihedral angle in Arp3 from x-ray crystal structures, Arp3 from the branch junction EM reconstruction, or filamentous or monomeric actin structures. The pdb accession number is indicated for each data point. Orange: Arp3, x-ray crystal structures; cyan: monomeric actin x-ray crystal structures; black: actin filament fiber diffraction or cryoEM structures, green: Arp3 (rou-A) from branch junction reconstruction. Twist angle was defined as the dihedral made by connecting the center of mass off each subdomain 1-4. Nucleotide cleft distance was defined as the distance between Cα atoms in residues 67 and 222 (Arp3) or 59 and 207 (actin)
Ubqln1 interacts with Ubqln4.Figure 1(A) Inducible GFP‐S‐Ubqln1 293 cell line was treated with 1 μg/ml of doxycycline for 24 h. GFP‐S‐Ubqln1 was immunoprecipitated and sent for mass spectrometry analysis. Red sequences indicate Ubqln4 peptides identified by mass spectrometry. (B) 293 cells were co‐transfected with GFP‐S or GFP‐S‐Ubqln1 and HA‐Ubqln4. GFP was immunoprecipitated and analysed by western blot using anti‐GFP and anti‐HA antibodies. Lower molecular weight GFP‐tag‐containing bands that appear in the GFP‐S‐Ubqln1 lane might represent degradation products. (C) Endogenous Ubqln1 in 293 cells was immunoprecipitated using a rabbit Ubqln1 antibody or a control rabbit IgG. The immunoprecipitates were analysed by western blotting using anti‐Ubqln1 and anti‐Ubqln4 antibodies. (D) Confocal images of Hela cells transfected with GFP‐S‐Ubqln1 and RFP‐Ubqln4. Linescan of the white line in the merged image indicates the intensities of the fluorescent molecules in the cross‐section. GFP, green fluorescent protein; HA, haemagglutinin; IP, immunoprecipitation; Ubqln, Ubiquilin; WCL, whole‐cell lysate.
Dyn2 associates with autolysosomal tubules.Figure 7.(A and B) Subcellular density gradient fractionation of Hep3B hepatocytes starved for 2 h in HBSS and treated with 40 µM Dynasore, to induce tubule formation, as in Fig. 5. Cells were lysed (WCL), and the post-nuclear supernatant (PNS) was pelleted by centrifugation to produce a crude lysosomal fraction (CLF) and high-speed supernatant (HSS). The CLF was subsequently loaded onto an 8-27% discontinuous iodixanol (OptiPrep) gradient for separation by ultracentrifugation. Nine fractions were collected from the top of the gradient and blotted for Dyn2 and the LAMP1 resident protein, LAMP1. Lysosomal acid phosphatase activity roughly correlates with LAMP1 protein levels in each fraction. Levels of Dyn2 are highest in fraction 2, corresponding with both the peak levels of LAMP1 and lysosomal activity. (A) The data shown are from a single representative experiment out of three repeats. (B) Blotting for subcellular components shows that Dyn2 is specific for these same fractions, unlike other organelle markers such as EEA1 (early endosomes) and COXIV (mitochondria). (C and D) Dyn2 localizes to the surface of LAMP1-positive compartments. Fluorescence imaging of Hep3B hepatocytes transfected with Dyn2-GFP and LAMP1-mCherry under resting (C and C′) or starvation (D) conditions. (C′′) Dyn2-GFP localizing to LAMP1-labeled lysosome structures under starvation conditions. Arrows indicate regions of protein colocalization. Dyn2 (arrows) is present at the site of scission of LAMP1-positive tubules (arrowhead) from large autolysosomal structures. Bars: (C) 5 µM; (C′, C′′, and D) 3 µM.
Figure 4A Mice (n = 5 or 6 per group) were infected with IAV with indicated pfu. Survival was monitored daily for 2 weeks. B Mice (n = 6-9 per group) were infected with each IAV as indicated. Body weight was recorded daily after infection for 2 weeks. C Mice (n = 5 or 6 per group) were infected with viruses (1000 pfu) carrying PR8 PB1-F2 with the I68T, L69P, or I68T+L69P mutation. Body weight changes and survival rate were monitored daily. Detailed analyses on the virulence necessitated the monitoring of body weight below the standard level (80%) of mortality. Three cohorts of mice (5 or 6 per group) were infected with 500 pfu of one of the three IAVs. Viral protein expression in mouse lung was determined by Western blotting 2 days after infection. E Mice (6 per group) were infected with 500 pfu of one of the three IAVs. Viral titration by plaque assay was performed at 2 days post-infection using lung homogenates. F Mice (5-9 per group) were infected with 500 pfu of each virus. At 2 days post-infection, IFNβ mRNA levels were determined by qPCR. G Mice (5-9 per group) were infected with 1 × 105 pfu of each virus. At 2 days post-infection, IFNβ protein levels in lung homogenates were measured by ELISA.
Figure 3(A) Super-resolution STORM images of neuronal membrane after exposure to ATTO647N labeled Fib-Tau (0.36nM). Primary neurons were fixed 10, 60, 120 and 240 min after exposure to ATTO647N labeled Fib-Tau. Images rendered with a pixel size of 5nm show both clustered and diffused (non-clustered) distribution of exogenous Fib-Tau molecules. Scale bar, 1µm. (B-F) Quantification of various parameters from the super-resolution images. Averaged values per image is plotted. Increased "total detections per µm2" (B) and "cluster-density" (C) of Fib-Tau bound to neurons with increasing exposure time is observed. No change in the "size of cluster" (D) or "number of molecules within a cluster" (E) is seen with increased exposure time. The proportion of single molecule events detected within clusters remained nearly constant (~ 50%) in all conditions (F) indicating an equilibrium between clustered and non-clustered Fib-Tau single molecules. Unpaired t-test to compare difference with 10 min, n is number of images (8 images). *p<0.05; **p<0.01; ***p<0.001; ns= not significant.
Figure 2(A) Mean and 95% confidence interval is shown for SCNA scores among samples at each tumor stage across 6298 TCGA samples with stage annotation and SCNA score available. (B) Mean and 95% confidence interval for CYT scores among samples at each tumor stage across 6458 TCGA samples with stage annotation and CYT score available. (C) Pan-cancer correlation between SCNA score and CYT score with tumors grouped by stage. Spearman rank correlation coefficient and statistical significance (calculated using the student's t distribution with degrees of freedom = n - 2) is shown at the top right of each panel. (D) Spearman correlation coefficients linking SCNA and CYT scores across 32 tumor types. Black circles denote significant correlation (FDR < 0.05) after Benjamini-Hochberg multiple testing correction.
changes in gene expression and decrease in Notch activity after Dll4 blockage.Figure 5Experimental design: E10.5 embryos were injected with IgG or αDll4, incubated for 5h and then dissected. The indicated IAHC subpopulations (kit+CD45+; kit+CD45-;kit-CD45-CD41+ were purified and RNA was sequenced. PCA of the different subpopulations from IgG or αDll4 treated embryos based on normalized gene counts. PCA considering the DEG according to the treatment specific for each subpopulation. Function enrichment analysis of DEG in the Kit+CD45- population according to the treatment(E) Experimental design and sorting gate for Kit+CD45- cells for single cell RNAseq (n=483 cells). Experimental design and sorting gate for Kit+CD45- cells for single cell RNAseq (n=483 cells). tSNE distribution of IgG and αDll4-treated Kit+CD45- cells from single cell RNA-seq data. (F) cells corresponding to each treatment are indicated and (G) different clusters identified by ICGS (F) are represented.1-3 are endothelial-like cells and 6-7 are hematopoietic-like cells. Violin plots of Notch family elements expression in IgG and αDll4 treated cells (adj p val: *<0.1 and *** p<0.00001). Percentage of cells in each cell cycle phase extracted from single cell RNA-seq analysis. Bars represent the average±SE (n=483).
Figure 2A) 8-µm cross section of 15-week-old WT and Dup18-30 TA and triceps were analyzed for dystrophin localization by immunofluorescence. Asterisk indicates clusters of revertant fibers. Scale bars, 100 μm. B) The muscle architecture of the same muscles was investigated by H&E staining. A representative image is shown. Scale bars, 100 μm. Asterisk indicates areas with necrotic fibers and fibrosis. Arrows indicate fibers with central nuclei. C) Percentage of dystrophin-positive fibers in the TA and triceps of WT and Dup18-30 mice. WT, n = 4; Dup18-30 n = 6-7. D) Percentage of myofibers with centrally located nuclei in the TA and triceps of WT and Dup18-30 mice. WT, n = 4; Dup18-30 n = 6. E) Forelimb grip strength was measured in 15-week-old WT and Dup18-30 mice. WT, n = 9; Dup18-30 n = 12. F) Specific tetanic force was measured in 15-week-old mice using an in vivo muscle-function analyzer. WT, n = 11; Dup18-30 n = 15 mice. G, H) Mice were tested in an open-field chamber in which total distance travelled (G) and vertical activity (H) were assessed. WT, n = 9; Dup18-30 n = 12.
a) Western blots showing autophagy induction by monitoring LC3 levels in whole-cell extracts from U2OS cells treated or not with 5-FU alone or in combination with Mx for the indicated time.f6(a) Western blots showing autophagy induction by monitoring LC3 levels in whole-cell extracts from U2OS cells treated or not with 5-FU alone or in combination with Mx for the indicated time. Staurosporin was used as a positive control for apoptosis induction shown by the appearance of cleaved fragments of caspase-3 (17 and 19 kDa) and poly (ADP-ribose) polymerase 1 (PARP1; ΔPARP, 85 kDa). α−Actin was used as loading control. (b) Quantification of LC3-II levels in cells treated as in a. Intensities of the lower LC3 band (LC3-II) was normalized to those of actin. . (c) Autophagy induction demonstrated by accumulation of LC3-positive puncta in U2OS cells treated or not with 5-FU alone or in combination with Mx for 48 h. The cells were fixed and prepared for immunofluorescence of LC3 (green). DAPI staining is shown in blue. Scale bar, 20 μm. (d) Quantification of LC3-positive puncta in cells treated as in c. In each separate experiment, 200-1,600 cells were scored per condition. e) Western blots showing absence of LC3-II accumulation in U2OS cells transfected with siRNA against MSH-2. The same blot probed with anti-MSH-2 antibodies confirm efficient knockdown. (f) Quantification of LC3-II levels in cells transfected with control or MSH-2 siRNA with or without FU treatment for 24 h. (g) Immunofluorescence showing reduced accumulation of LC3 puncta in U2OS cells transfected with siRNA against MSH-2. (h) Quantification of LC3-positive puncta in cells treated as in g. (i) Quantification of normalized RPS3 protein level (black bars) or RPS3 mRNA level (white bars) in U2OS cells after 48 h 5-FU treatment. RPS3 mRNA levels were determined by quantitative real-time reverse transcriptase-PCR.
Figure 8Schematic diagram illustrating how elevated ESM1 expression promotes development of PCa by raising cancer stem cell populations. The interaction between ESM1 and β-catenin leads to the stabilization of β-catenin-TCF4 complex and recruitment of this complex to the DNA. Stabilized β-catenin and ESM1 promotes the binding of TCF4 to DNA, Wnt activation of downstream gene expression, cancer cell stemness, as well as tumor metastasis.
Figure 3A. Subset of 4,544 myoblast cells in UMAP plot of the reference single cell atlas dataset. B. Dot plots showing the expression levels of the top variable genes between IFM and DFM myoblasts across 7 clusters of the reference cell atlas dataset. Confocal single plane images of third instar larval wing discs stained with: (C) anti-Ct (red) and anti-Zfh1 (green), (D) kirre[rp298]-lacZ (anti-β-gal, green) and anti-Zfh1 (red), (E) Ten-a[MI04411]::GFP (green), anti-Ct (red) and anti-Zfh1 (cyan), (F) anti-Nrt (red) and anti-Zfh1 (cyan), (G) Argk[CB3789]::GFP (green), anti-Ct (red) and anti-Zfh1 (cyan), (H) nkd[MI00209]::GFP (green), anti-Ct (red) and anti-Zfh1 (cyan), (I) Ama[NP1297]>GFP (green), anti-Ct (red) and anti-Zfh1 (cyan) and (J) Mef2>CD8-GFP (green), and fluorescent in situ hybridization of Ama-RNA probe (white). Confocal single plane images of third instar larval wing discs stained with: K. Ama[NP1297]>gTRACE showing the lineage of Ama-GAL4 (green) and the active GAL4 (red), and fluorescent in situ hybridization of Ama-RNA probe (white). L. Confocal single plane images of adult DFM of Ama[NP1297]>gTRACE showing the lineage of Ama-GAL4 (green) and the active GAL4 (red) stained with DAPI (white) dorsal right, anterior up. DFM are numbered in white following (Lawrence, 1982).
Confocal images of the GFP fluorescent patterns in seedlings expressing ATG5-GFP or GFP-ATG8.f1Confocal images of the GFP fluorescent patterns in seedlings expressing ATG5-GFP or GFP-ATG8. (a,b) Control conditions: cytosolic signal associated with rare fluorescent puncta. (c,d) High increase of fluorescent puncta after 2 h of nutrient starvation. Note ring-like ATG5 structures in c. (e,f) Wortmannin treatment blocks the occurrence of fluorescent ATG5 or ATG8 structures. (g,h) Maximum intensity projections of vacuolar focal planes, allowing visualization of autophagic bodies after 4 h of nutrient starvation in the presence of concanamycin A. Vacuolar ATG5-GFP fluorescence is not detected (g), while GFP-ATG8 signals are abundant (h). An inset in g, corresponding to a cortical focal plane, shows that formation of cytosolic ATG5 structures in the cytosol is not inhibited by concanamycin A treatment. Maximum intensity projections of 10-μm-deep deconvolved stacks, of cortical (a-f, and inset in g) or vacuolar focal planes (g,h). Scale bar, 10 μm.
Figure 41 or 2 μM of GST or GST-TRF1 were incubated with the indicated concentrations of mouse ERK2 kinase in the presence of 5μCi [γ-32-P]ATP. The mixture was resolve by SDS-PAGE followed by autoradiography. 1 or 2 μM of GST or GST-TRF1 were incubated with the indicated concentrations of human b-Raf kinase (WT or V600E) in the presence of 5μCi [γ-32-P]ATP. The mixture was resolve by SDS-PAGE followed by autoradiography. 1 or 2 μM of GST or GST-TRF1 were incubated with the indicated concentrations of mouse MEK1 kinase in the presence of 5μCi [γ-32-P]ATP. The mixture was resolve by SDS-PAGE followed by autoradiography. 1 μM of GST-TRF1 and 0.2 μM of mouse ERK2 kinase were incubated in the presence of ERK and MEK inhibitors. 2 μM of GST-TRF1 and 0.1 μM of human b-Raf kinase were incubated in the presence of the bRaf inhibitors dabrafenib and vemurafenib. 1 or 2 μM of GST or GST-TRF1 were incubated with the indicated concentrations of human mTOR kinase 1 or 2 μM of GST or GST-TRF1 were incubated with the indicated concentrations of human mTOR kinase in the presence of the mTOR inhibitors rapamycin and Ku0063794 Phosphopeptide peak intensity normalized to total TRF1 signal in samples containing only TRF1 or TRF1 plus ERK2 Phosphopeptide peak intensity normalized to total TRF1 signal in samples containing only TRF1 plus bRAFWT or bRAFV600E Phosphopeptide peak intensity normalized to total TRF1 signal in samples containing only TRF1 plus mTOR Schematic representation of TRF1 protein with the phosphorylation sites by ERK2, bRAF, mTOR and AKT. Representative image (down) and quantification (up) of in vitro phosphorylation assays with the indicated GST-TRF1 wild-type or mutated forms in the presence of mouse ERK2 kinase.
Figure 6Detection of nascent protein synthesis in the mitochondrial fraction of the ovary. A. Nascent protein synthesis was monitored by AHA incorporation, and detected by anti-biotin antibody. Tom20 served as a loading control. Note that the synthesis of nuclear-encoded mitochondrial proteins was decreased in the mdi1 background and was restored by overexpressing Tom20-Larp (mdi1/TL). Detection of nascent protein synthesis in the mitochondrial fraction of the ovary.B. Western blots of mitochondrial proteins in ovarioles of wt, mdi1 and mdi1 flies expressing Tom20-Larp (mdi1/TL) fusion protein. Note that levels of Tamas, TFAM, mRpL19 and COX4 were reduced in mdi1 flies, but restored in mdi1/TL flies. Detection of nascent protein synthesis in the mitochondrial fraction of the ovary. C. Nascent protein synthesis of Tamas and TFAM was decreased in mdi1 ovary, whereas mtSSB was not affected. Tfamgfp, Tamasgfp and mtSSBgfp were expressed in wt or mdi1 background. Nascent proteins were labeled by AHA incorporation and then the GFP-tagged protein was immunopurified with a GFP antibody and the nascent protein synthesis was detected by anti-biotin antibody. Detection of nascent protein synthesis in the mitochondrial fraction of the ovary. D. Representative profile of 254 nm absorbance of wt, mdi1 and mdi1/TL ovary extracts. Detection of nascent protein synthesis in the mitochondrial fraction of the ovary. E. Polysome mRNA profiling for tamas, tfam, mtSSB, cox4 and mRPL19 in wt, mdi1 and mdi1/TL ovary. The percentage of mRNA for each gene in non-polysomal fractions (N.P, including ribosomal subunits and monosome-associated) and polysomal fractions (poly) was calculated and plotted. The fractions of tamas, tfam, cox4 and mRPL19 mRNAs in the polysomal fractions were significantly decreased in mdi1 compared to wt, but were restored in mdi1/TL flies. N=4 for all samples. P values of comparing wt to mdi1: tamas, P= 0.0055; tfam, P= 0.001; cox4, P=0.0066; mRPL19, P=0.0097. P values of comparing mdi1/TL to mdi1: tamas, P= 0.0206; tfam, P= 0.0346; cox4, P=0.0036; mRPL19, P=0.0016. Detection of nascent protein synthesis in the mitochondrial fraction of the ovary. F. The nascent protein synthesis of COX4 and mRPL19 were decreased in mdi1 ovary and were restored mdi1/TL flies. The proteins were immunopurified with antibodies against the endogenous proteins.
Tumor-specific CCR9 interacts directly with T cells inducing prominent changes in the gene expression signature.Figure 6A ELISA showing CCL25 levels in cell lysates from indicated tumorcell lines. CCR9 knockdown (k.d.) in MCF7 cells was achieved using specific shRNA (see Materials and Methods). B Cr-release assay showing % specific lysis of MCF7 cells by survivin TC upon CCL25 (□) or CCR9 (○) inhibition using specific siRNAs in comparison to the control siRNA (▪). Mean ± SEM are depicted herein. C MCF7 cells were transfected with control or CCR9-specific siRNAs, and 48 h later, the supernatants (CCR9lo or CCR9hi SSN, respectively) were used to culture survivin TCs overnight. Supernatant-treated TCs were then used as effector cells against CCR9lo or CCR9hi MCF7 tumor cells in the Cr-release assay along with wild-type MCF7 cells. Mean ± SEM are depicted herein. D Cr-release assay showing % specific lysis of MCF7 cells that were pre-treated with or without pertussis toxin (PTX), or knocked down for CCR9 using specific siRNA. Mean ± SEM are depicted herein. E, F MCF7 cells transfected with control siRNA (CCR9hi) or CCR9 siRNA (CCR9lo) were cocultured with survivin TCs for 12 h. Gene microarray was performed with the total RNA extracted from purified T cells after the coculture. Volcano plot (E) illustrating fold change (FC; log2) in gene expression intensities compared with P-value (−log2) between CCR9hi- and CCR9lo-treated TCs. Horizontal bar at y = 4.32 represents a statistical significance of P = 0.05 (genes in gray below this line did not reach significance). LogFC cutoff at ± 0.5 is represented by the vertical lines. Heatmap representation of the top upregulated (LogFC > 0.5) and downregulated (LogFC < −0.85) genes (F) with P ≤ 0.05. Individual replicates per sample group are shown herein. E, F MCF7 cells transfected with control siRNA (CCR9hi) or CCR9 siRNA (CCR9lo) were cocultured with survivin TCs for 12 h. Gene microarray was performed with the total RNA extracted from purified T cells after the coculture. Volcano plot (E) illustrating fold change (FC; log2) in gene expression intensities compared with P-value (−log2) between CCR9hi- and CCR9lo-treated TCs. Horizontal bar at y = 4.32 represents a statistical significance of P = 0.05 (genes in gray below this line did not reach significance). LogFC cutoff at ± 0.5 is represented by the vertical lines. Heatmap representation of the top upregulated (LogFC > 0.5) and downregulated (LogFC < −0.85) genes (F) with P ≤ 0.05. Individual replicates per sample group are shown herein.
| Abnormal cortical structures in offspring brains derived from aged fathers in the early postnatal period.Figure 6A. Representative images of the cerebral cortex of YFO (left) and AFO (right) at postnatal day 6. Scale bar: 1 mm. B. Cortical thickness in the postnatal brain of YFO (n=4, from different mothers) and AFO (n=5, from different mothers). Data information: All data are presented as the mean ± SEM. *p<0.05, **p<0.01, determined using Student t-test. C. Immunostaining for cortical layer markers in the postnatal brain of YFO and AFO (representative magnified images of the white boxes in panel A). Satb2+/Ctip2-: layer 2-4, Ctip2high: layer 5, Satb2-/Ctip2-: layer 6. Scale bar: 100 μm. F. Cell number in each cortical layer in the postnatal brain of YFO and AFO. Data information: All data are presented as the mean ± SEM. *p<0.05, **p<0.01, determined using Student t-test. D. Immunostaining for another cortical layer markers in the postnatal brain of YFO and AFO (representative magnified images of the white boxes in panel A). Tbr1-/Ctip2-: layer 2-4, Ctip2high: layer 5, Tbr1+/Ctip2-: layer 6. Scale bar: 100 μm. G. Cell density in each cortical layer in the postnatal brain of YFO and AFO. Data information: All data are presented as the mean ± SEM. *p<0.05, **p<0.01, determined using Student t-test. E. Layer thickness (mm) in each cortical layer in the postnatal brain of YFO (n=4, from different mothers) and AFO (n=5, from different mothers). Data information: All data are presented as the mean ± SEM. *p<0.05, **p<0.01, determined using Student t-test.