Journal: PLoS Pathogens

Article Title: The Viral Chemokine MCK-2 of Murine Cytomegalovirus Promotes Infection as Part of a gH/gL/MCK-2 Complex

doi: 10.1371/journal.ppat.1003493

Figure Lengend Snippet: (A–E) Lysates of cells, supernatant virus, or precipitated proteins were separated on SDS-polyacrylamide gels and analyzed by Western blot using the anti-MCK-2 antibody 5A5 to detect MCK-2 or the anti-HA antibody 3F10 to detect gH-HA. The positions of the molecular weight markers (kDa) are indicated. (A) Infected cells and supernatant virus from NIH3T3 cells infected with wildtype MCMV were harvested 5 days after infection and lysed in reducing sample buffer. (B) Gradient purified wildtype MCMV was lysed in reducing (red.) and non-red. sample buffer. The MCK-2 specific bands are indicated by arrows. (C) Supernatant virus from NIH3T3 cells infected with MCMV-gH-HA was either directly lysed in red. or non-red. sample buffer (extract) or proteins were precipitated from lysates with anti-gH or anti-MCK2 antibodies and then analyzed under non-red. conditions. Monomeric gH-HA (black arrow) and gH/gL/MCK-2 (white arrow) are indicated by arrows. (D) Supernatant virus from NIH3T3 cells infected with MCMV-gH-HA or MCMV-gH-HA/129stop was lysed in red. or non-red. sample buffer. The upper panel shows extracts prepared under red. and non-red. conditions and stained with an anti-MCK-2 antibody. The lower panel shows the same extracts stained with an anti-HA antibody to detect gH-HA. The gH/MCK-2 band formed under non-red. conditions is indicated by an arrow. The MCMV-gH-HA/129stop extracts showed an at least fivefold higher protein content. (E) NIH3T3 cells were infected with MCMV-gH-HA and supernatant virus harvested 5 days after infection. Lysates of supernatant virus were either directly analyzed for gH-HA expression (supernatant virus) or after immunoprecipitation using a mouse IgG control antibody, an anti-HA antibody, an anti-gH antibody, or an anti-MCK2 (2H9) antibody recognizing the m131 region of MCK-2. The gH-HA specific protein band is indicated.

Article Snippet: Primary mouse embryonal fibroblasts from BALB/c mice (MEF), NIH3T3 cells ( ATCC : CRL-1658), the endothelial cell line MHEC5-T , the epithelial cell line TCMK-1 ( ATCC : CCL-139), the macrophage cell line J774 ( ATCC : TIB-67), and peritoneal exudates cells (PEC) from BALB/c mice were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum.

Techniques: Virus, Western Blot, Molecular Weight, Infection, Purification, Staining, Expressing, Immunoprecipitation, Control

Journal: PLoS Pathogens

Article Title: The Viral Chemokine MCK-2 of Murine Cytomegalovirus Promotes Infection as Part of a gH/gL/MCK-2 Complex

doi: 10.1371/journal.ppat.1003493

Figure Lengend Snippet: (A) Supernatant virus from NIH3T3 cells infected with MCMV-gH-HA or MCMV-gO-HA was lysed in red. or non-red. sample buffer. gO-HA and gH-HA were detected with an anti-HA antibody. Monomeric gO-HA (black arrow) and gH/gL/gO (white arrow) are indicated by arrows. Two exposures of the Western blots showing non-red. extracts are depicted. As a control, gO-HA was precipitated from a lysate of MCMV-gO-HA infected cells with an anti-HA antibody and analyzed under non-red. conditions. (B) MEF were infected with MCMV-gO-HA, and three days after infection, cells were harvested and either directly analyzed for gO-HA expression (total cell extract) or after immunoprecipitation using an anti-HA antibody, an anti-gH antibody, an anti-MCK2 (2H9), or a mouse IgG control antibody. (A–B) The gO-HA specific protein band runs at about 70 kDa. The positions of the molecular weight markers (kDa) are indicated.

Article Snippet: Primary mouse embryonal fibroblasts from BALB/c mice (MEF), NIH3T3 cells ( ATCC : CRL-1658), the endothelial cell line MHEC5-T , the epithelial cell line TCMK-1 ( ATCC : CCL-139), the macrophage cell line J774 ( ATCC : TIB-67), and peritoneal exudates cells (PEC) from BALB/c mice were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum.

Techniques: Virus, Infection, Western Blot, Control, Expressing, Immunoprecipitation, Molecular Weight

Journal: PLoS Pathogens

Article Title: The Viral Chemokine MCK-2 of Murine Cytomegalovirus Promotes Infection as Part of a gH/gL/MCK-2 Complex

doi: 10.1371/journal.ppat.1003493

Figure Lengend Snippet: Cells were infected at an m.o.i. of 0.1, supernatants were harvested every 24 hours and titrated. Shown are means +/− SD of three independent growth curves for each virus. The insert depicts the loss of MCK-2 by staining extracts of NIH3T3 cells infected with wildtype or 131stopB virus with an anti-MCK-2 (5A5) antibody. p.i., post infection.

Article Snippet: Primary mouse embryonal fibroblasts from BALB/c mice (MEF), NIH3T3 cells ( ATCC : CRL-1658), the endothelial cell line MHEC5-T , the epithelial cell line TCMK-1 ( ATCC : CCL-139), the macrophage cell line J774 ( ATCC : TIB-67), and peritoneal exudates cells (PEC) from BALB/c mice were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum.

Techniques: Infection, Virus, Staining

Journal: PLoS Pathogens

Article Title: The Viral Chemokine MCK-2 of Murine Cytomegalovirus Promotes Infection as Part of a gH/gL/MCK-2 Complex

doi: 10.1371/journal.ppat.1003493

Figure Lengend Snippet: (A) MEF, NIH3T3, MHEC-5T and TCMK-1 cells were plated in 96 well plates, infected with wildtype MCMV and 131stop mutants of MCMV at an m.o.i. of 0.5, and 6 h after infection stained for MCMV immediate early 1 (IE1) protein by indirect immunofluorescence. Each infection was done in triplicates and for each well IE1 + cells were counted and the means of these counts related to the mean of IE1 + MEF. The value for MEF was set to 100%. Shown are means +/− SEM of at least 4 independent experiments. Infection of TCMK-1 with m131stop mutants was significantly enhanced compared to wildtype infection. The P values (Student's t-test) are indicated in the histograms. (B) Immortalized macrophages ANA-1 (left and middle panel) and J774 (right panel) were infected in suspension with wildtype MCMV or MCMV-gH-HA carrying a wildtype MCK-2 or with the MCK-2 mutants m131stopB or D, MCMV-gH-HA/129stop, or pSM3fr BAC-derived virus. The numbers of infected macrophages were determined by intracellular FACS staining for IE1 + cells. All infections with MCK-2 mutants showed significantly lower numbers of infected macrophages than infections with wildtype virus. The P values (Student's t-test) are indicated in the histograms. Shown are means +/−SEM of 3 to 4 independent experiments. (C) Primary BMDM (left panel) and cells in the macrophage-enriched gate of PEC (PEC/M) (right panel) were infected with wildtype virus or the MCK-2 mutant 131stopD as described under (B). The numbers of infected cells were significantly lower for the 131stop mutant. The P values (Student's t-test) are indicated in the histograms. Shown are means +/−SEM of 3 independent experiments. (B and C) All virus preparations have been titrated on MEF and for each experiment MEF were infected in parallel to confirm that wildtype and mutant viruses infected comparable percentages of MEF.

Article Snippet: Primary mouse embryonal fibroblasts from BALB/c mice (MEF), NIH3T3 cells ( ATCC : CRL-1658), the endothelial cell line MHEC5-T , the epithelial cell line TCMK-1 ( ATCC : CCL-139), the macrophage cell line J774 ( ATCC : TIB-67), and peritoneal exudates cells (PEC) from BALB/c mice were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum.

Techniques: Infection, Staining, Immunofluorescence, Suspension, Derivative Assay, Virus, Mutagenesis

Journal: PLoS Pathogens

Article Title: The Viral Chemokine MCK-2 of Murine Cytomegalovirus Promotes Infection as Part of a gH/gL/MCK-2 Complex

doi: 10.1371/journal.ppat.1003493

Figure Lengend Snippet: NIH3T3, NIH3T3-MCK2, and NIH3T3-gO cells were infected at an m.o.i. of 0.1 with the double mutant Δm74/131stop reconstituted in NIH3T3-gO cells (A-C). (A) Spread in culture was followed by staining cells for IE1 expression 3 and 6 days after infection. (B) Infection was enhanced by a centrifugation step at 2,000× g for 30 min at RT. Cells were stained for IE1 24 h after infection to show the comparable initial infection of NIH3T3, NIH3T3-gO, and NIH3T3-MCK-2 cells (left panel). Release of infectious virus was monitored in multistep growth curves (right panel). (C) The release of DNAse-protected viral DNA was followed by real-time PCR using supernatants from the growth curves under (B, right panel) plus an additional time point at 6 h post infection. The column labeled “in” shows the amount of DNAse-protected viral DNA in the Δm74/131stop inoculum used to infect the cells. (A–C) p.i., post infection.

Article Snippet: Primary mouse embryonal fibroblasts from BALB/c mice (MEF), NIH3T3 cells ( ATCC : CRL-1658), the endothelial cell line MHEC5-T , the epithelial cell line TCMK-1 ( ATCC : CCL-139), the macrophage cell line J774 ( ATCC : TIB-67), and peritoneal exudates cells (PEC) from BALB/c mice were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum.

Techniques: Infection, Mutagenesis, Staining, Expressing, Centrifugation, Virus, Real-time Polymerase Chain Reaction, Labeling

Journal: PLoS Pathogens

Article Title: The Viral Chemokine MCK-2 of Murine Cytomegalovirus Promotes Infection as Part of a gH/gL/MCK-2 Complex

doi: 10.1371/journal.ppat.1003493

Figure Lengend Snippet: (A) MEF (left panel) and ANA-1 cells (right panel) were infected with MCMV mutants 131stopB and Δm74 and MEF additionally with Δm74/m74trans. The latter mutant was trans-complemented with gO by growth in NIH3T3-gO cells. Virus was either preincubated with anti-MCK2 rabbit antiserum or with an anti-pUL131A rabbit antiserum, which served as a control rabbit antiserum, at a dilution of 1∶10, or with medium as a mock control. Infection of cells was monitored by indirect immunofluorescence staining for IE1 + cells 6 h after infection. The percentage of IE1 + cells is expressed relative to the percentage of IE1 + cells of mock-treated infections. As indicated, both, Δm74 MCMV and Δm74/m74trans MCMV were significantly (Student's t-test) inhibited by the MCK-2 antiserum when compared to the control rabbit antiserum. The P values are indicated in the histograms. Shown are means +/−SEM of 3 to 4 independent experiments. (B) MEF were infected with a Δm74/131stop mutant either grown in NIH3T3-gO (gO-trans) or NIH3T3-MCK-2 (MCK-2 trans) cells in the presence or absence of energy depletion medium, bafilomycin A1, or NH 4 Cl. Three hours after infection, cells were fixed and stained for IE1 expression. The percentage of IE1 + cells is expressed relative to the percentage of IE1 + cells of mock-treated infections. For all inhibitors, inhibition of the gO trans-complemented and of the MCK-2 trans-complemented mutant was significantly different (Student's t test). The P values are indicated in the histograms. Shown are means +/− SEM of 4 to 6 independent experiments.

Article Snippet: Primary mouse embryonal fibroblasts from BALB/c mice (MEF), NIH3T3 cells ( ATCC : CRL-1658), the endothelial cell line MHEC5-T , the epithelial cell line TCMK-1 ( ATCC : CCL-139), the macrophage cell line J774 ( ATCC : TIB-67), and peritoneal exudates cells (PEC) from BALB/c mice were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum.

Techniques: Infection, Mutagenesis, Virus, Control, Immunofluorescence, Staining, Expressing, Inhibition

Journal: ACS nano

Article Title: Dynamic Biodistribution of Extracellular Vesicles In Vivo Using a Multimodal Imaging Reporter

doi: 10.1021/nn404945r

Figure Lengend Snippet: (a, b) Stable HEK293T cells expressing sshBirA with GlucB or Gluc. (a) Live-cell imaging of HEK293T cells stably transduced with GlucB-IRES-GFP or Gluc-IRES-GFP vectors, both with sshBirA-IRES-mCherry. Bar, 100 μm. (b) Western blot analysis showing enhanced biotinylation of cells stably expressing GlucB with sshBirA, as compared to GlucB alone. No biotinylation was detected in cells expressing Gluc alone or Gluc with sshBirA. Immunoblotting with anti-Gluc antibodies showed Gluc and GlucB at expected sizes (Gluc: 20 kDa; GlucB: 42 kDa). A low level of biotinylated BAP domain (22 kDa) was also detected in GlucB and GlucB + sshBirA samples. Mock transduced HEK293T were used as a negative control. GAPDH was immunoprobed as a loading control. (c, d) Transmission electron micrograph (TEM) demonstrating biotinylation and Gluc labeling of EV-GlucB on the membrane. (c) Sectioned EVs were immunolabeled with either anti-CD63, an exosome marker,21 or anti-Gluc antibody followed by gold-conjugated secondary antibody to visualize GlucB labeling of EVs on the membrane, with EV-Gluc showing no Gluc signal but having the CD63 signal. Bar, 100 nm. (d) EVs in suspension were immunolabeled with an anti-biotin antibody followed by 10 nm gold-conjugated secondary antibody and biotinylation of EV-GlucB, but not EV-Gluc surface was detected. (e) Dot blot detection of biotinylated EVs. EVs isolated from HEK293T cells stably expressing sshBirA with either GlucB (top) or Gluc (bottom) were dot blotted on nitrocellulose membranes in a dose range followed by probing with streptavidin-HRP and chemiluminescence detection. EV-GlucB showed quantity-dependent biotinylated EVs, whereas EV-Gluc control exhibited no biotinylation background signal. (f) Nanoparticle tracking analysis (NTA) of EVs. Similar size distribution between EV-Gluc (peaks: 67, 100 and 177 nm) and EV-GlucB (81, 104 and 175 nm) vesicles was detected.

Article Snippet: Twenty μg of total protein was boiled for 2 min in SDS sample buffer, resolved by 10% SDS-PAGE gel with molecular weight standards (Precision Plus ProteinTM All Blue Standards, Bio-Rad), and transferred onto nitrocellulose membranes.

Techniques: Expressing, Live Cell Imaging, Stable Transfection, Transduction, Western Blot, Negative Control, Control, Transmission Assay, Labeling, Membrane, Immunolabeling, Marker, Suspension, Dot Blot, Isolation

Journal: Communications Biology

Article Title: IL-33 enhances Jagged1 mediated NOTCH1 intracellular domain (NICD) deubiquitination and pathological angiogenesis in proliferative retinopathy

doi: 10.1038/s42003-022-03432-7

Figure Lengend Snippet: a Schematic diagram representing the murine OIR model. C57BL/6 mice pups with dams were exposed to 75% oxygen from P7 to P12 and returned to room at P12. At P17, eyes were enucleated, retinas isolated. b Total cellular RNA was isolated from retina and quantified for IL-33 and GAPDH mRNA levels by QRT-PCR. c Retinal tissue extracts were prepared and analyzed for IL-33 by Western blotting and normalized to β-tubulin. n = 6 mice per group. Quiesced HRMVECs were treated with and without IL-33 (20 ng/mL) and cell proliferation was measured by thymidine incorporation ( d ), and BrdU cell proliferation assay ( e ). f , g Everything is same as in d , except that cell migration was measured using Boyden chamber method ( f ) and wound healing assay ( g ). h HRMVECs cells were labeled, coated onto cytodex beads, embedded in a 3D-fibrin gel and sprouting was observed after 3 days under Zeiss LSM800 microscope. i Everything is same as in d , except that tube formation was assessed using growth factor reduced Matrigel. The bar graphs show the quantitative analysis of three independent experiments, expressed as Mean ± SD. * P < 0.05 vs control or normoxia. Scale bar represents 50 μm in h .

Article Snippet: The TaqMan Gene Expression Assays for mouse IL-33 (Mm00505403), and mouse GAPDH (Mm99999915_g1) were used for PCR amplification.

Techniques: Isolation, Quantitative RT-PCR, Western Blot, BrdU Cell Proliferation Assay, Migration, Wound Healing Assay, Labeling, Microscopy, Control

Journal: Communications Biology

Article Title: IL-33 enhances Jagged1 mediated NOTCH1 intracellular domain (NICD) deubiquitination and pathological angiogenesis in proliferative retinopathy

doi: 10.1038/s42003-022-03432-7

Figure Lengend Snippet: C57BL/6 mice pups were exposed to OIR and at P15 eyes from pups in normoxia or OIR were enucleated, retinas isolated, minced, digested and single cell retinal suspension were prepared in FACS buffer. The retinal cells were first incubated with indicated dyes and antibodies. After washings, the cells were resuspended into sorting buffer, added SYTOX blue and subjected to FACS analysis. a Live cells from both normoxia and P15 were gated as calcein blue positive cells. CD11b positive and CD45 low cells were gated as microglia. CD31 positive and ACSA1 negative cells were gated endothelial cells. ACSA1 positive and CD31 negative cells are gated as astrocytes. CD146 positive and CD140b positive cells are gated as pericytes. b Calcein blue positive cells from both normoxia and OIR were gated as live cells and the NeuO + cells were gated as neurons. c Total cellular RNA was isolated from all the sorted cells and analyzed for IL-33 and GAPDH mRNA levels by QRT-PCR. The bar graphs show the quantitative analysis of three independent experiments ( n = 6 animals/group/experiment), expressed as Mean ± SD. * P < 0.05 vs normoxia.

Article Snippet: The TaqMan Gene Expression Assays for mouse IL-33 (Mm00505403), and mouse GAPDH (Mm99999915_g1) were used for PCR amplification.

Techniques: Isolation, Suspension, Incubation, Quantitative RT-PCR