EMBO/sd-character-level-ner huggingface dataset:
Figure 2The mFc-tagged ACE2s were captured by anti-mIgG Fc antibodies immobilized on the CM5 chip, and sequentially tested the binding with serially diluted SARS-CoV-2 RBD, GX/P2V/2017 RBD and GD/1/2019 RBD. The raw and fitted curves are displayed in dotted and solid lines, respectively. The binding affinity between the RBDs (SARS-CoV-2 RBD, GX/P2V/2017 RBD and GD/1/2019 RBD) and the 13 ACE2s are shown. Mean ± SD represents the mean and standard deviation of three independent experiments.
Figure 2A, B, G, and M. Sequence alternations in WT and 7SK RNA mutants mut1, mut2 and mut3, were shown in shaded boxes. U250 is marked in red. The tag refers to a 6-nt sequence replacement to enable discrimination from endogenous 7SK by primer extension. C-F; H-K; and N-Q. The tagged WT or mutant 7SK RNA was co-transfected with the indicated Flag-tagged proteins into HeLa cells. Anti-Flag or immunoprecipitates (IP) were analyzed by immunoblotting for the indicated proteins and primer extension for the bound 7SK RNA. C-F; H-K; and N-Q. The tagged WT or mutant 7SK RNA was co-transfected with the indicated Flag--tagged proteins into HeLa cells. Anti-Flag immunoprecipitates (IP) were analyzed by immunoblotting for the indicated proteins and primer extension for the bound 7SK RNA. C-F; H-K; and N-Q. The tagged WT or mutant 7SK RNA was co-transfected with the indicated Flag-tagged proteins into HeLa cells. Anti-Flag immunoprecipitates (IP) were analyzed by immunoblotting for the indicated proteins and primer extension for the bound 7SK RNA. C-F; H-K; and N-Q. The tagged WT or mutant 7SK RNA was co-transfected with the indicated HA-tagged proteins into HeLa cells. Anti-HA immunoprecipitates (IP) were analyzed by immunoblotting for the indicated proteins and primer extension for the bound 7SK RNA. L. Alignment between the 7SK sequences (black) flanking U250 (red) and a portion of the box H/ACA snoRNA (blue) showing base-pairing between the two. The altered nucleotides in shaded boxes were introduced into 7SK to disrupt the base-pairing. M. Sequence alternations in WT and 7SK RNA mutants mut1, mut2 and mut3, were shown in shaded boxes. U250 is marked in red. The tag refers to a 6-nt sequence replacement to enable discrimination from endogenous 7SK by primer extension. R. The indicated 7SK were in vitro transcribed in the presence of 32p-UTP and subjected to in vitro modification reactions. The nuclease P1-digested products were analyzed by 1D-TLC with pU and pΨ as controls.
Figure 6(A) Heat map indicates which TFs significantly affects rosette area, growth rate, shoot biomass, flowering time and seed yield and whether the effects of TFs are dependent on salt treatment. TFs are hierarchically clustered using Euclidean distance. (B) Heat map summarizing the relative effect of TF mutant alleles under control and salt treatment on rosette area, growth rate, shoot biomass, flowering time and seed yield. Mutant alleles are listed in rows and traits under both control and salt treatment are in columns. Cells of TF mutant alleles in heat map are colored if the AGI or AGI:Condition term in the linear model of each trait is statistically significant (P < 0.05, two-way ANOVA, N = 6-10 plants per genotype per condition). Mutant alleles are hierarchically clustered using Euclidean distance. (C) Rosette area of mutant allele of BPC4 from day 7 to day 21 post-germination. Linear model for two-way ANOVA considers AGI, salt treatment, day post-germination and their interactions. The AGI and AGI:Salt Condition terms are statistically signification (P < 0.001, P < 0.01, respectively). Heat map under line plot indicates which term in linear model is statistically significant (P < 0.05) using two-way ANOVA for each day from 9 biological replicates per condition. Solid lines, Col-0; dashed line, bpc4; circle, control condition; triangle, 50 mM NaCl. (D) Venn diagram of transcription factors in which the natural abundance of 13C and 15N and the ratio of 13C to 15N that were perturbed in their respective mutant alleles. Transcription factors are listed if the AGI or AGI:Salt Treatment linear model terms are statistically significant (P < 0.05) as determined by a two-way ANOVA from 3-5 biological replicates per genotype per condition.
Figure 2A. Survival rates of wild-type (n=33) and Angptl2-/- (n=33) mice supplied with drinking water containing 2.5 % DSS for 6 days and then untreated water for 6 more days, from two different experiments. ***p < 0.001, by log-rank test. B. Representative images of colons from wild-type and Angptl2-/- mice following DSS treatment for 6 days and then untreated water for 6 more days (at days 12).C. Colon length of wild-type (n=7) and Angptl2-/- (n=6) mice following DSS treatment for 6 days and then untreated water for 6 more days (at days 12). D, E, F. Representative images of colon crypts as assessed by H&amp;amp;E (D), Ki-67 (E), and pH3 (F) staining in wild-type and Angptl2-/- mice following DSS treatment for 6 days and then untreated water for 6 more days (at days 12). Scale bar = 100 μm.G, H. The number of regenerating crypts as quantified using Ki-67 (G) and pH3 (H) staining. Each data point represents the number of Ki-67-positive viable crypts or the number of pH3-positive cells in a single field of view. Multiple areas in at least 6 mice per genotype were quantified. Numbers below panels represent the average. I. IHC for expression of active β-catenin in colon or wild-type and Angptl2-/- mice following DSS treatment for 6 days and then untreated water for 6 more days (at days 12). Scale bar = 50 μm. J. mRNA levels of indicated genes in isolated crypts from wild-type (n=4) and Angptl2-/-(n=4) mice following DSS treatment for 6 days and then untreated water for 6 more days (at days 12).
Figure 4A Comparison of the CD81 model (after 1 µs simulation; colored by structural elements) and the CD53 crystal structure (green). Compared to CD53, the conformation of CD81 EC2 protrudes higher from membrane plane owing to the longer EC1 and different helix A orientation in CD81. The transmembrane regions of CD53 and CD81 are essentially unchanged during the 1 µs simulation. The MD simulation is performed using GROMACS (Van Der Spoel et al., 2005) and CHARMM36m force field (Huang et al., 2017). B RMSF plot shows motions of the TMs and EC2 during the MD simulation of CD81. The secondary structures are indicated below the curve. A, B and E helices are relatively stable (< 2 Å) during the 1 µs simulation time. In contrast, the C-D region is more flexible. C Structural flexibility of CD81 C-D region. The EC2 domains of the CD81 MD model after 1 µs simulation (colored by structural elements) and the CD81 crystal structure (blue) are superimposed.
Figure 5(A) Gel pictures of the saturation phase of RT-qPCR analysis for miR-204 in SEZ primary culture cells, ChP co-culture and ChP explants 7 day post preparation. (B) Gel electrophoresis of RT-qPCR analysis loaded at the saturation phase for miR-204 in SEZ culture medium and SEZ medium from the ChP co-culture. (C) Dot plot showing the proportion of neural stem cells (GFAP+) out of DAPI+ cells in primary SEZ/ChP co-culture with or without treatment with GW4869 inhibitor or AntimiR204. (D-G) Micrographs depicting the cellular compositions of SEZ cultures after co-culturing with control treated, GW4869 inhibitor treated or AntimiR204 treated ChP 7 days after plating. Boxed areas correspond to the higher magnification images in adjacent panels (D', E', F', G').
Fig.Figure 7(A-F) Maximum intensity projections of representative 232 x 232 μm2 fields of view of the endothelial layer from an infected LoC reconstituted without macrophages at 2 dpi with (A) and without Tocilizumab treatment (D). Actin and nuclear staining are indicated with the azure and electric indigo LUTs respectively, the actin staining is saturated for the endothelial cell clusters to highlight cells with lower levels of actin. (B, C) Zooms corresponding to the region in (A) highlighted with white and yellow boxes, respectively. (B) A region with normal vascular profile, (C) A region of endothelial cell cluster formation with increased actin staining. (E, F) Zooms corresponding to the regions in (D) representing regions highlighted with white and yellow boxes, respectively. (E) An example of reduced tight junctions between endothelial cells; bare patches are indicated with arrows. (F) A region of endothelial cell clusters. Scale bar = 20 μm. (G, H) 3D views from two 232 x 232 μm2 field of views of the endothelial layer of a Tocilizumab treated infected LoC at 2 dpi, immunostaining for the tight junction protein CD31 is shown in the Spring Green LUT. Endothelial cell clusters with low CD31 expression and altered actin staining are also visible and indicated by yellow arrows. (I) A plot of the proportion of pixels (shown on a logarithmic scale) with regions of low F-actin intensity identified via cut-off thresholds defined as a percentage of the maximum intensity in regions of interest from one LoC with and without Tocilizumab administration. ROIs were chosen from between 6 and 7 fields of view (technical replicates), each field of view was 232 x 232 μm2. The ROIs were defined to exclude the occurrence of endothelial cell clusters. A significantly larger fraction of the surface area of the ROIs for the untreated chip have low F-actin intensities. The solid bar represents the median, the boxes represent the 25-75 percentile range, and the whiskers represent the standard deviation from 6-7 regions of interest (technical replicates) from each chip. P-values are calculated using a Kruskal-Wallis One-Way ANOVA Test, * represents p≤0.05, ** represents p≤0.01, ** represents p≤0.001. E
Figure 6(A) ZWF1 is required for snf1Δmig1Δ growing in HU. Overnight cultures were spotted onto plates containing HU, and cultivated at 30°C for 48 h before photograph. Yeast spot assays were performed as in Fig 2A. (B) Rfa1-GFP foci were measured as in Fig 3. At least 200 cells were analyzed for each experiment of three independent experiments. Error bars represent SD from three biological repeats. Statistical significance was evaluated based on Student's t-test (*0.01 < P-values < 0.05). (C) Rfa1-13MYC ChIP assays after HU treatment. Strains were synchronized in G1 phase by α-factor prior to release into fresh media containing 200 mM HU for the indicated time points. Cell extracts were prepared as in Fig 3. Error bars represent SD from three biological repeats. (D) Phosphorylation of Mig1 T489 S495 completely restores the ZWF1 mRNA levels in snf1Δ. The experiments were done as in Fig 1. Error bars represent SD from three biological repeats. Statistical significance was evaluated based on Student's t-test (*0.01 < P-values < 0.05; **0.001 < P-values < 0.01).
a) BCBL cells were used for immunoprecipitation (IP) with mouse IgG or anti-Atg3, followed by immunoblotting (IB) with anti-cFLIP antibody (left panel).figf2(a) BCBL cells were used for immunoprecipitation (IP) with mouse IgG or anti-Atg3, followed by immunoblotting (IB) with anti-cFLIP antibody (left panel). NIH3T3-KSHV-vFLIP cells were used for IP with mouse IgG or anti-Flag antibody, followed by IB with anti-Atg3 antibody (right panel). (b) FLIP and Atg3 interaction after autophagy induction. HCT116 cells were treated with or without 2 μM rapamycin or Hank's solution (HBSS) for 4 h before IP with anti-Atg3 antibody, followed by IB with anti-cFLIP antibody. (c) Schematic diagram of human Atg3 (top). N, amino terminus; FR, flexible region; C, carboxy terminus. At 48 h post-transfection with GST or GST-Atg3 along with Flag-vFLIP (left) or GST or GST-Atg3 along with GFP-LC3 (right), HEK293T cells were used for GST pulldown, followed by IB with the indicated antibodies. (d) Schematic diagram of KSHV vFLIP (top). The black boxes indicate the K-α2 and K-α4 peptides. At 48 h post-transfection with V5-Atg3 and either GST-vFLIP DED1 mutants (middle) or GST-vFLIP DED2 mutants (bottom), HEK293T cells were used for GST pulldown, followed by IB with anti-V5 antibody. (e) At 48 h post-transfection with GST, GST-Atg3, and either Flag-vFLIP or Flag-vFLIP mAtg3, HEK293T cells were used for GST pulldown, followed by IB with anti-Flag antibody. (f) At 48 h post-transfection with GST-Atg3 and GFP-LC3 along with increasing amount of Flag-vFLIP, HEK293T cells were used for GST pulldown, followed by IB with anti-GFP or anti-Flag antibody. (g) NIH3T3 vector and NIH3T3 vFLIP cells were treated with 2 μM rapamycin for 4 h and used for IP with anti-Atg3 antibody, followed by IB with anti-LC3 antibody. Finally, whole-cell lysates (WCLs) were used for IB with the indicated antibodies to show expression (a-g).
Cells harbouring the EML4/ALK translocation are remarkably sensitive to alvocidib, dinaciclib and THZ1.Figure 3A) Proliferation assay of CrizR1, CrizR4, CrizR5, CeritR, and AlecR isogenic drug-resistant cell lines, treated with the indicated concentrations of THZ1 for 72h. P <0.05 was calculated for IC50 shift, as indicated in the methods. B) Crystal violet staining of EML4/ALK parental and drug-resistant cells. Cells were treated with the indicated drugs until the vehicle control reached confluence, then fixed and stained. C) CrizR1 cells were treated with the indicated drugs for 72h, then cells were stained with Annexin V/PI. Flow cytometry was then used to quantify Annexin V+ cells. Alvocidib: early apoptosis P=0.002, late apoptosis P=0.006, alive P=0.0008; Dinaciclib: early apoptosis P=0.005, late apoptosis P=0.02, alive P=0.002.
SARS-CoV S elicits antibodies neutralizing SARS-CoV-2 S mediated entry into host cells.Figure 5.Sequence conservation of sarbecovirus S glycoproteins plotted on the SARS-CoV-2 S structure. Entry of SARS-CoV-2 and SARS-CoV S pseudotyped viruses is potently inhibited by four SARS-CoV S mouse polyclonal immune plasma.
Figure 4(A) Wild-type and indicated K562 knock-out cells expressing tf-NBR1 were transfected with TagBFP or TagBFP-RavZ, and BFP-positive cells were analyzed for red:green ratio by flow cytometry. The median red:green ratio of each sample was used to calculate flux relative to WT+TagBFP. Bar graphs represent mean +/- SD from three independent experiments. n > 7,500 cells per sample. (B) K562-derived extracts prepared from wild-type (WT) and clonal deletion isolates were resolved by SDS-PAGE followed by immunoblotting (IB) with indicated antibodies. All samples were normalized by total protein using a BCA assay prior to loading. Bar graphs represent mean +/- SD from independent experiments (n=4 {NBR1, NDP52}; n=3 {SQSTM1}). p values for WT vs ATG9AKO was determined using a one sample test (theoretical mean = 1) with Bonferroni correction. (C) Schematized representation of protease protection assay for detecting vesicle closure. Cells were treated for 18h with 50nM Bafilomycin A1 prior to mechanical lysis. The corresponding cell extracts were treated as indicated prior to being resolved by SDS-PAGE and analyzed by immunoblotting. (D) Wild-type and indicated K562 knock-out cells were treated The corresponding cell extracts were resolved by SDS-PAGE and analyzed by immunoblotting (IB) with indicated antibodies. Shown are representative images from one experiment with mean intensity levels from (E) indicated below each image (NBR1: n = 4; TAX1BP1: n = 3, NDP52: n = 4, tubulin: n = 4). (E) Quantitation of protease-protection data from experiments in (D). Bar graphs show the mean +/- SD of each sample from independent experiments. Trypsin-treated samples were compared using a one-way ANOVA (NBR1: n = 4, p = 0.0001; TAX1BP1: n = 3, p = 0.0098; NDP52: n = 4, p < 0.0001) with Tukey HSD post-test. **, p < 0.01; *, p < 0.05; ns, not significant
PPM1H is a dimer.Figure 3(A) Model of the dimeric form of MnPPM1HWT-LD. Each monomer of PPM1H has an A or B prefix. Flap domains mediate the dimer and are shown in dark blue/wheat colors. The view is down a pseudo two-fold formed by α3 in the flap domain. Dotted lines denote the flexible loop (183-235) that connects α1/α2 in the catalytic domain. The dimeric organization of PPM1H positions this loop next to substrates, shown as docked models of pRab8a in green ribbons (transparent). The phosphoryated switch 2 helix of pRab8a is red. Yellow spheres indicate site of a double mutation in the flap domain (G357E+A359E) that disrupts a contact with α2 of the catalytic domain. (B) SEC-MALS analyses of PPM1HWT-LD and PPM1H2Glu-LD, showing that the WT enzyme is a dimer (104.5±2 kDa) while the double mutant is a monomer (53.6±1 kDa). The calculated molecular weight of His6-tagged PPM1HWT-LD used for the experiment is approximately 52.1 kDa. The errors were calculated from the SE of technical replicates from the data represented as a line across the central peaks in the gel filtration column. Light scattering from 15/18 possible angles (DAWN-EOS, Wyatt Corp) and the refractive index change relative to buffer (Optilab TrEX) were collected every second along these time points. Subsequently, data were processed using Astra 7.1 software (Wyatt Corp) to generate a weight-averaged molecular mass (y-axis) plotted against time (x-axis). There were approximately 130 technical data points for PPM1HWT-LD and 60 technical data points for PPM1H2Glu-LD. (C) His6-SUMO-tagged full-length variants of PPM1H were used for catalytic assays (left panel). 3 μg of recombinant wild-type or indicated mutant PPM1H proteins were resolved on 4-12% Bis-Tris gradient gel and stained with Instant Blue Coomassie. Right, mass photometry histogram for 40nM His6-SUMO-PPM1H WT (blue) and His6-SUMO-PPM1H 2Glu (brown), where WT is 130kDa (±15.8 kDa, with 1004 single molecules counted) and 2Glu is 83kDa (±13.9 kDa, with 4117 single molecules counted). The calculated molecular weight of the fusion protein is approximately 68.5 kDa. (D) In vitro malachite green assay time course of recombinant His6-SUMO-tagged PPM1H wild-type (blue), 2Glu (red), or D288A (grey) against 16 μM pThr72 phosphorylated Rab8a protein (GTPγS, left), 32 μM pThr72 phosphorylated Rab8a peptide (middle), or 32 μM pThr73 phosphorylated Rab10 peptide (right). The experiments were repeated 4 times. The error bars represent SE of mean of the technical replicates.
Figure 2(A) Representative immunofluorescence images of KT alignment efficiency in mitotic S2 cells expressing PoloWT-EGFP, PoloT182D-EGFP or lacking expression of any Polo transgene. Cells were treated with MG132 prior to fixation to prevent cells from exiting mitosis and increase the number of pre-anaphase figures. Insets display magnifications of the outlined regions, which highlight both low-tension misaligned (inset 1 and 2) and high-tension aligned KTs (inset 3). (B) Graph represents the percentage of cells in each indicated mitotic state, as shown in (A) (n≥ 532 cells for each condition, n=2 independent experiments). Data information: Data are shown as mean ± SD. Scale bar: 5 μm. (C) Representative immunofluorescence images of calcium-stable KT-MT attachments in metaphase S2 cells expressing either PoloWT-EGFP or PoloT182D-EGFP. Insets display magnifications of the outlined regions. Asterisk highlights either an aligned KT pair attached to MTs in an end-on fashion (PoloWT-EGFP) or an aligned KT pair in which a sister KT is laterally attached to the end of a MT fiber (PoloT182D-EGFP). Plotted profiles show the overlap between Polo-EGFP and tubulin signals for the highlighted KT. (D) Graph represents the percentage of metaphase cells showing at least 1 KT with a lateral interaction, as shown in (C) (asterisk) (n≥58 cells for each condition, n=2 independent experiments). Data information: Data are shown as mean ± SD. Scale bar: 5 μm. (E) Selected frames from live image analysis of chromosome segregation fidelity in neuroblasts expressing either PoloWT or PoloT182D. Neuroblasts without Polo overexpression were used as control. Jupiter-GFP was imaged for direct visualization of the mitotic spindle. Time 0 refers to anaphase onset. (F) Representative immunofluorescence images of mitotic neuroblasts from squashed brains showing distinct chromosome content. Spc105 was used as reference for KTs. Chromosome content is shown for each neuroblast. (G) Table shows the quantification of the percentage of asynchronous migration (n≥23 neuroblasts for each condition, n≥3 independent experiments) and of aneuploid cells respectively. For the quantification of aneuploidy, the whole brain was analyzed and prometaphase and metaphase neuroblasts were scored for chromosome number. An aneuploid cell was considered when >8 chromosomes (chr) were visualized (n≥360 neuroblasts from at least 7 larvae brains for each condition, n≥3 independent experiments). ND, not determined.
Figure 1Representative dual-channel kymographs showing that Rheb is recruited to mitochondria in axons and co-migrates with mitochondria in a predominant retrograde direction upon mitochondrial membrane potential (∆ψm) dissipation. The motility of mitochondria (Mito) and Rheb-associated mitochondria (Rheb-Mito) in axons were quantified, respectively. Vertical lines represent stationary organelles. Slanted lines or curves to the right (negative slope) represent anterograde movement; those to the left (positive slope) indicate retrograde movement. An organelle was considered stationary if it remained immotile (displacement ≤ 5 μm). Time-lapse images (B) showing colocalization and comigration (white arrows) of Rheb with mitochondria in a retrograde direction along axons during a 148-s recording period. Cortical neurons were transfected with GFP-Rheb and DsRed-Mito at 5-7 DIV and incubated with DMSO or 10 μM CCCP for 24 hr prior to imaging at 10-12 DIV. quantitative analysis showing that Rheb is recruited to mitochondria in axons and co-migrates with mitochondria in a predominant retrograde direction upon mitochondrial membrane potential (∆ψm) dissipation. The motility of mitochondria (Mito) and Rheb-associated mitochondria (Rheb-Mito) in axons were quantified, respectively. Vertical lines represent stationary organelles. Slanted lines or curves to the right (negative slope) represent anterograde movement; those to the left (positive slope) indicate retrograde movement. An organelle was considered stationary if it remained immotile (displacement ≤ 5 μm). Quantitative analysis showing robust association of Rheb with depolarized mitochondria within axons treated with CCCP. The percentages of mitochondria targeted by Rheb or Rheb-SSVM in the presence or absence of CCCP were quantified, respectively representative images showing robust association of Rheb with depolarized mitochondria within axons treated with CCCP. Rheb-SSVM, a farnesylation deficient mutant of Rheb, fails to be recruited to mitochondria upon dissipating ∆ψm. Mitochondria associated with Rheb were marked by white arrows. F-H Representative dual-channel kymographs (F) and quantitative analysis (G, H) showing that Rheb-mediated mitophagy activation depends on Rheb recruitment to mitochondria through Rheb farnesylation. Note that Rheb, but not Rheb-SSVM, is associated with autophagic vacuoles (AVs) within axons upon ∆ψm dissipation. Rheb-associated AVs move in a predominant retrograde direction along axons. The number of Rheb-associated AVs (Rheb-AVs) with or without CCCP treatment and the relative motilities of AVs and Rheb-AVs in CCCP-treated axons were quantified, respectively
Figure 8Knockdown (KD) of OPA1 in HeLa cells. WT and Mic60-KO cells were transfected with a scrambled control (Ctrl.) or siRNA pools against OPA1 (OPA1-KD) for 5 d. (A) Cell lysates were analyzed by western blotting. (B) TEM recordings from OPA1 depleted WT and Mic60-KO cells. Arrows point to a septum. (C,D) Quantification of the cristae morphology of OPA1 deficient WT (C) and Mic60-KO (D) cells. The number of CJs was normalized to the length of the OM (C). The number of septated mitochondria in Mic60-KO cells was estimated (D). (E,F) ET of mitochondria from OPA1-KD cells. WT cells were transfected with a scrambled control (E) or siRNA pools against OPA1 (F) for 48 h. Reconstructions of representative mitochondria are displayed. The OM is displayed in clear grey, the side of the IM that faces the matrix is shown in dark blue. The IM side that faces the IMS is shown in light blue.