EMBO/sd-character-level-ner huggingface dataset:
Apg9p localizes to large perivacuolar punctate structures.Figure 6Apg9p localizes to large perivacuolar punctate structures. Immunofluorescence microscopy of strains CTD1 (apg9Δ), KSL12C (vps4Δ), and SKD6-1D (apg6Δ) transformed with the multicopy plasmid 3×HA APG9. Cells were grown in YPD to log phase (vegetative), and incubated further in SD(−N) medium with 1 mM PMSF for 3 h (starvation). The cells were fixed with formaldehyde and examined by immunofluorescence microscopy as described in Materials and Methods. Anti-HA and FITC-conjugated antibodies mark Apg9p (left), and the vacuole can be visualized by Nomarski optics (middle). An overlay is shown in the right panels.
Figure 5Specific phospholipase A1 (PLA1) activity measurements of Tle1H, SVgrG-Tle1H complex, and isolated SVgrG. Thermomyces lanuginosus lipase (TLL) was used as positive standard for PLA1 activity. Mean values and standard deviation from at least three independent assays are shown. Statistical analysis relative to the Tle1 activity is indicated. ***P<0.001 (P=0,0001998), unpaired two-sample Fisher-Pitman permutation test. VgrG inhibition of Tle1H. Specific phospholipase A1 (PLA1) activity measurements of purified Tle1H in the presence of 1:10 molar ratio of lysozyme from chicken egg white (Lyso) and increasing molar ratio (0, 1, 5, and 10) of SVgrG or SVgrG1-490. Inhibition experiments with SVgrG were repeated three times with independent protein preparations, each measured in triplicate. Inhibition experiments with SVgrG1-490 were repeated twice with two independent protein preparations, each measured in triplicate. Mean values and standard deviation are shown. Statistical analysis relative to the Tle1 activity is indicated. ns, non-significant (P>0.05), *P<0.05, **P<0.01, ***P<0.001, unpaired two-sample Fisher-Pitman permutation test. (P values: Lyso, 0.7006; VgrG 1:1, 0.004946; VgrG 1:5, 1.448e-07; VgrG1:10, 1.448e-07; VgrG1-490 1:1, 0.5627; VgrG1-490 1:5, 0.2977; VgrG1-490 1:10, 0.002631). Representative SDS-PAGE / Coomassie blue staining of the Tle1H, SVgrG and SVgrG1-490 purified proteins used in B (5 μg/well).
Figure 9(A) Induction of ycbB under the control of the ParaBAD promoter in BW25113 ΔmepK pHV63(ycbB) was studied by the disk diffusion assay. The clear zone around the disk containing L-arabinose indicates that production of YcbB inhibited growth. (B) Functional complementation of the mepK deletion of BW25113 ΔmepK pHV63(ycbB) was performed with the pHV30 vector or recombinant plasmids encoding each of the eight endopeptidases under the control of the PrhaBAD promoter. Induction of ycbB and of endopeptidase (ED) genes was performed with 0.2% L-arabinose and 1% L-rhamnose, respectively. BHI agar plates contained chloramphenicol (20 µg/ml) to counter-select loss of pHV63(ycbB).
Figure 1(A) Identification of Ams1 and Ape4 as Nbr1 interactors. Nbr1-associated proteins were identified by affinity purification coupled with mass spectrometry (AP-MS) analysis using YFP-FLAG-His6-tagged Nbr1 as bait. (B-C) Coimmunoprecipitation of Ams1 (B) and Ape4 (C) with Nbr1. Endogenously TAP-tagged Nbr1 was immunoprecipitated (IP) using IgG Sepharose beads. Cell lysates and immunoprecipitates were examined by immunoblotting (IB). (D-E) Subcellular localization of Ams1 (D) and Ape4 (E). Wild-type (WT), nbr1Δ, sst4Δ, and atg1Δ cells were grown to mid-log phase in EMM medium and examined by live cell imaging. In (D), Ams1 was endogenously tagged at its C-terminus with mCherry. In (E), Ape4 was endogenously tagged at its C-terminus with GFP and in the same cells GFP-binding-protein (GBP) fused 3xUb was expressed under the P41nmt1 promoter. Cpy1 is a vacuole lumen marker. Scale bar, 3 μm.
Development of cytosolic CA activity in mouse CA1 pyramidal neurons is based on sequential expression of isoforms VII and II.Figure 2Original single-cell pHi traces and their time derivatives from P5 and P14 WT and P14 CA VII KO neurons (baseline pHi 7.21, 7.13 and 7.11, respectively). Superfusion with CO2/HCO3−-free HEPES solution (upper horizontal bars) evoked an intracellular alkalinization, which in P14 WT was large and suppressed by 100 μM acetazolamide (AZ, lower horizontal bar), indicating the presence of CA activity (see Supplementary Materials and methods). The possible effect of AZ on extracellular CAs was excluded by adding 10 μM benzolamide (a poorly permeant CA inhibitor). Inset shows an overlay of the BCECF fluorescence signal and Dodt gradient contrast image of CA1 pyramidal neurons in P14 WT (scale bar 10 μm). Summary of the results obtained using the cytosolic CA activity detection method shown in A and quantified as the percentage of cells showing cytosolic CA activity. Data from CA II KO and CA II/VII KO neurons were obtained only at >P35. The number of cells tested is indicated for each bar. The animal numbers for WT mice at the different age points was n=3 (P5-8), n=2 (P10-11), n=3 (P14-16), n=2 (P17-18), n=2 (P20-30) and n=2 (>P35); for CAVII KO mice the numbers were n=3 (P5-8), n=2 (P10-11), n=5 (P14-16), n=3 (P17-18), n=4 (P20-30) and n=2 (>P35); and for CA II KO and CA II/CAVII KO mice the number was n=2 (>P35). Typical single pyramidal neuron pHi responses evoked by withdrawal of CO2/HCO3− (horizontal bar, HEPES) in slices from >P35 WT, CAVII KO and CA II/VII KO mice. The rate of rise and the amplitude of the alkaline shift are identical in WT (P39, baseline pHi 7.01) and CAVII KO neurons (P40, 7.06), whereas alkalinization develops much more slowly in the CA II/VII KO (P46, 7.04)
Figure 6Male, GADD45β+/+ (WT; n=16) or GADD45β-/- mice (KO; n=15) fasted for 24h (fasted) with (AD-G45b OE) or without (AD-NC) liver-restricted GADD45β over-expression (n = 7-8/group). Liver mRNA expression of Gadd45b (A) as well as fatty acid metabolic genes (B) encompassing transport (Slc28a2, Slc27a5, Cd36), intracellular binding (Fabp1, Dbi) and metabolism (Acly, Dgat1, Atgl, Hsl). C: Representative immunoblots of FATP2, CD36, FABP1 and GADD45β from liver whole tissue lysate (W) as well as fractionated organelles/intraceuular structures including nuclei (N), mitochondria (MT), microsomes (MS) and cytoplasm (C), from GADD45β+/+ (WT) and GADD45β-/- (KO) mice. D: Quantified band densities of FABP1 enrichment from fractions in C (n = 4/group). E: Liver fraction enrichment of FABP1 from male, GADD45β+/+ (WT) or GADD45β-/- (KO) fasted for 24h with (AD-G45b OE) or without (AD-NC) liver-restricted GADD45β over-expression (n = 4/group). Insert shows a representative FABP1 immunoblot. F: Liver fraction enrichment of FABP1 from obese/diabetic male db/db mice fasted for 24h with (AD-G45b OE) or without (AD-NC) liver-restricted GADD45β over-expression (n = 4/group). Insert shows a representative FABP1 immunoblot. G: FABP1 and GADD45B immunoblots from Flag-immunoprecipitations (IP-FLAG) or mock IP (IP-HA) from liver input samples from mice with (AD-G45b OE) or without (AD-NC) liver-restricted GADD45β over-expression. Shown is a representative immunoblot from 3 separate experiments using 3 different input samples per condition. H: Liver long chain acyl-CoA (LC-acyl-CoA) concentrations were determined in GADD45β+/+ (WT) or GADD45β-/- (KO) mice (H; n=6/ group) with (AD-G45b OE) or without (AD-NC) liver-restricted GADD45β over-expression (I; n =5/group). Liver LC-acyl-CoA concentrations were determined in wildtype (WT; C57Bl/6J) or obese/diabetic (db/db; BKS.Cg-m+/+ Lepr DB/J) with (AD-G45b OE) or without (AD-NC) liver-restricted GADD45β over-expression (J; n=4/group).
Figure 2(A) Overview of a reconstructed superresolution image of GFP-Bax overexpressing HeLa cells stained with AF647-anti-GFP-nanobodies. Image was acquired on fixed cells 3 h after apoptosis induction with STS. Dotted line shows the cell shape (see Fig EV3). Scale bar, 5 µm.(B) Magnified reconstructed superresolution image corresponding to the white rectangle in A showing the shapes of GFP-Bax WT structures (white arrows). Scale bar, 500 nm.(C) Gallery of typical GFP-Bax WT structures during apoptosis found in all the analyzed cells. Scale bars, 100 nm.(D) Overview (left) and 3 enlarged insets (right) of HeLa cells overexpressing GFP-Bax 1-2/L-6 stained with AF647-anti-GFP-nanobodies. Figure shows reconstructed superresolution images acquired without apoptosis induction. Scale bars, 5 µm (overview) and 500 nm (insets).
integrin.Figure 4Bright-field image of an E13.5 Flk1-Cre;Ilk∆/+;Itgb1∆/+ mouse embryo (referred to as "Control") with a heterozygous deletion of both Ilk and Itgb1 in endothelial cells, and a LSM image of a stained cross-section through its jugular lymph sac / primordial thoracic duct (jls/pTD). Scale bars: 500 and 100 µm, respectively. C, D Bright-field image of an E13.5 Flk1-Cre;Ilk∆/∆;Itgb1∆/+ embryo (referred to as "ILK & β1 integrin K.O.") with a homozygous deletion of Ilk and heterozygous deletion of Itgb1 in endothelial cells, and a LSM image of a stained cross-section through its jls/pTD. Scale bars: 500 and 100 µm, respectively. E-H LSM images of cross-sections through the jls/pTD of E13.5 control and ILK & β1 integrin K.O. embryos stained for the proliferation marker phospho-Histone H3. Arrows point to phospho-Histone H3-positive LEC LSM images of PLA dots composed of VEGFR3 and phosphorylated tyrosine (p-Tyr) on stained cross-sections through the jls/pTD of control and ILK & β1 integrin K.O. embryos. Arrows point to PLA dots within the Lyve1 stained area. Scale bars: 10 µm Number of LECs per jls/pTD section in E13.5 control or ILK & β1 integrin K.O. embryos LEC proliferation as determined by the number of phospho-Histone H3-positive LECs per jls/pTD section in E13.5 control or ILK & β1 integrin K.O. embryos Quantification of the PLA dots indicating VEGFR3 with phosphorylated tyrosine (p-Tyr) per LEC of E13.5 control or ILK & β1 integrin K.O. embryos
Ede1 and Aip1 are phospho‐regulated cytokinesis factorsGenes corresponding to the indicated proteins were replaced by wild‐type (wt) or Cdk phospho‐site mutant (xA) sequences and then conditionally fused to Clb2m or Clb2mΔCdk.Figure 6A Genes corresponding to the indicated proteins were replaced by wild‐type (wt) or Cdk phospho‐site mutant (xA) sequences and then conditionally fused to Clb2m or Clb2mΔCdk. Cytokinetic defects were scored by FACS analysis of DNA content as in Fig C. The individual values from two independent clones (red dots) as well as their average (bars) are shown. Student's t‐test was applied to compare matched wt and xA strains. c, control cells. B Cell chain formation in the samples from (A). The fraction of cells with > 2 cell bodies for each clone are represented with red dots and their averages by the black bar (n > 150). Significance is determined by Student's t‐tests for the indicated conditions. Scale bars, 5 μm. C The indicated strains were spotted in serial dilutions onto auxin‐containing plates and grown for 2 days. D Auxin was added to cultures of the indicated strains, and after 6 h, cells were sonicated and the number of attached cell bodies was counted (n > 200). Significance was calculated using a Wilcoxon rank‐sum test. E The fraction of cells in asynchronously proliferating cultures of the indicated strains with a DNA content of > 2C was determined by FACS analysis (n = 3). Error bars represent SD.
STARD3-mediated cholesterol transport in endosomes depends on S209.Figure 6A-E: HeLa/Ctrl (A), HeLa/STARD3 (B), HeLa/STARD3 S209A (C), HeLa/STARD3 S209D/P210A (D) and HeLa/STARD3 Conv-FFAT (E) cells were labeled with anti-STARD3 antibodies (magenta), anti-Lamp1 antibodies (green) and with the fluorescent cholesterol probe filipin (Cyan Hot). Nuclei are shown in gray (TO-PRO-3). Higher magnifications images (2x) of the area outlined in white are shown on the right. The filipin, STARD3 and Lamp1 merged image is labeled Overlay. Scale bars: 10 µm. Inset scale bars: 5 µm. F: Pearson's correlation coefficients between STARD3 and Lamp1 staining in HeLa/STARD3 cells. Each dot represents a single cell (34 cells from 4 independent experiments). Mean and error bars (SD) are shown. Note that STARD3 and Lamp1 signals are highly correlated. G: Relative fluorescence intensity of intracellular filipin signals in endosomes of HeLa/Ctrl, HeLa/STARD3, HeLa/STARD3 S209A, HeLa/STARD3 S209D/P210A and HeLa/STARD3 Conv-FFAT cells. Data are displayed as a Superplot (Lord et al, 2020) showing the relative filipin fluorescence intensity in endosomes of individual cells (small circles) from 6 independent experiments (mean of each experiment shown as a large circle). Independent experiments are color-coded. Means and error bars (SD) are shown as black bars. Kruskal-Wallis with Dunn's multiple comparison test (*P < 0.05; **P < 0.01; n=6 independent experiments).
a is dispensable for AID-induced SHM and DNA breaks.Figure 3(A-C) SHM analysis of the variable (V) region of the human BL2 cell line expressing an AID C-terminal mutant (JP8Bdel) fused to ER. Scheme shows the time points of siRNA transfection, the activation of JP8Bdel-ER by OHT, and the genomic DNA isolation for mutation analysis (A). Mutation frequency at the V region after 48 h of AID (JP8Bdel) activation; plot represents the summary of three independent experiments (B). The values are presented as mean ± sd (n=3). Statistical significance was assessed by two tailed unpaired Student's t-test (*** p ≤ 0.001). Western blot shows the knockdown efficiency by individual siRNAs (C). (D-E) Detection of Sμ DNA double-strand breaks by LM-PCR in CH12F3-2A cells transfected with the indicated siRNA combinations. The DNA samples were either left untreated (none) or treated with indicated end processing enzymes prior to linker ligation. (D-E) Detection of Sμ DNA double-strand breaks by LM-PCR in CH12F3-2A cells transfected with the indicated siRNA combinations. The DNA samples were either left untreated (none) or treated with indicated end processing enzymes prior to linker ligation. (F) Detection of ssDNA breaks by DNA break end labeling. The DNA breaks were end-labeled in situ by incorporating biotin-16-dUTP and TdT. The DNA was fragmented and labeled fragments were enriched by streptavidin pull down, followed by quantification by qPCR. The signal from the β₂-Macroglobulin (β₂m) locus served as a negative control. The values are presented as mean ± sd (n=3). Statistical significance was assessed by two tailed unpaired Student's t-test (*** p ≤ 0.001). The two DNA break assays were illustrated in Supplementary (Appendix Fig S3E, B and F).
Figure 4(A) Schematic of the experimental timeline (left). Lentivirus containing TET1 shRNA and GFP or random shRNA and GFP is injected in to the contralateral dentate gyrus of hippocampus. 2 months old wild type or DSCR1 knockout mice were used for analyses. (B) 3 weeks after lentivirus injection, neural progenitor cells are identified by double staining of GFP and Sox2 in dentate gyrus region. The white arrow indicates Sox2 and GFP double positive cells. Scale bar, 10 μm. (C) The number of GFP and Sox2 double positive cells is restored to that of wild type. Scale bar: 10μm. Values are shown as mean ± SEM and tested for statistical significance by paired t-test. N = 3 animal for each condition, *P< 0.05.
Figure 4A. Cells were grown in microfluidic chambers (see Movies EV1-4). To better visualize the fluorescence signals different intensity settings were used, so that the intensities here are not comparable. Bars, 4 µm. B. Still frames after different times of growth indicate a uniform fluorescence in Q25-GFP-expressing cells. Q97-GFP expression in wild type leads to many small and scattered aggregates per cell. In GAL-Mia40 cells, Q97-GFP accumulates to much larger intensities but then suddenly collapses into one single aggregate per cell. The arrow depicts aggregate formation. Bars, 4 µm. C. Quantified signal intensities in cells of the indicated mutants. In top and middle graphs, each curve corresponds to a single cell. Averaged signals (mean + SEM) in bottom graph (N=20 for WT, N=17 cells for GAL-Mia40). D. Still frames directly before and after the Q97-GFP aggregate formation in GAL-Mia40 cells. Total cellular fluorescence intensity before aggregation and total intensity in the aggregate (red arrow) were quantified and are shown in the graph on the right hand side, indicating that the entire cellular fluorescent signal collapses into the one single aggregate, irrespective of whether cells contain little, high or very high amounts of GFP signal (groups indicated by three red circles on the quantification). M, mother; D, daughter. Bars, 4 µm. E. Cells were shifted from galactose to glucose to stop expression of Q97-GFP. The quantified image shows mean values and SEM from 20 cells with aggregates and 15 cells without aggregate, indicating that soluble Q97-GFP is degraded whereas the aggregated protein is of much higher stability. Bars, 4 µm. F. Schematic inheritance patterns of polyQ proteins in wild type and GAL-Mia40 cells. Compare also Fig. EV2 for more details.
Figure 5(A) Surface plasmon resonance (SPR) assay of the direct interaction of AG with galectin-9. Curve fittings to a 1:1 Langmuir binding model calculated with TraceDrawer are shown as smooth black lines. (B) Coomassie blue stained SDS-PAGE of carbohydrate recognition domain CRD1 and CRD2 of galectin-9. Data are representative of n=3 independent experiments. (C) SPR assay of the interaction of AG with CRD2 of galectin-9. Curve fittings to a 1:1 Langmuir binding model calculated with TraceDrawer are shown as smooth black lines. (D) FACS assay showing interactions of Mtb H37Rv with different galectins including galectin-3, galectin-8, and galectin-9. The blank control was H37Rv staining with APC-anti-rabbit antibody alone. (E) FACS assay of interactions of galectin-9 with mycobacteria including M. bovis BCG and M. smegmatis mc2155 as well as E. coli. The blank control was BCG staining with APC-anti-rabbit antibody alone. Data shown are representative of n=3 independent experiments. (F) FACS assay of interactions of galectin-9 with wild -type M. marinum (Mm_WT) and MMAR-5356 and MMAR-5357 mutants of M.marinum treated with Tet. The blank control was Mm_WT staining with APC-anti-rabbit antibody alone. Data shown are representative of n=3 independent experiments.
Figure 7Macroscopic and histological analysis of the lungs of 140 days old MMTV-PyMT mice; lung metastatic nodules were visualized 8 weeks post-transplantation (5 mice per group). Right panel is the quantification of pulmonary metastases. Immunohistochemistry (IHC) staining of p-AKT of mammary tumors from MMTV-PyMT;CIP2A-BP+/+ or MMTV-PyMT;CIP2A-BP-/- mice. Lower panels show higher-magnification images of insets in upper panels. Right panel is the quantification of IHC staining p-AKT . MMTV-PyMT mice were injected with CIP2A-BP or svCIP2A-BP via mammary fat pad. Macroscopic and histological analysis of the lungs of 140 days old MMTV-PyMT mice; lung metastatic nodules were visualized 8 weeks post-transplantation (5 mice per group). Right panel is the quantification of pulmonary metastases. Representative IHC images of p-AKT of mammary tumors from MMTV-PyMT mice that were injected with CIP2A-BP or svCIP2A-BP via mammary fat pad; Lower panels show higher-magnification images of insets in upper panels. Right panel is the quantification of IHC staining p-AKT. Kaplan-Meier survival curves of nude mice transplanted with MDA-MB-231 cells and injected with CIP2A-BP or svCIP2A-BP (10 mice per group). The horizontal line indicates the time after the mice were injected with the cells via the tail vein. Macroscopic and histological analysis of the lungs of nude mice injected with MDA-MB-231 cells and CIP2A-BP or svCIP2A-BP via tail vein; lung metastatic nodules were visualized 8 weeks post-transplantation (5 mice per group). Right panel is the quantification of pulmonary metastases.
Figure 2(A) Western blot analysis of the CTH level in PC3-T2 cells with CTH overexpression by pCMV-CTH-HA or PC3-B2 cells with CTH knockdown by siCTH-1. (B) Top: The H2S production capacity of cell lysates from PC3-T2 cells with CTH overexpression by pCMV-CTH-HA or PC3-B2 cells with CTH knockdown by siCTH-1. Lead acetate-soaked paper strips show a PbS brown stain as a result of reaction with H2S. Bottom: The level of H2S production was quantified by densitometry, and the histograms represent the means ± SD (n=3 biological replicates). ANOVA followed by Tukey's post-hoc test was used for the statistical analysis (*P<0.05). (C) PC3-B2 cells were transfected with control or siCTH-1 and then were subjected to migration (16h) and invasion (24h) assays. Data information: Data shown represent the means ± SD (n=3 biological replicates). Student's t-test was used for the statistical analysis (*P<0.05; **P<0.01; ***P<0.001). (D) Cell proliferation rates of PC3-B2 cells with CTH knockdown. Cell proliferation assay was performed by MTS reagent. Data information: Data shown represent the means ± SD (n=3 biological replicates). Student's t-test was used for the statistical analysis (*P<0.05; **P<0.01; ***P<0.001). (E) Western blot analysis of the CTH levels in PC3 cells transfected with HA-CTH or pCMV-HA vector control. (F) PC3 cells were transfected with HA-CTH or pCMV-HA vector control and then incubated for migration (16h) and invasion assay (24h). Data information: Data shown represent the means ± SD (n=3 biological replicates). Student's t-test was used for the statistical analysis (*P<0.05; **P<0.01; ***P<0.001). (G) PC3 cells were pre-treated with 10μM PAG for 16 hrs, and incubated for migration (16h) and invasion assay (24h) in the presence of 10μM PAG. Data information: Data shown represent the means ± SD (n=3 biological replicates). Student's t-test was used for the statistical analysis (*P<0.05; **P<0.01; ***P<0.001).