EMBO/sd-character-level-ner huggingface dataset:
Figure 5(A) Average allelic ratio of single cells projected onto the UMAP plot. n = 220 X-linked genes per single cell. X-inactivation (average ratio <0.135) in red and X-reactivation (average ratio from 0.135 > 0.8) in green. Labels indicate the 5 different previously identified clusters. Dashed lines indicate the position of cluster borders (clusters obtained from Fig. 4D-F). (B) Distribution of single cells based on fluorescence intensity of XGFP reporter quantified by BD FACSDiva Software, plotted against the X chromosome average allelic ratio per cell. R and p-values calculated by Pearson's correlation are shown. Black line represents linear regression fitting. (C) Heatmaps of allele-specific ratios of X-linked genes in ESC, EpiLC, XGFP- PGCLC, Pre-meiotic, Meiotic and Mitotic clusters. X-inactive genes are shown in red (ratio ≤ 0.135), X-active genes in green (ratio > 0.135) and mono-allelic Xcas expression in blue (ratio between 0.5 and 1). Colour gradients used in between and above these two values as shown in the legend. Genes are ordered by genomic position and grouped according to the category to which they belong, indicated on the left side of the heatmap. n = 161 genes. (D) Average allelic ratios of X-linked genes within each category (Escapees, Early XCR, Late XCR) in ESC, EpiLC, XGFP- PGCLC, Pre-meiotic and Meiotic clusters. Shading denotes lower and upper Gaussian confidence limits based on the t-distribution. (E) Each dot indicates the average allelic ratio of a single X-linked gene belonging to the indicated category in XGFP- PGCLCs. The numbers above the bars indicate p-values (two-sample unpaired Wilcoxon-Mann-Whitney test with R defaults). Box plots depict the first and third quartiles as the lower and upper bounds of the box, with a band inside the box showing the median value and whiskers representing 1.5x the interquartile range.
Figure 2A Normalised cumulative nucleosome (150 ± 30 bp size class) position frequency profiles for 4013 S. pombe genes aligned at the transcription start site (TSS) plotted from low MNase (biorep1) and high MNase (biorep 2) datasets. P-values (calculated using a two-tailed unpaired t-test) for the difference in means between random n = 100 subsets of the frequency values at the points indicated by the dotted lines ("-4" nucleosome and "+4" nucleosome) are shown. B Nucleosome position frequency values for the coding regions of 4013 S. pombe genes were k-means clustered (k = 9) using the abo1Δ data from biorep 1 (low MNase) and displayed with positive values coloured yellow and other values coloured blue (left-hand panel). The cluster order was then used to display the equivalent wild type frequency values in the right-hand panel. C Level of histone gene mRNAs was determined by qRT-PCR. Data are the mean of four independent biological repeats and error bars are ±SEM. Two-tailed unpaired t-tests showed no significant differences (P > 0.05) between wild type and abo1Δ cells. D Whole cell extracts were subjected to western blotting with histone H3 (Abcam), histone H2A (Abcam) and tubulin antibodies. Examples of the primary data are shown (left) along with a quantification of histone H2A and H3 levels normalized to tubulin (right). Data are the mean of at least three independent repeats and error bars represent ±SEM. P-values were calculated using a two-tailed unpaired t-test.
Fig.Figure 1Schematic summary of tomogram analysis (not drawn to scale). SVs-pools: ribbon-associated (RA) SVs: first row, ≤ 80 nm from the ribbon surface (R, with filaments: dark green; without filaments: light green), membrane-proximal (MP) SVs: ≤ 50 nm membrane-to-membrane distance in the first row from AZ-membrane (blue) and ≤ 100 nm from the PD (pink) (with tethers: orange; non-tethered: yellow; docked: light orange). Tomographic 3D reconstructions of the RA-SV pool at wild-type (B-D) and OtofPga/Pga (E-G) in inhibitory (inhib, B,E), resting (rest, C,F) and stimulatory (stim, D,G) conditions (color codes see A). MP-SVs, PD and AZ-membrane in transparent. Scale bar, 100 nm. Comparable mean SV number per ribbon in wild-type at the distal (Ds., H) and the proximal (Pr., I) part of the ribbon. More SVs are observed at inhibitory OtofPga/Pga ribbons compared to inhibitory wild-type synapses at both ribbon parts, and further at OtofPga/Pga synapses under rest in the Pr. part. Data are presented as mean ± SEM; the red dotted line highlights the wild-type mean SV number per ribbon under rest as a reference point; Representative virtual sections of wild-type (J) and OtofPga/Pga (K) ribbon synapses under inhibition. Counted RA-SVs are marked with white asterisks. Magnification, 12,000x; scale bar, 100 nm. Exemplary virtual sections of wild-type and OtofPga/Pga under inhib (L,M), rest (L',M') and stim (L'',M''). At OtofPga/Pga SVs are densely packed. Blue arrowheads indicate proteinaceous filaments between wild-type SVs. Magnification 12,000x; scale bar, 100 nm.
Figure 5A: In biological membranes, there is mismatch between the natural thickness of the lipid bilayer and the hydrophobic length of membrane proteins. B: In order to cover hydrophobic protein regions, the bilayer has to deform (to bend and to stretch). C: If multiple proteins are present, the mismatch imposes curvature stress on each monolayer. The higher the protein concentration the higher is the curvature. D: We hypothesize that remodeled CL stabilizes the deformation of the bilayer because it has both intrinsic negative curvature (wedge-shaped molecule) and high protein affinity. By stabilizing bilayer deformations, remodeled CL increases the cohesion of protein-crowded membranes.
a) Three-dimensional surface rendering of deconvolved stacks of ATG5 (green) and ATG8 (magenta) domains on the forming autophagosome.f4(a) Three-dimensional surface rendering of deconvolved stacks of ATG5 (green) and ATG8 (magenta) domains on the forming autophagosome. Four representative objects are shown, disposed left to right according to a temporal sequence deduced from integration of real-time data on individual structures. Additional views are presented below structures I, II and IV. Note the planarity of structure I, the first morphologically informative object in the ATG5 ring formation phase, evident in two profile views. In structure II, the ATG5 torus is disposed around the aperture of the phagophore, as clearly seen with a top view of the reconstruction of the upper third of the object (arrow, green mesh for the ATG5 surface). In structure IV, corresponding to a late step in the ATG5 ring decay phase, the cavity of the neo-formed autophagosome is detectable in the additional mid-section. (b) Two-dimensional horizontal views of representative structures of type I and II, corresponding to the planes of maximum ATG5 intensity of the z-stacks. Fluorescence intensity profiles along a line crossing the confocal image confirm the disposition of ATG5-GFP and mCh-ATG8 visible in the 3D surface reconstructions. Scale bar, 2 μm.
a–c) At 12–16 h post-transfection with GFP–LC3, NIH3T3 (a, b), MEF (b) or HCT116 (c) cells containing vector or the FLIP gene were treated with Hank's solution for 2–4 h (a) or with 2 μM rapamycin for 1–3 h (b, c).figf1(a-c) At 12-16 h post-transfection with GFP-LC3, NIH3T3 (a, b), MEF (b) or HCT116 (c) cells containing vector or the FLIP gene were treated with Hank's solution for 2-4 h (a) or with 2 μM rapamycin for 1-3 h (b, c). Subsequently, GFP-LC3 was detected using an inverted fluorescence microscope. Scale bar, 10 μm (a). The number of GFP-LC3-positive dots per cell was counted using a fluorescence microscope. Data are mean ± s.e.m.; n = 200-300 cells; three independent experiments; *P 0.01; **P 0.05) (b, c). At 60 h post-transfection with control siRNA or cFLIP siRNA, MEF cells were transfected with GFP-LC3 (c). (d) Autophagosomes were visualized through scanning electron microscopy. Scale bar, 50 nm. The arrows indicate autophagosomes. Immunoblots (IB) of cFLIP and actin expression are shown in Supplementary Information, Fig. S1c. (e, f) NIH3T3 vector and NIH3T3-KSHV-vFLIP cells (e) or TREX-BCBL-Vector and TREX-BCBL-vFLIP cells (f) were treated with Hank's solution or 2 μM rapamycin for 2-4 h, respectively, followed by IB with anti-LC3 and anti-actin antibody. (g) At 12-16 h post-transfection with GFP-LC3, TREX-BCBL-vector and TREX-BCBL-vFLIP cells were treated with doxycycline for 24 h, followed by incubation with 2 μM rapamycin for an additional 12 h. GFP-LC3 was detected using an inverted fluorescence microscope. Scale bar, 5μm. The number of GFP-LC3-positive dots per cell was counted using a fluorescence microscope. Data are mean ± s.e.m.; n = 200-300 cells; three independent experiments; *P 0.01). (h) Autophagosomes were visualized by scanning electron microscopy. Scale bar, 50 nm.
Figure 9A. STING protein expression of 58 colon adenocarcinoma specimens within the TCGA COAD collection were obtained from the CPTAC project website. Samples were partitioned into STING-Low (non-detectable STING protein, N=15) and STING-Hi (detectable STING protein, N=43) groups. Kaplan Meier curve is shown with P value indicated. B. STING RNA expression from the same 58 specimens were partitioned into STING-Low (bottom 15 samples) and STING-Hi (top 43 samples) groups. Kaplan Meier curve is shown with P value indicated. C. Stage II and Stage III patients from the TCGA COAD dataset were partitioned into STING-Hi (top 1/3 of samples, N=87) and STING-Low (bottom 1/3 of samples, N=87). Kaplan Meier curve is shown with P value indicated. D. From a published colorectal cancer chemotherapy response study, STING RNA expression in responders and non-responders were compared, with each dot representing a tumor. E. A model summarizing our findings.
Figure 5Mass spectrometric analysis of co-immunoprecipitation experiments. LINC00665 ORF-His plasmid was transfected into MDA-MB-231 cells. Whole-cell lysates were subjected to co-immunoprecipitation using anti-His antibody and immunoblot analysis with CIP2A antibody to verify that CIP2A bound to CIP2A-BP. LINC00665 ORF-His plasmid was transfected into MDA-MB-231 cells. Whole-cell lysates were subjected to co-immunoprecipitation by CIP2A antibody and immunoblot analysis with CIP2A-BP antibodies to verify that CIP2A-BP bound to CIP2A. Endogenous CIP2A-BP and CIP2A were co-immunoprecipitated in MCF-10A cell line. LINC00665 ORF-His plasmid was transfected into MDA-MB-231 cells. Whole-cell lysates were treated with 30 mg/mL RNase A for 30 min, the indicated complexes were co-immunoprecipitated by anti-His or anti-CIP2A antibody, and CIP2A or micropeptide CIP2A-BP were detected. Upper: A diagram of the domain structure of full length CIP2A protein. Red: RIP=Required for Interaction with PPP2R5C; Orange: RH=Required for Homodimerization; Blue, CC=Coiled coil. Lower: Co-immunoprecipitation coupled western blot analysis assays revealed the interaction of CIP2A-BP with CIP2A via the N-terminus domain of CIP2A. Upper: A diagram of the domain structure of CIP2A protein fragment (1-245). Red: RIP=Required for Interaction with PPP2R5C. Lower: Co-immunoprecipitation coupled western blot analysis assays revealed the interaction of CIP2A-BP with CIP2A via the N-terminus domain of CIP2A (amino acid 159-245). Co-immunoprecipitation assays showed that the interaction between CIP2A and B56γ in TNBC cells was affected by CIP2A-BP expression.
Figure 5Schematic of the cytoskeletal inhibitors and their targets used in the experiments. SIM imaging of mouse T cells activated for 5' on APS, then treated with the indicated inhibitors or vehicle control. Shown are the representative SIM images (B), and quantification of actin foci (from the phalloidin images), talin, or pCasL in the contact interface (normalized to the mean value in the control case, C), and measured cell morphology aspect ratios (D, E). Since CK666 treatment reduces F-actin intensity Kumari et al., 2015(), the 'actin' image of the CK666 treated cell in (B) was contrasted differently than the 'control' and 'Blebb.' cases for a clearer visualization of F-actin. p values, *P =0.02, **P =0.001, n.s. >0.05. For the rest of the comparisons between the control and treatment cases, p <0.0001. For the bar graph in (B), n for foci groups are 214, 83, 92, 98; for pCasL groups are 100, 68, 91, 92; and for Talin groups are 102, 70, 89, 93 respectively. p value for the n.s. cases is > 0.05, *P =0.02. In the foci, pCasL and talin groups, when not indicated, the p values are <0.0001 using Mann-Whitney two-tailed test. Central values in the graphs represent Mean and error bars represent ± SEM. These experiments were repeated at least thrice with similar results. Scale bar, 5µm. Simulation of synaptic F-actin network following localized myosin inactivation and inactivated myosin-mediated F-actin cross-linking within the area marked with dotted line in the left image. The graph shows the predicted alteration in mechanical stress profile, as myosin is inactivated crosslinked in the defined region (marked by an arrow), (G). Experimental confirmation of localized actomyosin crosslinking and symmetry breaking. LifeAct-GFP expressing T cells were incubated with activating substrates, treated with vehicle control or 1µM Azido-blebb and their contact interface imaged using low excitation laser power (0.5 mW), using a spinning disc confocal microscope (H). The montage shows the shape change and movement that synapse undergoes after it is exposed to a brief flash of blue light crosslinking Azido-Blebb and actomyosin network within the illuminated area (shown with a box in the image). The deflection in center of mass (COM) within a minute was calculated and plotted for at least 10 cells in each case (graphs in I). The left graphs in (I) show individual COM trajectory, and the graph on the right shows average COM movement of cells within a minute post photo-inactivation of myosinII. This experiment was repeated twice with similar results. Scale bar, 5µm.
Figure 2(A) The indicated cell lines were treated with either 1µM MK-2206, 1 µM AZD5363, 1µM GDC0941 or 1µM BKM120 for the indicated times. Cell lysates were subjected to immunoblot analysis with indicated antibodies. (B) The indicated cells were treated as in (A) and SGK3 was immunoprecipitated from the lysates using an anti-SGK3 antibody. The immunoprecipitates (IP) were subjected to in vitro kinase assay by measuring phosphorylation of the Crosstide substrate peptide in the presence of 0.1 mM 32PATP in a 30 min 30oC reaction (upper panel) followed by immunoblot analysis with the indicated antibodies (lower panel). Kinase reactions are presented as means ± SD for triplicate reaction.
Figure 4A, Schematic of endogenous ORP2 tagging and auxin-inducible degron system. B, Control (endogenously GFP-tagged ORP2 without AtAFB2) and degron ORP2 cells were co-plated and treated with IAA for 1 h followed by live-cell confocal imaging. Dotted line separates GFP-ORP2 containing and depleted cells, asterisk indicates the area of ORP2-depleted cells, inset shows no prominent PM enrichment of endo-GFP-ORP2. C, Confocal image of endo-GFP-ORP2 and pFAK immunofluorescence staining in saponin pre-permeabilized cells loaded with LDL for 2 h before fixation. GFP antibody was used to enhance endo-GFP-ORP2 signal. Insets show examples of "no contact", "contact" and "overlap" of organelles. Dashed line indicates cell edge. D, Quantification of pFAK, integrin β1, and NPC1 association with endo-GFP-ORP2 in C. n = 380 pFAK; 353 integrin β1; 232 NPC1 organelles in 2 cells. E, Representative confocal images of triple labeling for endogenous ORP2, NPC1-mCherry and integrin β1 after 2 h of LDL loading, and saponin permeabilization prior to fixation. Dashed line indicates cell edge. F, Schematic outline for plasma membrane D4H labeling in combination with ORP2 degron system, and representative widefield epifluorescent images of co-plated cells after 4 h of LDL loading. Asterisk indicates ORP2-depleted degron cells. Dashed lines separate ORP2 expressing and depleted cells. G, Quantification of F. Mean ± SD, n = 20 fields of cells pooled from 2 independent experiments. Student's t-test. H-J, Control and degron-GFP-ORP2 cells were incubated in 5 % LPDS for 1 day, loaded with LDL + IAA for the indicated times, and LDLR (H) and hydroxymethylglutaryl- CoA reductase (HMGCR) (I) mRNA levels were quantified with qPCR or [3H]-oleic acid (OA) incorporation into cholesteryl esters (CE) was analyzed (J). Mean ± SD, n = 2-3 RNA samples from independent experiments with triplicate measurements (H,I); n = 3 (-LDL and 6 h); n = 6 (all other time points) pooled from 2 independent experiments (J).
Correlation between intra-parasitic hGS levels and schizont size on day 3 and 5 post invasion.Figure 5A) The total hGS signal within intracellular NF135 and B) NF175 schizonts on day 3 and 5. Each graph represents a biological replicate where each dot represents an individual schizont. C) The slope of the linear regression of NF135 and NF175 on day 3 (circle) and day 5 (square) p.i.. Each dot represents one biological replicate. D) The R squared value of the predicted linear relationship of hGS levels versus schizont size on day 3 and day 5 p.i.. Each dot represents one biological replicate. E) The Spearman's correlation coefficient between hGS levels and schizont size of NF135 and NF175 on day 3 and day 5 p.i.. Each dot represents one biological replicate.
Figure 2Top - Representative RAB7 expression in base of stomach units (ZCs) in vehicle or 24h HDT by immunohistochemistry. Arrowheads point to RAB7 vesicle aggregates. Scale bar 20µm. Bottom - Representative ATF3 expression in base of stomach units (ZCs) by immunohistochemistry. ATF3 appears in nuclei following 24h high-dose tamoxifen (HDT). Scale bar 20µm. Stomach unit base outlined by dashed black line. Atf3 mRNA expression by qRT-PCR in vehicle, 6 hour, 24 hour, and 72 hour HDT treatment. Each data point represents the mean of triplicate technical replicates from a single mouse, n=3-4 mice in independent experiments. Significance determined by One-way ANOVA, Tukey. Error bars denote standard deviation. Western blot of ATF3 expression over HDT and CER timecourses with β-Actin loading control. Top­ - Representative RAB7 staining in pancreatic acinar cells. Arrowheads indicate RAB7 vesicles. Scale bar 20µm. Bottom - Representative ATF3 expression in pancreatic acinar cells. Nuclei express ATF3 after 24h cerulein (CER) injury. Scale bar 20µm. ChIP-PCR on mouse stomach tissue at vehicle and 6 hour HDT. Chromatin probed with ATF3, Histone H3 antibodies, or a Rabbit IgG control. Also included is the 2% chromatin input and water to control for DNA contamination. Rab7b amplicon is from a conserved, putative ATF3-binding site in the first intron. Amplicon from Atf3 is a positive control from a previously characterized ATF3-binding site; negative control is from a site in Rab9a lacking putative ATF3 binding motifs.
Shits induced cell-autonomous glia degeneration in optic lamina.Fig 1H&amp;amp;E staining of adult head sections of (A) repo>GFP.nls and (B) repo>Shits1 at 29°C for 3 days. Lamina degeneration was identified as vacuoles in (B). Cryosection of adult (C) repo>H2B-RFP exhibited the expression of the nuclear red fluorescent protein (RFP) in the glia (epithelialglialnucleus: arrowhead) and (D) repo>Shits1 at 28°C for 12 days. Vacuoles were identified in the lamina neuropile (D). Epithelial (arrowhead) (E), marginal (arrow) and distal satellite (arrowhead) glianuclei (F) are labeled by HisCl-GAL4 and NP2109-GAL4, respectively. Note that HisCl-GAL4 is not expressed in all epithelialglia. (G, H) Weak lamina vacuolization was identified in HisCl>Shits1 (G, 0.92%) and NP2109>Shits1 (H, 1.46%) at 29°C for 14 days. (I, J) A single MARCMglia clone (GFP, green) expressed Shits1 at 21°C (I) and 29°C (J). (J) A vacuole occurred within a glial clone. DAPI (white) stains the nuclei (C, D, and G-J). (K) The percentage of the vacuole area in the lamina of the repo>Shits1flies at 28°C progressively increased. n indicated in each column. P-values were calculated using one-way ANOVA with Bonferroni's post-test. (L) When repo>Shits1flies were shifted to 28°C for 12 days and then shifted to 17°C for 9 additional days, the vacuolization was not alleviated. P-values were calculated using one-way ANOVA with Tukey's post-test. n indicated in each column.
WDR26/RanBP9-containing hGID complexes assemble ring-shaped tetramers A.Figure 3A. Chromatogram of the SEC-MALS analysis at a flow rate of 0.5 ml/min, showing the UV curve and the Rayleigh ratio (1/cm) at a scattering angle of 90 degrees (left y-axis), together with the molar mass (MDa) of the peaks determined by MALS (right y-axis). The peak fraction with a homogenous size distribution at around 1.1 MDa is labeled with gray dotted lines. B. XL-MS analysis of the 5-subunit hGID complex (RanBP9, WDR26, RMND5a, MAEA and TWA1). Cross-links within different complex subunits are indicated by green lines, and cross-links within the same subunit with purple lines. The predicted domain boundaries of the different subunits are colored as follows: LisH domain in light orange, CTLH domain in dark orange, RING domains in blue, TWA1's CRA domain in light blue and RanBP9's CRA domain in light grey, WD40 in dark cyan, and SPRY in light magenta. C. Rotational views of the cryo-EM map of the 5-subunit hGID complex (RanBP9, WDR26, RMND5a, MAEA and TWA1) at 11.2 Å resolution. The higher resolution cryo-EM map at 9 Å produced by particle symmetry expansion is shown in blue. The dotted rectangle highlights the positions of the fitted WD40 domains from two tetrameric building blocks. D. Western blotting of samples following ectopic overexpression of HBP1 either alone, or with full-length (FL) or WD40-truncated WDR26 (ΔWD40) in HEK-293T cells. HBP1 levels were monitored in cells treated with MG132 or DMSO for 12-14 h (n=3). E. Western blot analysis of in vitro ubiquitinated HBP1 in the presence of wild type WDR26 (WT) or the WDR26 (ΔWD40) mutant (n=2).
PHD1 hydroxylase is required for p53 phosphorylation upon chemotherapy treatment.Figure 4Western blot for p53 pS15, p53, and vinculin (vinc) in PHD1-silenced HCT116 upon treatment with 300 μM 5-FU for 8 h with or without 0.5 mM DMOG for 26 h. Vinculin (vinc) is used as a loading control. Western blot detection of PHD1-Flag and p53 in whole cell extracts (WCE) or after immunoprecipitation against the Flag-tag of overexpressed PHD1-Flag in HEK293T cells. Western blot detection of PHD1-Flag and p53 after immunoprecipitation against the Flag-tag of recombinant PHD1-Flag incubated with recombinant p53. Western blot for p53 and PHD1 in WCE or after immunoprecipitation of endogenous p53 in p53wt/wt HCT116 cells, silenced with a Scr control, siPHD1, or sip53, or after immunoprecipitation of p53wt/wt HCT116 cell lysates with an IgG control.
a, b) Interaction of MutStab mutants of Beclin 1 with Atg14L or UVRAG in vivo.f6(a, b) Interaction of MutStab mutants of Beclin 1 with Atg14L or UVRAG in vivo. HEK 293T cells were transfected with GFP-tagged Beclin 1 variant alone (b) or together with FLAG-UVRAG (b). The lysates were immunoprecipitated by GFP antibody, followed by western blot analysis with anti-Atg14L antibody for detecting binding to endogenous Atg14L (a) or with anti-UVRAG antibody for detecting binding to the FLAG-tagged UVRAG protein (b).