EMBO/sd-character-level-ner huggingface dataset:
Time course of COPI depletion shows that Golgi disperses before AVs are formed, but Golgi dispersal does not cause AV formation.Figure 2.Time course of COPI depletion shows that Golgi disperses before AVs are formed, but Golgi dispersal does not cause AV formation. (A) COPI subunits were depleted in 293/GFP-LC3 cells and analyzed 24, 36, and 48 h after addition of siRNA. Immunoblotting for β′- and α-COP confirms loss of COP subunits. (B) GFP-LC3-I and -II were monitored by immunoblots in parallel lysates using anti-GFP antibodies at the indicated times. (C) After 24 h siRNA treatment, depletion of β′-COP caused morphological changes and a reduction in perinuclear population of GM130. Box indicates enlarged area. (D) Rab1a/b was depleted using siRNAs for 48 h. Lysates were probed with anti-Rab1 to confirm depletion. (E) β′-COP or Rab1a/b were depleted as in D, and analyzed by indirect immunofluorescence using anti-GM130 and GFP fluorescence. In β′-COP panel, the asterisk indicates cells that did not show an accumulation of GFP-LC3-positive AVs.
Heat shock and recovery leading to thermotoleranceA.Figure 1A. Schematic diagram of the temperature shift procedure to render MCF7 cells into thermoresistant MCF7aTT cells. B. Sensitivity of MCF7 and MCF7aTT cells to heat shock at 45 oC as determined by trypan blue staining. The graph shows means +/- SD, n = 3. Numbers indicate p values. C. Lysate of MCF7 and MCF7aTT cells (rendered thermoresistant through the protocol described in Fig. 1A) were separated by sucrose density gradient centrifugation, and P/M ratios were determined by integration of monosomal and polysomal peak areas (bar graph, mean P/M ratios +/- SD, n = 6). Numbers above bars indicate p values calculated with the two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli.
Figure 5A Co-staining of SNX10-Flag and LPS in Caco-2 cells transfected with SNX10-Flag after treatment with OMVs (100 μg/mL) for 24 h. Scale bar: 20 μm. B LPS levels in the cytosolic and residual fractions of Caco-2 cells treated with OMVs (100 μg/mL) for the indicated time were detected by LAL assay (n = 6 independent experiments). Data information: Data are means ± SD. One-way ANOVA followed by Bonferroni post hoc test was used for statistical analyzes. *p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant. C Caco-2 cells were transfected with Ad-vector or Ad-SNX10, followed by treatment with or without OMVs (100 μg/mL) for 24h. LPS levels in the cytosolic and residual fractions of Caco-2 cells were evaluated (n = 6 independent experiments). Data information: Data are means ± SD. One-way ANOVA followed by Bonferroni post hoc test was used for statistical analyzes. *p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant. D WT and SNX10 KO Caco-2 cells were transfected with Ad-vector or Ad-SNX10, followed by treatment with or without OMVs (100 μg/mL) for 24 h. LPS levels in the cytosolic and residual fractions of Caco-2 cells were detected (n = 6 independent experiments). Data information: Data are means ± SD. One-way ANOVA followed by Bonferroni post hoc test was used for statistical analyzes. *p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant. E, WT and SNX10 KO Caco-2 cells were treated with OMVs (100 μg/mL) for the indicated time and stained with antibodies against LPS and Rab5 (E). Scale bar: 20 μm. F WT and SNX10 KO Caco-2 cells were treated with OMVs (100 μg/mL) for the indicated time and Colocalized LPS and Rab5 were quantified by ImageJ software (F) (n = 3 independent experiments, n = 18 fields analysed). Data information: Data are means ± SD. One-way ANOVA followed by Bonferroni post hoc test was used for statistical analyzes. *p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant. G LPS levels in the cell culture supernatants of WT and SNX10 KO Caco-2 cells stimulated by OMVs (100 μg/mL) for the indicated time were detected by LAL assay (n = 6 independent experiments). H LPS levels in the cytosolic and residual fractions of Caco-2 cells stimulated by OMVs (100 μg/mL) with or without chloroquine (CQ) (200 nM) or bafilomycin A1 (Baf A1) (50 nM) for the indicated time were detected by LAL assay (n = 6 independent experiments). Data information: Data are means ± SD. One-way ANOVA followed by Bonferroni post hoc test was used for statistical analyzes. *p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant.
Figure 4NF-κB and JAK/STAT signalling drive innate immune responses. (A-C) Fold induction at 24 hpi of (A) CXCL10, (B) IFIT1 or (C) IL-6 mRNA (qRT-PCR) after Calu-3 infection with SARS-CoV-2 over a range of MOIs (0.004, 0.04, 0.4 TCID50VERO/cell) in the presence of 10 μM TPCA-1 or DMSO as shown. (D-F) Fold induction at 24 hpi of (D) CXCL10, (E) IFIT2 or (F) IL-6 mRNA (RT-qPCR) after Calu-3 infection with SARS-CoV-2 over a range of MOIs (0.0004, 0.004, 0.04, 0.4 TCID50VERO/cell) in the presence of 2 μM Ruxolitinib (Rux) or DMSO as shown. (G-H) Viral genomic and subgenomic E RNA at 24 hpi (RT-qPCR) with DMSO or TPCA (G) or Rux (H) treatment. Data information: (A-H) Mean +/- SEM, n=3, statistical comparisons are made by unpaired t test comparing inhibitor-treated to mock-treated SARS-CoV-2 infected conditions at each MOI and each timepoint. * (p<0.05), ** (p<0.01), *** (p<0.001), **** (p<0.0001). (I) Live/dead stain count from non-adherent cells recovered from supernatants of SARS-CoV-2 infected Calu-3 cultures (MOI 0.04 TCID50VERO/cell) 48hpi (flow cytometry). Mean +/- SEM, (n=3). One Way ANOVA comparison of inhibitor-treated infected cells to mock-treated infected cells. *** (p<0.001).
Transcriptional and functional characterization of in vitro PrE cells.Figure 3Principal component analysis of indicated samples. GFP sorted cells express the Nanog>GFP reporter and mCherry sorted cells the Gata6::mCherry reporter. Morula (MOR). Scatter plots of log2 fold change (FC) gene expression changes (∆mRNA) between naïve Hdac3-/- or Dax1-/- mutants and WT mESCs (left), between Nanog>GFP-expressing Hdac3-/- or Dax1-/- mutants and WT cells (middle), and between Gata6::mCherry-expressing Hdac3-/- or Dax1-/- mutants and Nanog>GFP-expressing WT cells (right). Cluster 5 genes are colored and selected PrE TFs labeled. Representative control embryo, and embryos that were injected with labeled SLRA-differentiated WT cells purified for Nanog>GFP-expression and mutant cells purified for Gata6::mCherry-expression. Embryos are outlined with continuous line. Dashed lines indicate embryonic EPI and PrE compartments. Arrowheads point to injected cells. Scale bar is 25 µm. Quantification of the cell fate (based on expression of Nanog and Sox17) and the localization of injected cells. Average and SD of two independent experiments with at least 5 embryos per condition and experiment. tSNE maps of scRNAseq of WT, Dax1 and Hdac3 mutants after 3.5d in SLRA. Cells are color-coded by k-means clusters (top left), genotype (top right), and expression of indicated genesets (bottom). Expression distribution of Gata6 and cluster 5 genes in single cells of indicated scRNAseq clusters, and E4.5_EPI and E4.5_PrE (Mohammed et al, 2017).
Administration of corticosteroids promotes the persistence of H.Figure 1Administration of corticosteroids promotes the persistence of H. influenzae in miceA-D C57BL/6 mice were infected with H. influenzae in PBS with or without supplementation with beclomethasone. Animals were killed at 1, 3, 5 and 7 days after infection, and 10-fold serial dilutions of the lung homogenates (A) and spleen homogenates (B) were plated to determine the bacterial load. Airway inflammation represented by total and differential cell counts in bronchoalveolar lavage fluid (BALF). The number of total leukocytes and in particular of neutrophils, monocytes and lymphocytes recruited in the airways was analyzed in BALF after day 3 (C) and day 7 (D) of infection.Data information: Values represent the mean ± standard deviation (SD). The data are pooled from three independent experiments. Statistical significance by two-tailed Student's t-test is indicated: *P < 0.05, **P < 0.01.
Figure 3(A) FACS profile of cells transfected with nondegradable KEN-box mutant sororin-FLAG (sorKBM-FLAG) after synchronization in G1-phase and propidium iodide staining. (B) Immunofluorescence microscopy experiment to determine transfection efficiency. (C) Western blot of fractionated lysates to detect sororinKBM-FLAG. (D) Quantification of SMC3-LAP residence time on chromatin after transfecting cells with sorKBM-FLAG in G1-phase. Error bars denote s.e.m.; n > 15 cells per condition. (E-F) Analysis of cell cycle distribution and transfection efficiency using cells synchronized in G2-phase.(G) Quantification of stably chromatin-bound SMC3-LAP from cells expressing nondegradable sororin in G2-phase. Cells were treated with siRNA as indicated. (H) Western blot showing fractionated and nuclease digested chromatin from cells synchronized in G1 and G2-phase after immunoprecipitation with FLAG antibodies.
Figure 1(a) Widefield immunofluorescence images of PFA fixed HeLa cells showing endogenous FLCN and LAMP1 staining in (top) normal growth or (bottom) starvation conditions. Orange arrows highlight association of FLCN with LAMP1 positive membranes in the peri-nuclear region. White dotted line shows cell periphery in starved condition. Scale bar = 10μm.(b) (left) Quantification of FLCN/LAMP1 association in growth and starvation conditions. A cell is scored as positive if 5 or more discrete FLCN/LAMP1 puncta were observed at 100x magnification using a widefield microscope. Data represents 60 cells from 3 independent experiments, error bars show S.E.M, *** = p<0.001. (right) Western blot shows levels of phosphorylated of S6K and 4EBP in whole cell extracts under the same conditions. (c) Western blot showing typical efficiency of FLCN knockdown in HeLa cells using a pool consisting of 4 oligonucleotides and 2 independent oligonucleotides from the pool. Graph shows relative FLCN expression (n=3, error bars show S.E.M.) (d) Representative confocal fluorescence microscopy images showing LAMP1 (magenta) distribution in HeLa cells under normal growth or starvation conditions in cells depleted of FLCN or control cells transfected with a non-targeting siRNA. The location of the Golgi (green) is highlighted by giantin staining.(e) Schematic illustrating application of the cumulative intensity distribution method for quantification of lysosome distribution.(f) Graph showing cumulative distribution of LAMP1 intensity in normal growth and starvation conditions in HeLa cells. P-value is determined by the extra sum of F-squares test following non-linear regression and curve fitting. Error bars show S.E.M. from 30 cells in 3 replicates.(g) Graph showing cumulative distribution of LAMP1 intensity in starvation conditions for control cells transfected with non-targeting siRNA or cells depleted of FLCN. Error bars show S.E.M. from 30 cells in 3 replicates.
Working model of centriolar cartwheel architecture (A) Occurrence of CID or fCID in species analyzed in this study (T.Figure 6(A) Occurrence of CID or fCID in species analyzed in this study (T. spp., T. agilis and T. mirabilis, blue background), and in select other organisms, not to scale, mapped on a taxonomic tree generated using phyloTv2 based on NCBI taxonomy - https://phylot.biobyte.de/. See Discussion in main text for references. (B) Schematic of cartwheel hub in T. spp. and T. agilis with 9-fold symmetrical CID (grey) connecting to the hub, equidistant between two spokes. (C) The CID (dark grey) in T. spp. and T. agilis is polarized distally relative to the hub element comprising two tightly superimposed ring elements - shown in different shades for clarity. (D) Schematic of cartwheel hub in T. mirabilis with continuous fCID (dark grey) along the proximal-distal axis, with potential thin connections to the hub, equidistant between two spokes. (E) Working model of cartwheel stacking, with alternation of two modes of SAS-6 ring stacking: pairs of rings are tightly superimposed with a rotational offset (dashed line) and such offset double rings are then stacked in register (solid line); the offset direction is consistent in T. spp., T. agilis and T. mirabilis, yet may be different in other species, as suggested in Paramecium (Klena et al). Surface representation of SAS-6 rings in different shades for clarity; homodimers highlighted by black contour. (F) Consensus model of periodicities in centriolar cartwheel along the proximal-distal axis. Surface representation of offset double rings tightly superimposed with ~3.2 nm periodicity, in register with the next such offset double ring located ~5.2 nm away, thus amounting to an overall periodicity of ~8.4 nm. Note that these values are based on the T. mirabilis 36% class, which has the most clearly resolved periodicities, yet does not have a CID as shown in this composite model, with related values in other species; see text for details. The CID exhibits a related ~8.4 nm mean periodicity in the two Trichonympha species. Two spoke periodicities are observed: close to the hub, pairs of spokes are ~8.4 nm apart, whereas spoke pairs merge towards the periphery, where their periodicity is hence ~16.8 nm, matching that of the pinhead and connected microtubules. spoke angles are merely illustrative and limited by the resolution of the available STA maps. alternative modes of spoke merging may be revealed by analyzing much larger sub-volumes and be present in other species
Figure 3(A) Structure of the reported PP2A-B56γ-hSgo1 binding interfaces and residues mutated in the hSgo1 3A and 4A mutants are shown. 3A refers to Y57A, N60A, and K62A mutations at the PP2A-C binding interface. 4A refers to L83A, K87A, Y90A, and C94A mutations at the B56 binding interface of hSgo1. (B-C) IP of YFP-hSgo1 from cells stably expressing the hSgo1 WT, 3A, and 4A using different salt conditions (50mM NaCl (B) or 150 mM (C)) followed by immunoblotting of indicated proteins and quantification by LiCor. Representative blots are shown. B56α signals were normalized to YFP and plotted. Error bars represent SD (n=3 for B and n=4 for C). (D) Localization of B56α in cells depleted of hSgo1 and expressing the indicated YFP-hSgo1 variants. Representative immunofluorescent images are shown. Scale bar, 5μm. (E) B56α signal intensity was quantified. B56α signal from each cell was determined from 5 kinetochore pairs and normalized to YFP-hSgo1 signal. Each circle represents an individual cell, and the average and SD are indicated. (F) Mitotic U2OS LacO Haspin CM cells expressing hSgo1-LacI-GFP variants or LacI-GFP (control) were stained for PP2A-C. Scale bar, 5μm. (G) PP2A-C signal intensity was quantified, normalized to GFP, and then plotted. Each circle represents an individual cell, and the mean fluorescent intensity is indicated. Representative of at least 3 independent experiments. (H) Schematic of the TurboID-hSgo1 approach. (I) Table summarizing the Log2 differences between hSgo1 WT, 3A and 4A.
Figure 4(A) Immunoblotting of XO8 GSCs and DFCs. (B) Immunoblotting analysis of Ub in XO8 GSCs and XO8 GSC-derived cells in the course of differentiation (P1-P7). (C) Similar to (B) except that XO6 GSCs were used. (D) Immunoblotting analysis of ubiquitin ligases and proteasomal subunits in XO8 GSCs and XO8 GSC-derived cells in the course of differentiation (P1-P7). (E) Similar to (E) except that XO6 GSCs were used. (F-I) In vitro proteasome activity assays were performed as described in Materials and Methods. Bars represent means +S.D. from three independent experiments. For statistical analysis, student t-test (two-sided, one type) were used. * P value: (F), 0.0039. (F-I) In vitro proteasome activity assays were performed as described in Materials and Methods. Bars represent means +S.D. from three independent experiments. For statistical analysis, student t-test (two-sided, one type) were used. * P value: (F), 0.0039; (G), 0.0052; (H), 0.0009, (I) DFC, 0.00032; (I)-HEK293, 0.0039; (I)-HeLa: 0.00051. (J) XO8 GSCs and XO8 DFCs treated with MG132 as described in Materials and Methods were labeled with annexin V and visualized using fluorescent microscopy. Scale bar, 100 μM. (K) Cells from (J) were subjected to flow cytometry analysis. Annexin V-positive cell numbers were obtained from three independent experiments and normalized to those of controls cells treated with DMSO. Bars, means +S.D. from three independent experiments. For statistical analysis, student t-test (two-sided, one type) were used. * P value: 6 hrs, 0.0012; 17 hrs, 0.0041. (L) Cells treated with 50 nM MG132 were subjected to immunoblotting analysis of apoptotic markers. (M and N) Similar to (L) except that XO6 and XO10 cells were used.
Figure 7A PGAM5 overexpression partially rescues the mitochondrial defects by Stx17 loss. Transmission electron microscope images of the indirect flight muscle in the indicated genotypes (a, Control; b, Stx17-/-; c, Stx17-/- and Stx17 overexpression; d, Stx17-/- and PGAM5 overexpression) of 7-day-old adult flies are shown. The bar graph on the right shows frequency of healthy and abnormal mitochondria presented as percentages (mean ± SEM) using the scoring system : Class 0, normal; Class 1, fuzzy or dilated cristae; Class 2, fragmented cristae and loss of electron density. *p < 0.05, #p < 0.001 vs. the same class of control (Dunnett's test). n = 50-119 mitochondria from three or four independent samples. Genotypes used were: +/y; Act5c-GAL4/+ (a), +/y; Act5c-GAL4/+; Stx17LL06330/Stx17LL06330 (b), +/y; Act5c-GAL4/UAS-FLAG-Stx17; Stx17LL06330/Stx17LL06330 (c), +/y; Act5c-GAL4/UAS-PGAM5; Stx17LL06330/Stx17LL06330 (d). Scale bars, 200 nm. B Simultaneous reduction of Stx17 and PGAM5 results in mitochondrial degeneration. a, Stx17+/-; b, PGAM5-/-; c, Stx17+/-, PGAM5-/-. The bar graph on the right represents the mitochondrial phenotypes classified p < 0.05, **p < 0.01 vs. the same class of Stx17+/- (Dunnett's test). n = 62-195 mitochondria from three independent samples. Scale bars = 1 µm. Genotypes used were: +/y; Act5c-GAL4/+; Stx17LL06330/+ (a), PGAM51/y; Act5c-GAL4/+ (b), PGAM51/y; Act5c-GAL4/UAS-Stx17-FLAG; Stx17LL06330/+ (c).
Figure 5Control or CSAG2 expressing H460 cells were treated with the indicated concentrations of doxorubicin (A) for 24 hr before cell viability was determined by alamarBlue (n=6 biological replicates). Control or CSAG2 expressing H460 cells were treated with the indicated concentrations of H2O2 (B) for 24 hr before cell viability was determined by alamarBlue (n=6 biological replicates). shCTRL, shCSAG2-1, or shCSAG2-2 stable HCT116 cells were treated with the indicated concentrations of doxorubicin (C) for 24 hr before cell viability was determined by alamarBlue (n=6 biological replicates). shCTRL, shCSAG2-1, or shCSAG2-2 stable HCT116 cells were treated with the indicated concentrations of H2O2 (D) for 24 hr before cell viability was determined by alamarBlue (n=6 biological replicates). (E-F) HCT116 cells were transiently transfected with the indicated shRNAs for 48 hr before treatment with 1 μM doxorubicin/ 0.4 μM TSA for 6 hr. Cell lysates were collected and blotted for the indicated proteins (E). Quantitation (n=3 biological replicates) of normalized cleaved PARP levels is shown (F). (G) H460 cells were transfected with wild type, Δ37, or Δ63 CSAG2 for 48 hr. Cells were then treated with the indicated concentrations of H2O2 for 24 hr before cell viability was determined by alamarBlue (n=6 biological replicates). (H) Inhibition of SIRT1 activity abolished CSAG2 induced cell survival under genotoxic stress. CSAG2 over-expressing H460 cells were treated with the indicated concentrations of H2O2 with or without 1 μM EX-527 for 24 hr before cell viability was determined by alamarBlue (n=6 biological replicates). Control or Myc-CSAG2 expressing HCT116 p53 -/- cells were treated with the indicated concentrations of H2O2 (I) for 24 hr before cell viability was determined by alamarBlue (n=6 biological replicates). Control or Myc-CSAG2 expressing HCT116 p53 -/- cells were treated with the indicated concentrations of doxorubicin (J) for 24 hr before cell viability was determined by alamarBlue (n=6 biological replicates). shCTRL, shCSAG2-1, or shCSAG2-2 stable HCT116 p53 -/- cells were treated with the indicated concentrations of H2O2 (K) for 24 hr before cell viability was determined by alamarBlue (n=6 biological replicates). shCTRL, shCSAG2-1, or shCSAG2-2 stable HCT116 p53 -/- cells were treated with the indicated concentrations of doxorubicin (L) for 24 hr before cell viability was determined by alamarBlue (n=6 biological replicates). Control or Myc-CSAG2 expressing H1299 (p53 mutant) cells were treated with the indicated concentrations of H2O2 (M) for 24 hr before cell viability was determined by alamarBlue (n=6 biological replicates). Control or Myc-CSAG2 expressing H1299 (p53 mutant) cells were treated with the indicated concentrations of doxorubicin (N) for 24 hr before cell viability was determined by alamarBlue (n=6 biological replicates).
Figure 2A, Schematic of high-efficiency infection of neuronal cultures by AAV1 viruses followed by detection of AMPA receptor subunits. B, Western blot analysis of total GluA1 expression in Ctrl or Fxr1 overexpression (Fxr1) condition during upscaling (Ctrl/Veh n=5, Ctrl/TTX n=5, Fxr1/Veh n=6, Fxr1/Veh n=6). One way Anova with Dunnett's Multiple Comparison Test ***p<0.001. C, Western blot analysis of total GluA1 expression in Ctrl or Fxr1over condition during downscaling (Ctrl/Veh n=4, Ctrl/TTX n=4, Fxr1/Veh n=4, Fxr1/Veh n=4). D, RT-qPCR measurement of Gria1 mRNA in Ctrl or Fxr1over condition during upscaling (Ctrl/Veh n=3, Ctrl/TTX n=3, Fxr1/Veh n=3, Fxr1/Veh n=3). One way Anova with Bonferroni's Multiple Comparison Test ***p<0.001. E, Schematic representation of tagging of Luciferase cDNA with 5'UTR, 1-900CDS, 900-1800CDS, 1800-2700CDS, and 3'UTR of GluA1 gene. F, Measurement of relative luciferase signal after co-transfection of different tagged luciferase constructs along with GFP (Ctrl) or GFP-Fxr1 (Fxr1) plasmids. n=5 in each condition, Student's T-test *p<0.05, ***p<0.001. G, Immunostaining for surface GluA1 in GFP (Ctrl) or GFP-Fxr1 (Fxr1 over) infected cultures after treatment with Veh, TTX or BIC for 48h. H, Percentage of surface GluA1 relative to the mean of Ctrl/Veh condition (Ctrl condition: Veh n=63, TTX n=34, BIC n=29, Fxr1 condition: Veh n=56, TTX n=30, BIC n=29 ). One way Anova with Bonferroni's Multiple Comparison Test *p<0.05, **p<0.01, ***p<0.001.