EMBO/sd-character-level-ner huggingface dataset:
Figure 5A. MDCK-SIAT1 cells were pretreated with MH-S EVs (EV:cell = 2, 'EV pretreat') or control for 8 h, washed, and subsequently incubated with Luc-WSN/33 (MOI = 1). Data represent mean luminescence AUC from individual wells normalized to the means of the control condition from 3 independent experiments. MLE-12 cells were incubated with WT PR/8 + AM-EVs at 4 °C (to permit surface binding but not entry) for 1 h, washed, and processed for (B) RNA isolation (B) Data represent mean M gene transcripts relative to the means of the corresponding untreated condition, each normalized to β actin from 3 independent experiments. C. MLE-12 cells were incubated with WT PR/8 + AM-EVs at 4 °C (to permit surface binding but not entry) for 1 h, washed, and processed for (C) immunofluorescence microscopy. (C) Cells in both conditions were fixed and stained with anti-NP or isotype control Ab. Images are representative of two independent experiments. Scale bars = 50 μm D. A549 cells were incubated with WSN/33 or HK/14 + AM-EVs for 40 min, washed, fixed, and permeabilized. Cells were subsequently stained with Anti-NP (green), Anti-Rab-7 (purple) Abs, and DAPI (blue). Images shown are representative of 8 independent experiments. Scale bars = 5 μm E, F. Co-localization analysis of (E) NP:DAPI and (F) NP:Rab7 for each condition was performed on Z-stack confocal images images of WSN/33 (black) or HK/14 (red) infected A549s. Data represent the mean NP:DAPI co-localization ratios and the mean NP:Rab7 Mander's co-localization coefficient for individual cells from these 8 experiments.
XIAP regulates serum starvation‐induced autophagy.Figure 4(A) HCT116 XIAP WT and XIAP KO cells were treated with EBSS for the indicated periods of time. GFP‐LC3 puncta formation was observed by a microscope. Quantification of LC3 punctate cells was shown on the right. The data are represented as means±s.d. of three independent experiments. (B) HCT116 XIAP WT and XIAP KO cells were treated with EBSS for 4 h followed by western blot analysis with the indicated antibodies. The data are representative of three biological replicates. The ratio of LCII/LC3I to actin is presented in Supplementary Figure S6B. (C) HCT116 XIAP WT and XIAP KO cells were treated with 1 μM API‐2 for 6 h. Cell lysates were analysed by western blotting. The data are representative of three biological replicates. The ratio of LCII/LC3I to actin is presented in Supplementary Figure S6C. (D) HCT116 cells were treated with or without EBSS for 4 h. Cell lysates were subjected to immunoprecipitation with anti‐XIAP antibody followed by western blot analysis. The data are representative of two biological replicates. (E) Lysates from HCT116 cells were immunoprecipitated with anti‐Mdm2 antibody. Immunoprecipitates and cell lysates were analysed by western blotting with anti‐p‐XIAP antibody. The data are representative of two biological replicates. (F) HCT116 XIAP KO cells were transfected with Flag‐XIAP, Flag‐XIAP S87A, Flag‐XIAP S87D or control vector. Twenty‐four hours after transfection, cells were harvested. LC3 conversion was evaluated by western blot analysis. The data are representative of three biological replicates. The ratio of LCII/LC3I to actin is presented in Supplementary Figure S6D. (G) p53−/−Mdm2−/− MEF cells were transfected with HA‐Mdm2 plus Flag‐XIAP, Flag‐XIAP S87A or Flag‐XIAP S87D. Twenty‐four hours later, cells were treated with 20 μM MG-132 for another 4h. Cell lysates were subjected to immunoprecipitation with anti‐HA antibody. Immunoprecipitates were analysed by western blotting. The data are representative of two biological replicates.
Figure 7A. Model showing the hierarchy between naïve and primed pluripotency as well as germ layer speciﬁcation. B. Regulators of the balance between mesendoderm and ectoderm speciﬁcation at the exit of pluripotency.
Histological analysis of intracranial U87-derived glioblastoma after therapy.Figure 5Hematoxylin and eosin staining (H&E) and immunostaining for the proliferation marker protein Ki67 of representative tumor sections from all experimental groups, Scale bars, 200 μm for H&E and 100 μm for Ki67. T, tumor; B, brain. The low-magnification inserts represent negative controls with the secondary antibody alone on serial sections used for both Ki67 and caspase-3 immunostainings. Blood vessel staining with CD31 (red). DAPI staining of the sections is shown in blue. Scale bar, 200 μm. T, tumor; B, brain. The low-magnification inserts represent negative controls with the secondary antibody alone on serial sections used for both Ki67 and caspase-3 immunostainings. Tumor immunostaining for apoptosis using an anti- caspase-3 antibody. Scale bar, 100 μm.
Figure 1High-resolution imaging of mitochondria in live cells using the Airyscan module of Zeiss LSM880 confocal microscope. Images of IMM in living HeLa, L6, and H1975 cells, stained with 10-N-nonyl acridine orange (NAO). NAO preferentially binds phospholipids in the IMM, such as cardiolipin. Arrowheads indicate cristae in the IMM. Scale bar = 500 nm. N ≥ 3 independent experiments for each cell type. High-resolution imaging of mitochondria in live cells using the Airyscan module of Zeiss LSM880 confocal microscope. Mitochondrion cropped from HeLa cell shown in A (dashed line) and zoomed in to show cristae, inner boundary membrane (IBM), and matrix (arrows). Scale bar = 100 nm. High-resolution imaging of mitochondria in live cells using the Airyscan module of Zeiss LSM880 confocal microscope. H1975 cells transduced with matrix-targeted-DsRed and stained with NAO. Matrix-targeted Ds-Red differentiates matrix from cristae stained with NAO. Arrowheads point to cristae. Scale bar = 500 nm. N = 1 independent experiment. High-resolution imaging of mitochondria in live cells using the Airyscan module of Zeiss LSM880 confocal microscope. The structure of the IMM in Mic13-KO cells (HeLa), stained with NAO. The number of cristae is decreased in Mic13-KO compared to control cells shown in panel A, labeled with arrowheads. Scale bar = 500 nm. N = 3 independent experiments. High-resolution imaging of mitochondria in live cells using the Airyscan module of Zeiss LSM880 confocal microscope. Live-cell imaging of the IMM in control and PTPMT1 KD H1975 cells, a model of cardiolipin deficiency. A gallery of mitochondria from various control H1975 cells expressing scrambled shRNA and stained with NAO, showing cristae (arrowheads). Scale bars = 500 nm. N = 3 independent experiments. A gallery of mitochondria from various H1975 cells expressing PTPMT1 shRNA and stained with NAO. Note the derangement of the ultrastructure (arrowheads). Scale bars = 500 nm. N = 3 independent experiments.
Figure 3Analysis of nuclear RNA granules in A549 cells with RNase L targeted to the nucleus (NLS-RL-WT) either mock transfected or treated with poly(I:C) (PIC) for 4 to 5 hours. A. FISH analysis to detect poly(A)+ RNA and nucleolar-localized snoRD3A RNA. IF analysis to detect nucleolar granular component protein nucleophosmin (NPM1). B. Graph of the mean and value of the average volume of individual snoRD3A foci in nuclei. 17 nuclei mock treated cells, 31 nuclei PIC treated cells. C. Fraction of nuclei with NPM1 protein enriched in ring structures classified as granular component assemblies or dispersed in nucleoplasm. 17 nuclei mock treated cells, 31 nuclei PIC treated cells. Analysis of nuclear RNA granules in A549 cells with RNase L targeted to the nucleus (NLS-RL-WT) either mock transfected or treated with poly(I:C) (PIC) for 4 to 5 hours. D. IF analysis to detect dense fibrillar component protein, ribosome processing factor 1 (RPF1). E. Graph of the mean and value of the average volume of individual snoRD3A foci in nuclei. 43 nuclei mock treated cells, 69 nuclei PIC treated cells. F. Graph of the mean and value of the average volume of RPF1 foci in 43 mock treated cells and 69 nuclei PIC treated cells. Analysis of nuclear RNA granules in A549 cells with RNase L targeted to the nucleus (NLS-RL-WT) either mock transfected or treated with poly(I:C) (PIC) for 4 to 5 hours. G. FISH analysis to detect poly(A)+ RNA and nuclear speckle-localized MALAT1 RNA and IF analysis with anti-sc35 antibody to detect nuclear speckle protein SRRM2. H. Graph of the mean and value of the intensity of MALAT1 signal divided by the nuclear volume of individual nuclei. 51 nuclei mock treated cells, 56 nuclei PIC treated cells. I. Graph of the median and value of the volume of individual SRRM2 foci in nuclei. Mock 1526 SRRM2 foci in 51 nuclei. PIC 136 SRRM2 foci in 20 nuclei that contain <10 SRRM2 foci. Analysis of nuclear RNA granules in A549 cells with RNase L targeted to the nucleus (NLS-RL-WT) either mock transfected or treated with poly(I:C) (PIC) for 4 to 5 hours. J. FISH analysis to detect poly(A)+ RNA and IF analysis to detect Cajal body protein coilin. K. Graph depicting the fraction of nuclei in mock and PIC treated cells with decreased oligo(dT) with different distributions of coilin protein. 90 nuclei in mock treated cells, 192 nuclei in PIC treated cells.
Generation and characterisation of recombinant CSPSIINFEKL and UIS4SIINFEKL P.Figure 1(A) Schematic overview of transgenic parasite lines. To generate CSPSIINFEKL, SIINFEKL replaced amino acids SYIPSAEK in CSP. To generate UIS4SIINFEKL, SIINFEKL was adjoined to the carboxyl-terminus of the UIS4 protein. (B) Sporozoite immunofluorescent antibody staining of WT, CSPSIINFEKL or UIS4SIINFEKL sporozoites after gliding on BSA-coated glass slides. Shown are microscopic images of the respective sporozoites that were stained with anti-CSP (green), anti-UIS4 (red) and nuclear stain Hoechst 33342 (blue). Scale bars, 10μm. The numbers show mean percentage (±SD) of sporozoites with trails assessed from ≥220 sporozoites. (C) Fluorescent-microscopic images of EEF-infected Huh7 hepatoma cells. 24 and 48 hours after infection with WT, CSPSIINFEKL or UIS4SIINFEKL sporozoites, the cells were fixed and stained with anti-UIS4 (red), anti-HSP70 (green) and the nuclear stain Hoechst (blue). Scale bars: 10µm. The numbers show mean numbers (±SD) of intracellular parasites counted per well of 8-well Labtek slides. Data information: The data shown is representative from one of two independent experiments.
Bob1-deficient mice show an organ- and subset-dependent defect in the generation of Tfh cells.Figure 2Analysis of Tfh cell development in the spleen (SPL), mesenteric lymph nodes (MLN), and Peyer´s patches (PP) of Pou2af1-/- and Pou2af1+/-mice following immunization with sheepred blood cells. (A and B) Generation of Tfh cells characterized by the coexpression of CXCR5 with either ICOS, PD1, or BTLA on day 7 after immunization. (A) Representative flow cytometry plots indicating the frequency of CXCR5hiICOS+, CXCR5hiPD1+ and CXCR5hiBTLAhiTfh cells in Pou2af1-/- and Pou2af1+/-mice. Dot plots show gated CD4+7AAD-T cells. (B) Summarized data for the analysis shown in A (n=17 for ICOS+, n=14 for PD1+ and n=10 for BTLA+Tfh cells from at least 4 independent experiments; mean ± SEM). Analysis of Tfh cell development in the spleen (SPL), mesenteric lymph nodes (MLN), and Peyer´s patches (PP) of Pou2af1-/- and Pou2af1+/-mice following immunization with sheepred blood cells. (C and D) CXCR5 expression on B220+B cells (C) and CD4+T cells (D) in Pou2af1-/- and Pou2af1+/-mice. The graphs show the mean fluorescence intensity (MFI) for CXCR5 (n=5-7). Analysis of Tfh cell development in the spleen (SPL), mesenteric lymph nodes (MLN), and Peyer´s patches (PP) of Pou2af1-/- and Pou2af1+/- mice following immunization with sheep red blood cells. (E) Coexpression of ICOS, PD1, and BTLA on Tfh cells. CD4+CXCR5hi T cells were analyzed for the simultaneous expression of these costimulatory molecules. Venn diagrams illustrate the overlap of ICOS, PD1 and BTLA expression on CD4+ICOShi T cells. Data represent mean values from 4 independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001; P < 0.0001; unpaired Mann Whitney test.
Figure 6A HeLa cells were transfected with siRNA and plasmids encoding the indicated proteins, and then exposed to nocodazole for 3 hr. Mitotic cells were cytospun onto coverslips and subjected to immunofluorescence staining with DAPI, ACA and anti-GFP antibodies. Example images are shown. Scale bars, 10 μm. U2OS-LacO cells transiently expressing Myc-LacI-Bub1 and the indicated EGFP-TOP2A proteins were subjected to immunofluorescence staining with DAPI and antibodies for the Myc-tag and H2ApT120. Example images are shown (B). The relative enrichment of EGFP signal at the LacO repeats was quantified in 30 cells (C). Arrows point to the transgene loci. Data information: Means and error bars representing S.D. are shown ; unpaired t test). Scale bars, 10 μm. U2OS-LacO cells transiently expressing Myc-LacI-Bub1 and the indicated EGFP-TOP2A proteins Cell lysates were immunoblotted (D). E, F HeLa cells transfected with siRNA and plasmids encoding the indicated proteins were treated and immunostained Example images are shown (E). The immunofluorescence intensity ratio of centromeric EGFP-TOP2A/arm EGFP-TOP2A was determined on around 300 chromosomes in 15 cells (F). Means and error bars representing S.D. are shown unpaired t test). Scale bars, 10 μm. G, H HeLa cells transfected with siRNA and plasmids encoding the indicated proteins were treated and immunostained Example images are shown (G). The immunofluorescence intensity ratio of centromeric TOP2A-Myc-6xHis/arm TOP2A-Myc-6xHis was determined on around 200 chromosomes in 10 cells (H). Data information: Means and error bars representing S.D. are shown unpaired t test). Scale bars, 10 μm. U2OS cells stably expressing the indicated CENP-B fusion proteins were transfected with the plasmid encoding TOP2A-Myc-6xHis or TOP2A-ΔChT-Myc-6xHis, and then subjected to immunofluorescence staining with DAPI and antibodies for the Myc-tag and H2ApT120. Example images are shown (I). Scale bars, 10 μm. U2OS cells stably expressing the indicated CENP-B fusion proteins were transfected with the plasmid encoding TOP2A-Myc-6xHis or TOP2A-ΔChT-Myc-6xHis, and then subjected to immunofluorescence staining with DAPI and antibodies for the Myc-tag and H2ApT120. The relative enrichment of anti-Myc staining signal at the centromere region versus that in the nearby nuclear region was quantified on around 200 chromosomes in 10 cells (J). Data information: Means and error bars representing S.D. are shown unpaired t test). U2OS cells stably expressing the indicated CENP-B fusion proteins were transfected with the plasmid encoding TOP2A-Myc-6xHis or TOP2A-ΔChT-Myc-6xHis, and then subjected to immunofluorescence staining with DAPI and antibodies for the Myc-tag and H2ApT120. Cell lysates were immunoblotted (K). L MBP-TOP2A (429-1531)-10xHis and MBP-TOP2A (429-1531)-ΔChT-10xHis were subjected to pulldown by GST, GST-H2A, or GST-H2ApS120, followed by immunoblotting with antibodies for MBP or GST.
Fig.Figure 3HEK293 cells were transfected with α11.2, α2δ-1, and β2A and cultured for 22-24h before cell attached patch recording in 110 mM Ba2+. Single CaV1.2 channel recordings of CaV1.2 WT and K1647A. Holding potential (HP) was -80 mV and test potential (TP) 0 mV. Shown are 10 consecutive sweeps from representative experiments (see Fig. EV 4 for more sweeps). Dot plots and means + SEM for availability (i.e., likelihood that a sweep had at least one event; c), NPo (d), Po (e), unitary current amplitude i (e), and the mean of the current of a single channel at any point in time calculated from the ensemble averages of each experiment (g) (**p<0.01, ***p<0.001 compared to WT; one-way ANOVA with Bonferroni post-hoc test (c) or Welch ANOVA with Tamhane T2 test (d-g); n = 7 to 35 see Table 5 for more details).
Microtubules are essential for single cell durotaxis Left: Schematic diagram of manufacturing polyacrylamide gels with stiffness gradient.Figure 1Left: Schematic diagram of manufacturing polyacrylamide gels with stiffness gradient. The gel containing photoinitiator is solidified by ultraviolet light irradiation. Mask with a constant speed is used to control the irradiation time for a hardness gradient. Right: Representative stiffness gradient of polyacrylamide gels measured by atomic force microscope was plotted (34 kPa/mm and 29 kPa/mm respectively). Representative images of fluorescently labeled fibronectin at the gradient gel surface and the quantification of fluorescence intensity. 1-4 in turn represent hard to soft areas. Left: Representative time-lapse images showing the migration of single hRPE cells on the surface of polyacrylamide gels with uniform stiffness (top) and stiffness gradient (bottom), referring as random migration and durotaxis migration respectively. Yellow line is the cell outline and blue line is the cell migration trajectory. Scale bar: 50 μm. Middle: Angular displacement of hRPE cells on the uniform stiffness gels (top) and stiffness gradient gels (bottom). Right: y/x FMI (forward migration index), velocity and persistence of hRPE cells with random migration and durotaxis. Left: Representative immunofluorescent staining of F-actin in control and CK-666 (100 μM, 3 h) treated hRPEs. Scale bar: 20 μm. Right: Y FMI of control and CK-666 (100 μM, pre-treated for 1h and incubated for the whole experiment) treated hRPEs moving on stiffness gradient gel. Left: Representative immunofluorescent staining of α-tubulin in control and nocodazole (8 μM, 30 min) treated hRPEs. Scale bar: 20 μm. Right: Y FMI of control and nocodazole (8 μM, pre-treated for 10 min and incubated for the whole experiment) treated hRPEs moving on stiffness gradient gel.
MMS treatment leads to formation of Btn2‐dependent non‐canonical DNA stress foci at the INQ A S.Figure 8A S. cerevisiae wt cells were treated with MMS and Btn2 levels were determined at the indicated time points. Zwf1 levels are given as a loading control. B, C S. cerevisiae wt and btn2Δ cells expressing Hos2‐GFP (green, B) or GFP‐VHL (green, C) were grown at 30°C and treated with MMS. Changes in protein localizations were recorded at the indicated time points. DNA was stained by DAPI (blue). The total number of GFP‐VHL foci per cell and frequencies (%) of INQ formation were determined.
BMP activity regulates neural AP patterning A.Figure 3One-cell stage embryos were injected animally with mRNAs encoding the noggin (5/20 pg) and/or meis3 (0.5 ng) proteins. AC explants were removed from control and injected embryos at blastula stage 9, and explants were cultured to neurula stage 17. Total RNA was isolated from seven control embryos (lane 2), and from eighteen ACs from each group (lanes 3-8). Neural AP markers were examined by sqRT-PCR: forebrain (xanf1, otx2), hindbrain (krox20, hoxb1, hoxb3), spinal cord (hoxb9, cdx1-4) and panneural (soxd). Ef1α is the control for quantitating RNA levels. -RT-PCR was performed on total RNA isolated from control embryos (lane 1). One-cell stage embryos were injected animally with mRNA encoding bmp4 (0.2 ng) protein. Total RNA was isolated from five control embryos at neurula stage 17 (lane 1) and five embryos from the injected group (lanes 2). Neural and mesodermal markers were examined by sqRT-PCR: forebrain (otx2), hindbrain (krox20, hoxb3), spinal cord (hoxb9, hoxc10, cdx4), panneural (nrp1, ncam) and mesodermal (muscle actin - MA). ODC is the control for quantitating RNA levels. -RT-PCR was performed on total RNA isolated from control embryos (lane 1). One-cell stage embryos were injected animally with mRNA encoding bmp4 (0.2 ng) protein Embryos underwent in situ hybridization. Expression of the hindbrain marker hoxb3 was reduced/eliminated by bmp4 (0.2 ng) in 68% of the embryos (n=19). Expression of the spinal cord marker cdx4 was slightly increased/normal, in 100% of the embryos (n=19).
Figure 4H1299 cells transfected with His-SUMO2 and/or USP36 were subjected to Ni2+-NTA agarose beads pulldown (PD) followed by IB to detect SUMOylation of Nop58 (A) and Nhp2 (B). *indicates a non-specific anti-Nhp2 antibody-reacting band. USP36 promotes Nop58 and Nhp2 SUMOylation in the nucleolus. Nucleoli isolated from HeLa cells transfected with His-SUMO2 and/or USP36 were subjected to Ni2+-NTA PD, followed by IB. Knockdown of USP36 reduces Nop58 and Nhp2 SUMOylation in cells. Cells infected with scr or USP36 shRNA lentiviruses were assayed by IB. The SUMOylated Nop58 (D) and Nhp2 (E) are indicated. USP36 SUMOylates Nhp2 and Nop58 in vitro. In vitro SUMOylation assays were performed by incubating recombinant Nhp2 (F, G) or Nop58 (H) (0.1 μM) with SUMO E1 (30 nM), Ubc9 (50 nM), SUMO2 (4 μM) in the absence or presence of USP361-800 (50 nM, 1x; 100 nM, 2x) and/or ATP (2.5 mM) at 37ºC for two hours or the indicated times. The reactions were assayed by IB using anti-Nhp2 and anti-Nop58 antibodies, respectively.
Figure 7Tumor volume at different time points after co-injection of SCC13 cells with EV or dnActRIB fibroblasts. N=5 tumors. Representative immunofluorescence images of sections from ear skin tumors formed by SCC13 cells co-injected with EV or dnActRIB fibroblasts stained for K14 (left) or PDGFRα (right) (green) and mDia2 (red), counterstained with Hoechst (blue). Bar graph shows the percentage of mDia2-positive area. N=3. SCC13 EV or SCC13 Fst cells were injected into the ear skin of NOD/SCID mice and tumor weight was determined 12 weeks after injection. N=9-10 tumors. Representative immunofluorescence images of sections from ear skin tumors formed by SCC13 EV or SSC13 Fst cells stained for K14 or mDia2 (red), counterstained with Hoechst (blue). Bar graph shows the percentage of mDia2-positive area. N=3. Tumors formed by SCC13 Act cells were treated ex vivo for 6h with follistatin (FST) (50 ng/ml) and/or SMIFH2 (10 μM), or with activin A (20 ng/ml) and stained for Ki67. N=3 tumor explants, n=4 histological sections. Representative images of spheroids formed by SCC13 Act cells labeled with PKH67 (green) with primary human fibroblasts embedded in collagen gels and graph showing increase in the spheroid area between day 0 and day 3. Spheroids were either treated with FST (50 ng/ml) or SMIFH2 (10 μM) or left untreated. N=6-8 tumor spheroids. Increase in A431 tumor volume (mm3) compared to day 0 (time when the tumors were first injected with FST, approximately 3-4 weeks after injection of the cells) upon treatment of the tumors with FST (100 ng per injection) or vehicle (PBS). N=3-5 tumors per group. Representative immunofluorescence images the FST or vehicle-treated tumors, stained for mDia2 (green) and K14 (red), counterstained with Hoechst (blue). Proposed role of activin A secreted by cancer cells in inducing a pro-tumorigenic CAF phenotype in fibroblasts. Activin A signaling (blocked by follistatin or dnActRIB (not shown in the figure) induces upregulation of mDia2 (inhibited by SMIFH2), which in turn contributes to tumor development by reducing the nuclear p53 pool and promoting the expression of CAF marker genes, fibroblast proliferation and migration.
Figure 2A. T6SS secretion assay of A. tumefaciens strains: wild type C58, ΔtssL, Δtap-1, and Δatu3641 harboring a pTrc200 vector (V) or derivatives pTap-1 (tap-1 expressed from pTrc200), or p3641 (atu3641 expressed from pTrc200). B. T6SS secretion assay of A. tumefaciens C58 Δtap-1Δatu3641 containing an empty pTrc200 vector (V) or its derivatives expressing tap-1, atu3641, tde1-tdi1, or tde2-tdi2. C. T6SS secretion assay of A. tumefaciens C58ΔG2op mutant encoding full-length or truncated VgrG1 proteins.