EMBO/sd-character-level-ner huggingface dataset:
Figure 2A 293T cells were transfected with plasmids encoding HA-USP19 and Flag-tagged Beclin-1 complex members (Flag-ATG14L, Flag-VPS15, Flag-Beclin-1, Flag-VPS34, Flag-UVRAG and Flag-AMBRA1), followed by immunoprecipitation (IP) with anti-Flag beads and immunoblot analysis with anti-HA. Throughout was the immunoblot analysis of whole-cell lysates (WCL) without immunoprecipitation. B, C Extracts of A549 cells (B) or human PBMCs (C) incubated with EBSS for various time points (above lanes) were subjected to immunoprecipitation with anti-Beclin-1 and immunoblot analysis with indicated antibodies (shown on the left). D HeLa cells were incubated in EBSS medium for 1 h after co-transfection eGFP-USP19 and dsRed-Beclin-1 for 48 h. Cells were imaged for eGFP and dsRed. Scale bar, 200 μm.E Quantitative data are mean ± SEM of triplicate samples (20 cells per sample). *p < 0.05, **p < 0.01 and ***p < 0.001. F Co-immunoprecipitation and immunoblot analysis of 293T cells transfected with deletion mutants of USP19 plasmid along with vector encoding Flag-Beclin-1. G Co-immunoprecipitation and immunoblot analysis of 293T cells transfected with deletion mutants of Beclin-1 plasmid along with vector encoding HA-USP19.
pex3 atg1 cells contain enhanced numbers of Pex14-containing structures.Figure 2.(A) iEM analysis of pex3 cells using α-Pex14 antibodies, identifying structures (arrows) in the vicinity of mitochondria. CW, cell wall; M, mitochondrion. (B) Quantification of Pex14-mGFP spots in pex3 and pex3 atg1 cells. (C) FM images of pex3 atg1 or pex3 cells, producing Pex14-mGFP complemented with FM4-64 vacuolar staining. The inset (enlarged from the boxed region) shows optimized intensities for pex3 cells, highlighting the Pex14-mGFP spot and vacuolar mGFP. (D) WB analysis of cells grown for 16 h on MM-M/G using α-Pex11 or α-Pex14 antibodies. Pyruvate carboxylase 1 (Pyc1) was used as a loading control. Error bars indicate SEM. Bars: (A) 100 nm; (C) 1 µm.
Autoinjector - an image-guided microinjection platform: A.Figure 1Photograph of the manipulator end of the Autoinjector. Scale bar is 40 mm. Photograph showing the custom pressure controller used for programmatic pressure control during automated microinjection. Scale bar is 8 mm. Overall hardware schematic. A computer interfaces with all the components of the platform, including the pressure controller, manipulator, and microscope camera. Images from the microscope camera are used to control the position of the micropipette using the manipulator. The pressure controller is used to precisely deliver injection pressure to the micropipette during microinjection. House pressure is coarsely downregulated by a mechanical regulator, followed by fine downregulation using an electronic regulator controlled by a microcontroller. Delivery of pressure to the micropipette is digitally controlled using a solenoid valve. The trajectory of the micropipette is controlled by the manipulator and is guided using images acquired using the microscope. Black arrows indicate digital interface routes, blue arrows indicate pneumatic route.
hnRNPL protects select human transcripts with long 3'UTRs from NMD.Figure 2A RNA-seq analysis of cells treated with hnRNP L siRNA or a combination of siRNAs against hnRNP L and UPF1 identifies populations of transcripts that decreased in abundance upon hnRNP L knockdown in a UPF1-dependent manner. B CDF plot of transcript 3'UTR lengths for subsets identified in (A). Statistical significance was determined by two-sided K-S test. P value for decreased set is from comparison to the set of total transcripts. P value for rescued set is from comparison to decreased set. C Venn diagram (to scale) of populations of mRNAs putatively protected from NMD by hnRNP L or PTBP1.
Figure 3NEDD4L was knocked out by the CRISPR Cas9 method in NHEKs. (A) Cell proliferation induced by IL-17A and IL-6 was detected by CCK-8 assays. Data information: Data are shown as the mean ± s.e.m., and are representative of three independent experiments Significant differences were tested using a two-tailed Mann-Whitney U test P＜0.01, ** P＜0.01, *** P＜0.001. n=4 biological replicates. NEDD4L was knocked out by the CRISPR Cas9 method in NHEKs. Western blot (WB) analysis of protein expression in NHEKs induced by IL-17A Data information: Data are shown as the mean ± s.e.m., and are representative of three independent experiments NEDD4L was knocked out by the CRISPR Cas9 method in NHEKs. Western blot (WB) analysis of protein expression in NHEKs induced by IL-6 Data information: Data are shown as the mean ± s.e.m., and are representative of three independent experiments NEDD4L was knocked out by the CRISPR Cas9 method in NHEKs. WT and NEDD4L KO NHEKs were precoated with anti-IL-6 neutralization antibody or an isotype, and the cell proliferation induced by IL-17A was detected by CCK-8 assays. Data information: Data are shown as the mean ± s.e.m., and are representative of three independent experiments Significant differences were tested using a two-tailed Mann-Whitney U test P＜0.01, ** P＜0.01, *** P＜0.001. n=4 biological replicates. NEDD4L was knocked out by the CRISPR Cas9 method in NHEKs. WT and NEDD4L KO NHEKs were infected with lentivirus overexpressing NEDD4L-WT or NEDD4L-C943S mutants. Cell proliferation induced by IL-6 were detected by CCK-8 Data information: Data are shown as the mean ± s.e.m., and are representative of three independent experiments Significant differences were tested using a two-tailed Mann-Whitney U test P＜0.01, ** P＜0.01, *** P＜0.001. n=4 biological replicates. NEDD4L was knocked out by the CRISPR Cas9 method in NHEKs. WT and NEDD4L KO NHEKs were infected with lentivirus overexpressing NEDD4L-WT or NEDD4L-C943S mutants. cell signaling pathway activation induced by IL-6 were detected by WB analyses Data information: Data are shown as the mean ± s.e.m., and are representative of three independent experiments (G, H) Primary keratinocytes were separated from the WT and Nedd4l KO mice. (G) WB analysis of the protein expression in IL-6-induced cell signaling pathway activation. (H) WB analysis of the proteins in the IL-6 signaling pathway. Data information: Data are shown as the mean ± s.e.m., and are representative of three independent experiments (I) Cell proliferation assay of GP130 KO NHEKs transfected with siNEDD4L induced by IL-17A. Data information: Data are shown as the mean ± s.e.m., and are representative of three independent experiments Significant differences were tested using a two-tailed Mann-Whitney U test P＜0.01, ** P＜0.01, *** P＜0.001. n=4 biological replicates.
The Shp2‐regulated gene expression programs predict the survival of patients with breast cancers A Kaplan-Meier survival analysis of human patients (n = 3,935) with invasive breast cancers divided into two groups based on the gene expression level of Shp2 (see ).Figure 5A Kaplan-Meier survival analysis of humanpatients (n = 3,935) with invasive breast cancers divided into two groups based on the gene expression level of Shp2 (see ). B-D Kaplan-Meier survival analyses of the humanpatients (n = 3,935) divided into two groups based on the expression level of the genes of each overrepresented class determined in Fig A: cell cycle genes, DNA replication genes, or p53 target genes. B-D Kaplan-Meier survival analyses of the humanpatients (n = 3,935) divided into two groups based on the expression level of the genes of each overrepresented class determined in Fig A: cell cycle genes, DNA replication genes, or p53 target genes. E, F Kaplan-Meier survival analyses of humanpatients (n = 3,935) divided into two groups based on the gene expression level of Skp2 or Aurka. HR, hazard ratio.
The upper panel shows strength of association of Affymetrix 500K SNPs in the region, with combined P values shown in red for replicated SNPs.figf1The upper panel shows strength of association of Affymetrix 500K SNPs in the region, with combined P values shown in red for replicated SNPs. The lower panel is a recombination map showing how the signals are clearly demarcated by recombination hotspots. Nearby genes are shown in yellow.
activity in DNA damage response.Figure 5A U2OS cells were treated with 10nM actinomycin D and cell extracts were prepared at indicated time points. Proteins from extracts were analyzed by western blot using the indicated antibodies. B U2OS and H1299 cells were treated with actinomycin D for 12 hours. Cells were harvested and proteins from cell lysates were subjected to western blot probed with the indicated antibodies. C U2OS cells transfected with the indicated siRNAs were treated with actinomycin D and harvested at the indicated time points. Proteins from cell lysates were subjected to western blot. D HCT116 cells were treated with 10nM of actinomycin D or 4 μM of doxorubicin (Dox) for 12 hours. Cells were harvested and total proteins were immunoprecipitated with anti-NAT10 antibody. Immunoprecipitates were analyzed by western blot for the indicated proteins. E HCT116 cells were treated with actinomycin D for the indicated times. Cells were fixed and immuno-stained as indicated. Immunofluorescence images were taken by confocal microscopy. Scale bar represents 10 μM. F H1299 cells were transfected with the indicated vectors. After treated with actinomycin D, cells were harvested and the whole cell extracts were subjected to western blot using the indicated antibodies. G H1299 cells were transfected with GFP-NAT10 vectors. After treated with actinomycin D for 12 hours, cells were fixed and nuclei were stained with DAPI. Bar represents 10μM. H HCT116 cells transfected with the indicated constructs were treated with actinomycin D or doxorubicin. Western blot was performed with total cell extracts and Mdm2-specific immunoprecipitates for the indicated proteins. I HCT116 cells were transfected with the indicated constructs. Cells were harvested after treatment with actinomycin D. p53 protein was immunoprecipitated with an anti-p53 polyclonal antibody and immunoblotted with p53 monoclonal antibody (DO-1).
Figure 8A. Schematics of human LATS1 and LATS2. PY, PY motif. B, C. 293T cells were transfected with the indicated DNAs and immunoprecipitated with an anti-Flag antibody or pulled down with streptavidin-binding beads. D, E. 293T cells were transfected as indicated and immunoprecipitated with an anti-HA antibody. LATS2 immune complexes were subjected to a kinase assay with His-YAP and cold ATP. F. Lats1 -/-; Lats2 fl/fl; SV40 mouse embryonic fibroblasts complemented with vector, LATS2 WT or ΔUBA mutant were infected with empty (control) or Zeocin-Cre retroviruses and selected with 400 μg/ml of Zeocin for 4 days. Harvested cells were analyzed by Western blotting for the indicated proteins.
Fractions of RA-SV sub-pools at the distal and proximal ribbon part at wild-type and OtofPga/Pga ribbon synapses(A,B) Depicted are the fractions of SVs without filaments, ribbon-attached SVs (Ri.Figure 3Depicted are the fractions of SVs without filaments, ribbon-attached SVs (Ri.), interconnected SVs (In.) and SVs with Ri. + In. per ribbon, at (A) the distal (Ds.) and (B) the proximal (Pr.) part of the ribbon. Data are represented as mean ± SEM of wild-type and OtofPga/Pga IHCs under inhib, rest and stim. (A) Upon stimulation, in the Ds. part at wild-type, the fraction of SVs without filaments reduces, but of SVs with Ri. + In. increases significantly. At OtofPga/Pga IHCs the fraction of SVs without filaments is reduced when comparing stim vs. inhib. The fraction of interconnected SVs increases with stimulation compared to inhibition and rest. In the mutant (inhib), the fraction of interconnected SVs decreases compared to wild-type (inhib), and Ri. + In. SVs reduce vs. wild-type (stim). (B) The fraction of SVs without filaments increased at inhibited OtofPga/Pga synapses. The red dotted lines in (A) and (B) represent the mean fraction of RA-SVs/ribbon part of resting wild-type for each defined sub-pool. Schematic summary: the distal (Ds.) ribbon (R) part, at wild-type (left panel) and OtofPga/Pga (right panel) ribbon synapses upon stimulation. Not drawn to scale; SVs with filaments (dark green, blue filaments) and without filaments (light green).
Figure 3(A) Left: Representative images of in situ PLA between S9.6 and γH2AX antibodies in U2OS cells depleted of BRCA2 (siBRCA2), DDX5 (siDDX5) or control cells (siC). When indicated, cells were transfected with a plasmid expressing RNase H1 (RH) 24h before or treated with Cordycepin (Cordy) for 2h at 37°C before fixation. Single antibody controls from non-irradiated siC cells are shown. Scale bar indicates 10 µm. Nuclei as defined by auto threshold plugin on the DAPI image (Image J) our outlined in yellow. Right: Quantification of PLA spots per nucleus in each condition as indicated. At least 500 cells per condition were counted from three independent experiments. For statistical comparison of the differences between the samples we applied a Kruskal-Wallis test followed by Dunn's multiple comparison test and the p-values show significant differences. The red line in the plot indicates the median and each symbol represents a single PLA spot. (B) DRIP-qPCR signal values at RBMXL1 and HIST1H2BG loci in U2OS DIvA cells transfected with the indicated siRNAs and treated in vitro with RNase H1 (RH) pre-immunoprecipitation where indicated. The experiment was performed in both untreated cells (-OHT) and after tamoxifen addition (+OHT). The data represent the mean ± SEM from at least four independent experiments. The statistical significance of the difference was calculated with unpaired one-tail t-test and the p-values show the significant difference.
Domain structure and sequence features of SARS-CoV-2 nucleocapsid protein.Figure 1A) Domain structure (top), prion-like sequence propensity (PLAAC, middle), and predicted disorder propensity (DISOPRED3) vs. sequence of SARS-CoV-2 N. N contains three putatively disordered regions, a globular N-terminal and a globular C-terminal oligomerization domain. B) Schematic of the folded and disordered domains of a SARS-CoV-2 N dimer.
Cellular locations of native and virally mediated Kcnq1 expression in the cochlea.Figure 1Cellular locations of native and virally mediated Kcnq1 expression in the cochleaA Diagram showing the major landmarks of the cochlear section to facilitate comparison of data shown in (B-F).B-F Immunolabeling results of Kcnq1 (green) in cochlear cryosections are shown for uninjected WT (B), untreated Kcnq1−/− (C), injected WT (D), and Kcnq1−/− mice given injections into the ST (E) or SM (F). Cell nuclei were outlined by counterstaining with either DAPI (B-D, F) or Qnuclear deep red (E). Scale bars represent approximately 100 μm. Meaning of white arrows are given in the text.
HST is required for amiRSUL cell-to-cell and long-distance movement(A) amiRSUL northern analysis, in two independent replicates (rep.Figure 4amiRSUL northern analysis, in two independent replicates (rep.), in Meselect-separated leaf-vasculature versus -epidermis of pSUC2::amiRSUL plants with the specified genotypes (see also Appendix Fig.S12A). An enhanced contrast is shown for the epidermis. miR165/166 and U6 were probed as endogenous controls. U6-normalized band-intensity quantifications relative to HST are indicated for each tissue. Average epidermis/vasculature ratio of amiRSUL levels presented in (A) and Appendix Fig.S12A. Error-bars: SD. t-test p-values are indicated. n=4. amiRSUL northern analysis, in biological duplicates, in scions (S) and rootstocks (R) in the specified genotypes' grafting combinations (amiR: pSUC2::amiRSUL) (see also Appendix Fig.S12B). U6: as in (A). Average relative U6-normalized band-intensity quantifications are indicated for each tissue. Average rootstock/scion ratio of amiRSUL levels as measured in (C), Fig.1G, Appendix Fig.S3A and Fig.S12B. Error-bars: SD. t-test p-values are indicated. n≥4. amiRSUL northern analysis, in biological duplicates, from total RNA extracted from aphids fed on WT or amiR plants, in the specified genetic backgrounds. U6: as in (A). Average relative U6-normalized band-intensity quantifications are indicated.
Figure 4(A) Five µL from wild-type, XccbphP, pXccBphP and pC13S bacterial cultures (OD600 = 1) were plated onto PYM-glucose agar plates under light or dark conditions. Xanthan production was determined measuring colony diameters (n = 4 replicates) for 20 independent experiments. (A-B) Values are expressed as mean ± s.e.m. Statistical analysis was performed by a Kruskal-Wallis test and Dunn´s multiple comparison test (*P < 0.05). (B) Wild-type, XccbphP, pXccBphP and pC13S were grown in 20 ml PYM liquid medium under light or dark conditions. Extracellular xanthan was purified by KCl addition and ethanol precipitation, then dried and weighted (n = 2 replicates) for 4 independent experiments. (A-B) Values are expressed as mean ± s.e.m. Statistical analysis was performed by a Kruskal-Wallis test and Dunn´s multiple comparison test (*P < 0.05). (C) Representative confocal laser-scanning microscopy pictures of biofilm development of bacteria cultured for 4 d in minimal medium in chambered cover slides under light or dark conditions. The scale bar represents 30 µm.
Figure 2 (A) Electron micrograph of negatively stained Ams1 with particles and higher-order assemblies (arrows). Class averages exhibit two distinct two-fold views consistent with D2 symmetry. Scale bar 10 nm. (B) Electron cryomicrograph of Ams1 with particles highlighted (circles). (C) Selected particles (top row) are shown with corresponding class averages (bottom row). Scale bar 10 nm. (D) Fourier shell correlation indicates a resolution of 6.3 Å according to the 0.143 criterion.  (E) Top view of the cryo-EM structure, four Ams1 molecules were segmented and colored (dark blue, beige, red, light blue).  (F) Molecular architecture of Ams1 monomer. Primary structure, two map segments with fitted pseudo-atomic models are shown. Left. The N-terminal portion was built from distant homology models: a four-helix bundle (17 - 27, 209 - 271) in yellow and a jelly-roll fold (45 - 203) in red. Right. The α-mannosidase core (287 - 1083) with an α/β barrel (287 - 573) in blue, a three-helix bundle (574 - 671) in green, and the β-sandwich domain (672 - 1083) in purple.  (G) Density section through the Ams1 monomer with three-helix bundle in the center.  (H) Complete atomic model of Ams1 with active site indicated as yellow sphere fitted into the cryo-EM map. Corresponding 3D structure in Movie EV1.  (I) Schematic representation of Ams1 domain distribution highlighting the molecular interfaces within the tetramer.  (J) The β-sandwich domain (purple) makes up the transverse interface between chain A and chain B.  (K) The N-terminal four-helix bundle (orange) mediates the longitudinal interface. Asterisk denotes position of W234E mutation between chain A and C/D.  (L) Negatively stained W234E Ams1 mutant reveals predominantly smaller particles when compared with wild-type tetramers. Representative class averages show a two-lobed structure consistent with reprojections of chain A and B (dark blue and beige) dimers depicted in ribbon and surface representation below. Scale bar 10 nm.
Figure 3A qPCR analysis of marker molecules for CNS cell types. The hippocampus of WT and Siglechdtr/dtr mice 2 days after PBS or DT administration was analyzed (n = 3 animals per group, one-way ANOVA with post hoc Tukey's test). Results are normalized to Gapdh, and are shown as ratios to the value of WT mice injected with PBS. B Immunohistochemical detection of astrocytes in Siglechdtr/dtr mice. Hippocampal CA1 sections were prepared 2 days after PBS or DT administration, and stained with an anti-GFAP antibody. Images were acquired using the same laser power and sensitivity, and image processing was the same. Scale bar, 30 µm. C Astrocyte number in hippocampal CA1 of Siglechdtr/dtr mice 2 days after PBS or DT administration (n = 5 animals, two-tailed unpaired Student's t-test).
FAS-FASL signaling mediates subretinal MP clearance.Figure 5Quantitative RT-PCR of FasLmRNA normalized with β-actinmRNA of 2-month-, 12-month-, and 2-month-old mice after 4 days of light challenge of C57BL/6 and Cx3cr1GFP/GFP mouse RPE/choroid plexus (aging: n = 5-6/group; Mann-Whitney U-test: 12 months *P = 0.0129; 4 days light challenge: n = 7-8/group; *P = 0.029). Immunohistochemistry of FASL (red) and IBA-1 (green) of a RPE flatmounts and sections (insets, Hoechst staining blue) from a 12-month-old WT (B) and Cx3cr1GFP/GFP(C) mouse (representative of three independent experiments, immunostainings omitting the primary antibody served as negative controls). Quantitative RT-PCR of FasLmRNA normalized with β-actinmRNA of 12-month-old C57BL/6 RPE/choroid plexus extracts 3 h after subretinal injection of C57BL/6J macrophages or Cx3cr1GFP/GFP macrophages (n = 6 per group), Mann-Whitney U-test, *P = 0.0087. IBA-1-stained RPE flatmounts of 2-month-old C57BL/6J, Faslgld/gld, and Faslpr/lpr mice after 4 days of light challenge.Quantification of subretinal IBA-1+ mononuclear phagocytes in control (left) and 4-day light-challenged (right) 2-month-old mice of the indicated strains (n = 6-10/group ANOVA/Dunnett test at 4-day light challenge: C57BL/6J versus FasLgld/gld and C57BL/6J versus Faslpr/lpr both *P < 0.0001; Mann-Whitney U-test at 4-day light challenge: C57BL/6J versus FasLgld/gld *P < 0.0001; C57BL/6J versus Faslpr/lpr *P < 0.0001). Quantification of subretinal CFSE+F4/80+macrophages on RPE and retinal flatmounts 24 h after subretinal injection of C57BL/6JCFSE+macrophages into C57BL/6J and FasLgld/gldmice and Faslpr/lprmacrophages into C57BL/6Jmice (n = 10-15/group, one-way ANOVA/Dunnett test: C57BL/6Jmacrophages inj. into C57BL/6Jmice versus C57BL/6Jmacrophages inj. into Fasgld/gldmice *P = 0.0002; C57BL/6Jmacrophages inj. into C57BL/6Jmice versus Faslpr/lprmacrophages inj. into C57BL/6Jmice *P < 0.0001. Mann-Whitney U-test: C57BL/6Jmacrophages inj. into C57BL/6Jmice versus C57BL/6Jmacrophages inj. into FasLgld/gldmice *P < 0.0001; C57BL/6Jmacrophages inj. into C57BL/6Jmice versus Faslpr/lprmacrophages inj. into C57BL/6Jmice *P < 0.0001). Quantification of subretinal CFSE+F4/80+ macrophages on RPE and retinal flatmounts 24 h after subretinal injection of Cx3cr1GFP/GFP CFSE+ macrophages into C57BL/6J with or without the Fas agonist MegaFasL (calculated intraocular concentrations 1 ng/ml; n = 7-8; Mann-Whitney U-test: *P = 0.014). In vitro MegaFasL-induced apoptosis of monocytes and macrophages of the indicated genotypes cultured for 24 h. TUNEL+ nucleus quantification expressed as percentage of non-MegaFasL-exposed control.
Figure 4(A) Representative frame of a 16 hour time-lapse microscopy experiment; GFP+ MM cells (green) were co-cultured with mouse bone marrow derived macrophages (gray) in the presence of 200 nM BoxA. Arrows indicate GFP+ MM cells engulfed by macrophages. This experiment was repeated twice. (B) Fraction of phagocytosed GFP+ MM cells after 16 hours. Statistics: χ2 test, phagocytosed vs non phagocytosed MM cells (n= 68 to 158). (C) Surface CD47 in MM cells, untreated (blue) or treated for 24 hours with 800 nM BoxA (red). Control isotype (grey). (D) Surface CD47 on MM cells evaluated by flow cytometry after incubation for 24 hours with the indicated concentrations of BoxA. The experiment shown is representative of 3 performed. Error bars indicate standard deviation. Statistics: One-way Anova plus Dunnett's post-test. *p<0.05, **p<0.01, ***p<0.001. (E) Representative immunofluorescence staining of CD47 (red) in unpermeabilized and permeabilized MM cells treated or not with 800 nM BoxA for 24 hours. Nuclei are counterstained with DAPI. Scale bar 50 μm.