Journal: Journal of Virology

Article Title: Enterovirus 70 Receptor Utilization Is Controlled by Capsid Residues That Also Regulate Host Range and Cytopathogenicity

doi: 10.1128/jvi.01569-06

Figure Lengend Snippet: FIG. 1. Host range of EV70-Rmk14 and EV70-Dne in cultured cells. One-step growth analysis of viruses was performed in human (HeLa), monkey (LLC-MK2), and murine (L, L-hDAF) cell lines with EV70-Rmk14 (top) or EV70-Dne (bottom) virus at an MOI of 5. Infections were halted at different times postinfection, and virus titers were determined by plaque assay.

Article Snippet: EV70 prototype strain J670/71 was obtained from the American Type Culture Collection (Manassas, VA) and propagated in rhesus monkey kidney LLC-MK2 cells at 37°C.

Techniques: Cell Culture, Virus, Plaque Assay

Journal: Journal of Virology

Article Title: Enterovirus 70 Receptor Utilization Is Controlled by Capsid Residues That Also Regulate Host Range and Cytopathogenicity

doi: 10.1128/jvi.01569-06

Figure Lengend Snippet: FIG. 2. Host range in cell lines derived from human eye and brain. One-step growth analysis of viruses was performed using EV70-Rmk14 (top) or EV70-Dne (bottom) virus to infect (MOI 5) the following eye and brain derived cell lines: HeLa, T98, LLCMK2, U373MG, 15C4, SY5Y, or HCE. Infections were halted at different times postin- fection, and virus titers were determined by plaque assay.

Article Snippet: EV70 prototype strain J670/71 was obtained from the American Type Culture Collection (Manassas, VA) and propagated in rhesus monkey kidney LLC-MK2 cells at 37°C.

Techniques: Derivative Assay, Virus, Plaque Assay

Journal: Journal of Virology

Article Title: Enterovirus 70 Receptor Utilization Is Controlled by Capsid Residues That Also Regulate Host Range and Cytopathogenicity

doi: 10.1128/jvi.01569-06

Figure Lengend Snippet: FIG. 3. Effect on viral replication of enzyme or antibody treatment of cultured cells. HeLa cells were incubated with one of the following as indicated along the x axis: phosphate-buffered saline (PBS [mock treated]), neuraminidase, PI-PLC, or antibodies specific for hapten or the SCR1 or SCR2 domains of the human DAF molecule. Cells were washed and infected with EV70-Rmk14 or EV70-Dne at an MOI of 3. At 24 h postinfection, the total RNA was isolated from infected cells and transferred onto a nitrocellulose membrane for slot blot analysis. Positive-strand viral replication was assessed by hybridization with a radiolabeled negative-strand EV70 RNA probe. The amount of hy- bridized probe was determined with a PhosphorImager and Image- Quant software and reported as the optical density. The data were normalized to the mock (PBS)-treated sample (PBS treatment 100% replication).

Article Snippet: EV70 prototype strain J670/71 was obtained from the American Type Culture Collection (Manassas, VA) and propagated in rhesus monkey kidney LLC-MK2 cells at 37°C.

Techniques: Cell Culture, Incubation, Saline, Infection, Isolation, Membrane, Dot Blot, Hybridization, Software

Journal: Journal of Virology

Article Title: Enterovirus 70 Receptor Utilization Is Controlled by Capsid Residues That Also Regulate Host Range and Cytopathogenicity

doi: 10.1128/jvi.01569-06

Figure Lengend Snippet: FIG. 4. Role of amino acid differences between EV70-Rmk and EV70-Dne viruses in replication in HeLa cells. One-step growth anal- ysis was performed with mutants of EV70-Dne harboring single amino acid substitutions to the EV70-Rmk14 sequence. HeLa cells were infected at an MOI of 5, infections were halted at different times postinfection, and virus titers were determined by plaque assay. (A) Growth analysis of viruses DDDDD (EV70-Dne encoding amino acids K14, M238, L133, P178, and D226), DRRRR (K14K, I238, F133, R178, N226), RDRRR (E14, M238, F133, R178, and N226), RRDRR (E14, I238, L133, R178, and N226), RRRDR (R14, I238, F133, P178, and N226), and RRRRR (EV70-Rmk, E14, I283, F133, R178, and N226). (B) Growth analysis of viruses DDDDD (see above), RDDDD (E14, M238, L133, P178, and D226), DRDDD (K14, I238, L133, P178, and D226), DDRDD (K14, M238, F133, P178, and D226), DDDRD (K14, M238, L133, R178, and D226), and DDDDR (K14, M238, L133, P178, and N226).

Article Snippet: EV70 prototype strain J670/71 was obtained from the American Type Culture Collection (Manassas, VA) and propagated in rhesus monkey kidney LLC-MK2 cells at 37°C.

Techniques: Sequencing, Infection, Virus, Plaque Assay

Journal: Journal of Virology

Article Title: Enterovirus 70 Receptor Utilization Is Controlled by Capsid Residues That Also Regulate Host Range and Cytopathogenicity

doi: 10.1128/jvi.01569-06

Figure Lengend Snippet: FIG. 5. Role of amino acid differences between EV70-Rmk and EV70-Dne viruses in hDAF usage. One-step growth analysis was per- formed using mutants of EV70-Dne virus (described in the legend to Fig. 4). L-hDAF cells were infected at an MOI of 5, infections were halted at different times postinfection, and virus titers were deter- mined by plaque assay.

Article Snippet: EV70 prototype strain J670/71 was obtained from the American Type Culture Collection (Manassas, VA) and propagated in rhesus monkey kidney LLC-MK2 cells at 37°C.

Techniques: Virus, Infection, Plaque Assay

Journal: Journal of Virology

Article Title: Enterovirus 70 Receptor Utilization Is Controlled by Capsid Residues That Also Regulate Host Range and Cytopathogenicity

doi: 10.1128/jvi.01569-06

Figure Lengend Snippet: FIG. 6. Role of amino acid differences between EV70-Rmk and EV70-Dne viruses in cell killing. HeLa cells were infected at an MOI of 3, using EV70-Dne (DDDDD), EV70-Rmk14 (RRRRR), or mu- tant viruses described in the legend to Fig. 4. Infections were halted at different times postinfection, and cells were pelleted by low-speed centrifugation, resuspended in PBS with trypan blue dye, and exam- ined by light microscopy. The percent viability was determined by dividing the number of cells that excluded dye by the number of cells examined.

Article Snippet: EV70 prototype strain J670/71 was obtained from the American Type Culture Collection (Manassas, VA) and propagated in rhesus monkey kidney LLC-MK2 cells at 37°C.

Techniques: Infection, Centrifugation, Light Microscopy

Journal: Journal of Virology

Article Title: Enterovirus 70 Receptor Utilization Is Controlled by Capsid Residues That Also Regulate Host Range and Cytopathogenicity

doi: 10.1128/jvi.01569-06

Figure Lengend Snippet: FIG. 7. Predicted location in the viral capsid of EV70 amino acids that influence host range and cell killing. The known crystallographic structure of BEV-1 was used to predict the locations in the viral capsid of amino acid residues that differ between EV70-Rmk14 and EV70-Dne. Capsid protomer proteins are color coded, with VP1 blue, VP2 yellow, and VP3 red. VP4 is omitted for clarity. Amino acid changes are enumerated in white. (A) Exterior view of a pentamer, comprising five copies each of VP1, VP2, VP3, and VP4 (not shown), revealing the locations of four of the five strain-specific amino acid changes: in the VP1 DE loop, amino acid 133 of EV70 VP1 (F1133L, BEV 1128 in figure), and three in the canyon, including 238 of EV70 VP3 (I3238M, BEV 3240), 178 of EV70 VP1 (R1178P, BEV 1151), and 226 of EV70 VP1 (N1226D, BEV 1219). Residues on only one protomer are labeled. (B) Closer exterior view of canyon and fivefold axis of symmetry. Amino acid changes in all five protomers are labeled.

Article Snippet: EV70 prototype strain J670/71 was obtained from the American Type Culture Collection (Manassas, VA) and propagated in rhesus monkey kidney LLC-MK2 cells at 37°C.

Techniques: Labeling

Journal: Molecular cell

Article Title: Dietary supplement chondroitin-4-sulfate exhibits oncogene-specific pro-tumor effects on BRAF V600E melanoma cells

doi: 10.1016/j.molcel.2018.02.010

Figure Lengend Snippet: Key Resources Table

Article Snippet: Human: SK-MEL-24 cells , ATCC , Cat# HTB-71; RRID: CVCL_0599.

Techniques: Virus, Recombinant, RNA Extraction, Transfection, SYBR Green Assay, Protease Inhibitor, Enzyme-linked Immunosorbent Assay, Malachite Green Assay, Activity Assay, shRNA, Sequencing, Mutagenesis, CRISPR, Plasmid Preparation, Software, Expressing

Journal: Cell Reports Medicine

Article Title: CD97 maintains tumorigenicity of glioblastoma stem cells via mTORC2 signaling and is targeted by CAR Th9 cells

doi: 10.1016/j.xcrm.2024.101844

Figure Lengend Snippet:

Article Snippet: Murine CD3 + T cells were purified from C57BL/6J mice splenocytes using the EasySep Mouse T cell Isolation Kit (STEMCELL Technologies Inc.) and stimulated on plates coated with 2 μg/mL mCD3ε (BD Pharmingen) and 2 μg/mL mCD28 mAbs (Biolegend) for 48 h. The stimulated T cell population underwent lentiviral transduction to express a second-generation anti-CD97 CAR T cells were expanded in complete medium RPMI-1640 (ATCC), 10% FBS (Hyclone), 1% Penicillin-Streptomycin (Gibco) with hIL-2 (10 ng/mL; PeproTech.) changing medium every 2 days.

Techniques: Produced, Virus, Plasmid Preparation, Recombinant, Purification, Cell Culture, Cell Isolation, Reporter Gene Assay, cDNA Synthesis, Apoptosis Assay, Cytotoxicity Assay, Gene Expression, shRNA, Sequencing, Amplification, Software, Microscopy, Western Blot

Journal: eLife

Article Title: STING mediates immune responses in the closest living relatives of animals

doi: 10.7554/eLife.70436

Figure Lengend Snippet: ( A ) Immunofluorescence illuminates the diagnostic cellular architecture of M. brevicollis , including an apical flagellum ( f ) made of microtubules, surrounded by an actin-filled microvilli feeding collar (co). Staining for tubulin (green) also highlights cortical microtubules that run along the periphery of the cell body, and staining for F-actin (magenta) highlights basal filopodia (fp). DNA staining (blue) highlights the nucleus ( n ). ( B ) M. brevicollis exhibits truncated flagella after exposure to P. aeruginosa. M. brevicollis were exposed to E. coli or P. aeruginosa for 24 hr, and then fixed and immunostained. Arrows point to flagella. Green: anti-tubulin antibody (flagella and cell body), magenta: phalloidin (collar), blue: Hoechst (bacterial and choanoflagellate nuclei). Scale bars represent 10 μm. Flagellar length was quantified using Fiji, and statistical analysis (unpaired t-tests) was performed in GraphPad software. ( C ) Exposure to P. aeruginosa , but not other Gammaproteobacteria, results in M. brevicollis cell death. Bacteria were added to M. brevicollis culture at an MOI of 1.5 (at Hours = 0), and M. brevicollis cell density was quantified at indicated time points. Data represent mean ± SD for three biological replicates. Statistical analysis (multiple unpaired t-tests) was performed in GraphPad software; p - values shown are from comparisons between Flavobacterium and P. aeruginosa . ( D–F ) M. brevicollis does not ingest P. aeruginosa bacteria. ( D,E ) M. brevicollis were fed either fluorescent E. coli ( D ) or P. aeruginosa ( E ) for 1 hr, and then visualized by DIC (D,E, left) and green fluorescence (D, E, right). Fluorescent food vacuoles were observed in choanoflagellates fed E. coli, but not P. aeruginosa . ( F ) M. brevicollis was exposed to GFP-expressing E. coli , V. parahaemolyticus , C. jejuni , or P. aeruginosa (MOI = 50) for 1 hr, and then imaged by DIC and green fluorescence to quantify number of cells with internalized bacteria. Choanoflagellate cells with ≥1 GFP+ food vacuole were scored as GFP+, and cells without any GFP+ food vacuoles were scored as GFP–. Data represent cells quantified over three biological replicates. ( G,H ) P. aeruginosa does not broadly inhibit M. brevicollis phagocytosis. ( G ) Internalization of 0.2 μm fluorescent beads was used to quantify phagocytic activity after exposure to E. coli or P. aeruginosa bacteria. Although cells did not phagocytose P. aeruginosa, cells exposed to E. coli and P. aeruginosa had similar phagocytic uptake of beads. Data represent n = 600 cells from three biological replicates. Statistical analyses (multiple unpaired t-tests) were performed in GraphPad software. ( H ) Exposure to P. aeruginosa does not inhibit phagocytic uptake of E. coli . Internalization of fluorescent E. coli or P. aeruginosa bacteria was quantified after exposure to unlabeled P. aeruginosa (PAO1 strain). Data represent n = 200 cells from two biological replicates. Statistical analysis (unpaired t-test) was performed in GraphPad software. ( I ) Secreted P. aeruginosa molecules are sufficient to induce M. brevicollis cell death. 5 % (vol/vol) bacterial conditioned medium was added to M. brevicollis culture (at Hours = 0), and M. brevicollis cell density was quantified at indicated time points. Data represent mean ± SD for three biological replicates. Statistical analysis (multiple unpaired t-tests) was performed in GraphPad software, and p- values shown are from comparisons between Flavobacterium and P. aeruginosa . ( J ) Sustained exposure to secreted P. aeruginosa molecules is required to induce M. brevicollis cell death. P. aeruginosa or Flavobacterium conditioned medium (5% vol/vol) was added to stationary-phase M. brevicollis cultures. After indicated times, cultures were washed and resuspended in fresh media. M. brevicollis cell density was quantified after 24 hr. The % survival is a measure of the cell density of P. aeruginosa -treated cells relative to Flavobacterium -treated controls. Data represent mean ± SD for three biological replicates.

Article Snippet: Escherichia coli K12 , – , ATCC 10798 , Julie Pfeiffer.

Techniques: Immunofluorescence, Diagnostic Assay, Staining, Software, Bacteria, Fluorescence, Expressing, Activity Assay

Journal: eLife

Article Title: STING mediates immune responses in the closest living relatives of animals

doi: 10.7554/eLife.70436

Figure Lengend Snippet: Bacteria screened for pathogenic effects.

Article Snippet: Escherichia coli K12 , – , ATCC 10798 , Julie Pfeiffer.

Techniques: Bacteria, Isolation

Journal: eLife

Article Title: STING mediates immune responses in the closest living relatives of animals

doi: 10.7554/eLife.70436

Figure Lengend Snippet: ( A ) The genotypes of wild type and genome-edited STING – strains at the STING locus. ( B ) STING protein is not detectable by immunoblot in STING – cells. Shown is a representative blot from three biological replicates. ( C,D ) STING is necessary for 2’3’cGAMP-induced cell death. ( C ) Wild type and STING – strains were treated with increasing concentrations of 2’3’cGAMP, and survival was quantified after 24 hr. In contrast to wild type cells, 2’3’cGAMP does not induce cell death in STING – cells. Data represent mean ± SD for four biological replicates. ( D ) Wild type and STING – cells were transfected with STING-mTFP, and treated with puromycin to generate stable clonal strains. Stable expression of STING-mTFP in STING – cells partially rescued the phenotype of 2’3’cGAMP-induced cell death. Data represent mean ± SD for three biological replicates. Statistical analysis (multiple unpaired t-tests) was performed in GraphPad software. ( E ) Wild type and STING – strains have distinct transcriptional responses to 2’3’ cGAMP. Differential expression analysis was performed on wild type and STING – cells treated with 100 µM 2’3’cGAMP or a vehicle control for 3 hr. A heatmap comparing the log 2 fold change of genes identified as differentially expressed (FC ≥2; FDR ≤ 10 –4 ) in wild-type cells after 2’3’ cGAMP treatment, to their log 2 fold change in STING – cells after 2’3’ cGAMP treatment. RNA-seq libraries were prepared from two biological replicates. ( F ) Presence of STING in the transcriptomes of diverse choanoflagellate species. Data from . ( G ) Effects of 2’3’cGAMP on different choanoflagellate species. Choanoflagellates were grown to late-log phase, and treated with increasing concentrations of 2’3’cGAMP. Survival was quantified after 24 hr. 2’3’cGAMP only affected the survival of M. brevicollis and S. macrocollata , the two sequenced choanoflagellate species with a STING homolog. Data represent mean ± SD for three biological replicates. ( H ) Wild type and STING – cells have similar survival responses to LPS, suggesting that STING is not required for mediating a response to LPS. Wild type and STING – strains were treated with increasing concentrations of E. coli LPS, and survival was quantified after 24 hr. Data represent mean ± SD for four biological replicates. Statistical analysis (multiple unpaired t-tests) was performed in GraphPad software. ( I,J ) STING renders M. brevicollis more susceptible to P. aeruginosa -induced growth inhibition. ( I ) Wild type and STING – cells were exposed to control Flavobacterium or P. aeruginosa conditioned medium (5% vol/vol), and cell densities were quantified at indicated time points. Data represent mean ± SD for three biological replicates. ( J ) Percent survival calculated from growth curves in ( I ). Statistical analysis (multiple unpaired t-tests) was performed in GraphPad software.

Article Snippet: Escherichia coli K12 , – , ATCC 10798 , Julie Pfeiffer.

Techniques: Western Blot, Transfection, Expressing, Software, Quantitative Proteomics, Control, RNA Sequencing, Inhibition