Journal: Journal of pharmacological sciences

Article Title: Formyl peptide receptor 1 and 2 dual agonist inhibits human neutrophil chemotaxis by the induction of chemoattractant receptor cross-desensitization.

doi: 10.1254/jphs.10194fp

Figure Lengend Snippet: Fig. 1. Cpd43 induces cross-desensitization of the Ca2+ response in neutrophils. Human neutrophils were loaded with Fluo4-AM. The cells were prestimulated with various concentrations of Cpd43 for 30 min and subsequently stimulated with 1 nM IL-8, 0.1 nM C5a, or 10 nM LTB4. Calcium flux at the second stimulation in the cells was ana- lyzed using a flow cytometer. Data (mean ± S.E.M.) are expressed as the mean fluorescence intensity in the 5-s duration immediately before (white bar) and after (black bar) stimulation. The experiment was performed in duplicate and the representative data of four independent experiments with similar results are shown. AU, arbitrary units.

Article Snippet: Carboxyfluorescein-conjugated human CXCR1 monoclonal antibody (mAb) (clone 42705), phycoerythrin-conjugated human CXCR2 mAb (clone 48311), and carboxyfluoresceinconjugated human LTB4 receptor 1 (BLT1) mAb (clone 203/14F11) were purchased from R&D Systems.

Techniques: Flow Cytometry, Fluorescence

Journal: Journal of pharmacological sciences

Article Title: Formyl peptide receptor 1 and 2 dual agonist inhibits human neutrophil chemotaxis by the induction of chemoattractant receptor cross-desensitization.

doi: 10.1254/jphs.10194fp

Figure Lengend Snippet: Fig. 2. Pretreatment of Cpd43 inhibits neutrophil chemotaxis induced by several chemoattractants. Human neutrophils were pre- incubated with various concentrations of Cpd43 at 37°C for 30 min and washed twice to remove the compound. Chemotaxis of these cells was induced by 1 nM IL-8, 0.1 nM C5a, or 1 nM LTB4. Data (mean ± S.E.M.) are expressed as the percentage of net migrated cells. The experiment was performed in duplicate and the representa- tive data of four independent experiments with similar results are shown.

Article Snippet: Carboxyfluorescein-conjugated human CXCR1 monoclonal antibody (mAb) (clone 42705), phycoerythrin-conjugated human CXCR2 mAb (clone 48311), and carboxyfluoresceinconjugated human LTB4 receptor 1 (BLT1) mAb (clone 203/14F11) were purchased from R&D Systems.

Techniques: Chemotaxis Assay, Incubation

Journal: bioRxiv

Article Title: Inhibition of polo-like kinase 1 (PLK1) facilitates reactivation of gamma-herpesviruses in B-cell lymphomas and their elimination

doi: 10.1101/2020.10.08.330548

Figure Lengend Snippet: (A) Total RNAs were extracted from the fixed diffuse large B-cell lymphoma (DLBCL) tissue samples of HIV-negative (HIV-, n=3) and HIV-positive (HIV+, n=5) individuals. mRNA level of PLK1 in these tissue samples was measured by RT-PCR and normalized to GAPDH. (B) A PE-conjugated CD19 antibody was used for B cell enrichment from PBMCs of HIV-infected, aviremic patients (HIV+, n=5) or healthy donors (HIV-, n=5). Purity of isolated CD19 + B cells was confirmed by flow cytometry analysis. (C) mRNA level of PLK1 in above isolated B cells (B) was measured by RT-PCR and normalized to GAPDH. (D) Protein level of intracellular PLK1 in above isolated B cells (B) was measured by immunofluorescence. Percentage of PLK1-expressing cells was determined by flow cytometry analysis. (F) HIV-1 IIIB infected Jurkat cells were co-cultured with BJAB cells that were pre-stained with CFSE. Protein level of intracellular PLK1 in CFSE-labeled BJAB cells was measured by immunofluorescence. Percentage of PLK1-expressing cells was determined by flow cytometry analysis. (G-I) Protein level of PLK1 in BJAB (G), Atata-BX1 (H), or TREx BCBL1-Rta (I) cells treated with recombinant Nef (rNef) protein at the increasing dose or DMSO was measured by immunoblotting. GAPDH was used as a loading control. Results were calculated from n=3 independent experiments and presented as mean ± SD (* p<0.05; two-tailed paired Student t-test).

Article Snippet: Portion of isolated B cells was incubated with CD19 antibody (1/200 of stock, Milteny) for 30 mins to determine the purity using BD Accuri C6 Plus with corresponding optical filters.

Techniques: Reverse Transcription Polymerase Chain Reaction, Infection, Isolation, Flow Cytometry, Immunofluorescence, Expressing, Cell Culture, Staining, Labeling, Recombinant, Western Blot, Control, Two Tailed Test

Journal: Cancer Research

Article Title: Inhibition of Ephrin B2 Reverse Signaling Abolishes Multiple Myeloma Pathogenesis

doi: 10.1158/0008-5472.CAN-23-1950

Figure Lengend Snippet: Inhibition of ephrin B2 suppresses multiple myeloma growth in vivo . A, Representative immunofluorescence images of RPMI-8226 multiple myeloma (MM) cells stained for BCMA (green) and EFNB2; merged image with DAPI (blue) nuclear stain at right (red). Scale bar, 100 μm. B, Representative immunofluorescence images of VE-cadherin (Cdh5) expression (white) and Ephb4 colocalization (green) in BM blood vessels in NSG mice. Scale bar, 100 μm. C, Representative images of femur cross sections from NSG mice at 5 weeks posttransplant of RPMI-8226 multiple myeloma cells; sinusoidal blood vessel expressing VE-cadherin (Cdh5; white), multiple myeloma cells expressing BCMA (green), and EFNB2 (red) and merged image shown at right. Scale bar, 100 μm. Arrows, multiple myeloma cells adjacent to sinusoidal vessel. D, Left, representative flow cytometric analysis of human CD138 + multiple myeloma cells in the BM of NSG mice at 5 weeks posttransplant of 5 × 10 6 RPMI-8226 cells treated with sh-EFNB2 or sh-Control . SSC, side scatter. Right, mean percentages of CD138 + multiple myeloma cells in the BM of transplanted mice. n = 8/group. E, Percent survival of NSG mice after transplantation of 5 × 10 6 RPMI-8226 cells transduced with sh-EFNB2 or sh-Control . Log-rank test, P = 0.0012, n = 12/group. F, Mean percentages of Annexin V + RPMI-8226 cells at 24 hours posttreatment with EFNB2 scFv ( n = 6/group). G, Top, schematic representation of experiment. Bottom left, representative flow cytometric analysis of human CD138 + multiple myeloma cells in the BM of NSG mice at 4 weeks posttransplantation of 3 × 10 6 RPMI-8226 cells and intravenous administration of EFNB2 scFv or IgG on days +4, 6, 8, 10, and 12. Bottom right, mean percentages of CD138 + multiple myeloma cells in the BM of transplanted mice. One-way ANOVA was utilized for analyses of three or more groups; Student two-sided t test was utilized otherwise. Error bars, SD. **, P < 0.01; ****, P < 0.0001; # , P < 0.05; ### , P < 0.001. ( G, Created with BioRender.com.)

Article Snippet: Cells were stimulated with either human IgG1 mAb (R&D Systems, MAB9894-SP) or preclustered recombinant mouse EPHB4 Fc Chimera Protein (R&D Systems, 446-B4).

Techniques: Inhibition, In Vivo, Immunofluorescence, Staining, Expressing, Control, Transplantation Assay, Transduction

Journal: Cancer Research

Article Title: Inhibition of Ephrin B2 Reverse Signaling Abolishes Multiple Myeloma Pathogenesis

doi: 10.1158/0008-5472.CAN-23-1950

Figure Lengend Snippet: The effects of ephrin B2 on multiple myeloma are mediated by STAT5. A, Heat map of differentially expressed genes by RNA-seq of sh-EFNB2 versus sh-Control cells. B, Upstream regulator analysis from IPA of RNA-seq data. C, Selected STAT5 target genes in the RNA-seq data. D, Left, representative analysis of phospho-STAT5 in RPMI-8226 cells treated with sh-EFNB2 or sh-Control in presence of EPHB4-Fc. Right, mean fluorescence intensity (MFI) of pSTAT5 in the sh-EFNB2 - and sh-Control –treated multiple myeloma cells. E, Numbers of U266 cells in culture over time when transduced with gRNA1-EFNB2 and gRNA2-EFNB2 . F, Left, representative histograms of BrdU incorporation over 6 hours in U266 cells transduced with the labeled construct. Right, %BrdU + cells in each group. G, Left, representative flow cytometric analysis of Annexin V + 7AAD + (necrotic) and Annexin V + 7AAD − (apoptotic) cells. Right, mean levels of total Annexin V + cells within each treatment group. H, Mean percentages of Annexin V + U266 cells engineered with the indicated construct and exposed to bortezomib (Bor) or dexamethasone (Dex) for 24 hours. n = 6/group. I, Representative histograms of p-STAT5 in U266 cells transduced with gRNA-EFNB2 or gRNA-control , with and without stimulation by EPHB4-Fc. IgG (isotype) was used as a negative control for EPHB4-Fc. Right, mean MFI of pSTAT5 in each treatment population. J, Multiple myeloma cells transduced with gRNA-EFNB2 or gRNA-control were cultured with increasing concentrations of the STAT5 inhibitor 2-[(4-oxo-4H-1-benzopyran-3-yl)methylene]hydrazide 3-pyridinecarboxylic acid (STAT5i) for 24 hours, then BrdU incorporation analysis was performed. n = 6/group. One-way ANOVA used for analyses in which there were three or more groups, Student two-sided t test for the other analyses. Error bars, SD. ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Article Snippet: Cells were stimulated with either human IgG1 mAb (R&D Systems, MAB9894-SP) or preclustered recombinant mouse EPHB4 Fc Chimera Protein (R&D Systems, 446-B4).

Techniques: RNA Sequencing, Control, Fluorescence, Transduction, BrdU Incorporation Assay, Labeling, Construct, Negative Control, Cell Culture

Journal: Cancer Research

Article Title: Inhibition of Lymphotoxin-β Receptor–Mediated Cell Death by Survivin-ΔEx3

doi: 10.1158/0008-5472.can-05-2479

Figure Lengend Snippet: Figure 1. Interaction of caspase-3 with KSHV-K7 and survivin-DEx3. A, inhibition of caspase-3 activity in LIGHT/ IFN-g-mediated cell death. Wild-type Hep3BT2 (WT) and transfectants stably expressing survivin-DEx3, KSHV-K7, and Bcl-2 were incubated with IFN-g and LIGHT for 12 hours, and caspase-3 activities in cell lysates were determined using the fluorescent probe MCA-DEVD.APK (DNP). Columns, average of three independent experiments done in duplicate; bars, SD. *, P < 0.05, significantly different from untreated as determined by ANOVA. B, interaction between survivin-DEx3, KSHV-K7, and activated caspase-3. HEK293 cells were treated with TNF-a (10 ng/mL) plus cycloheximide (1 Ag/mL) for 8 hours, and cell lysates were then incubated with 1 Ag recombinant FLAG-KSHV-K7 or FLAG-survivin-DEx3 protein purified from pFLAG-KSHV- K7-transfected or pFLAG-survivin-DEx3- transfected HEK293T cells followed by immunoprecipitation using anti-FLAG mAb. The presence of caspase-3 in the immunoprecipitates was determined using an anti-caspase-3 (CPP32) mAb. C, inhibition of activated caspase-3. Recombinant activated caspase-3 was incubated with Z-DEVD or transfected cell lysates in the presence of MCA-DEVD.APK (DNP) for 1 hour at 37jC. Caspase-3 activities were determined using a fluorescence spectrophotometer.

Article Snippet: The blots were probed with mAbs against cytochrome c (6H2.B4; Imgenex, San Diego, CA), cytochrome oxidase IV (COX IV; Molecular Probes), actin (Sigma), and Smac/DIABLO (R&D Systems).

Techniques: Inhibition, Activity Assay, Stable Transfection, Expressing, Incubation, Recombinant, Purification, Transfection, Immunoprecipitation, Fluorescence, Spectrophotometry

Journal: Cancer Research

Article Title: Inhibition of Lymphotoxin-β Receptor–Mediated Cell Death by Survivin-ΔEx3

doi: 10.1158/0008-5472.can-05-2479

Figure Lengend Snippet: Figure 4. Survivin-DEx3 prevents the release of cytochrome c and Smac from Hep3BT2 cells after LIGHT/IFN-g treatment. A, mitochondrial integrity assay by subcellular fractionation. Cells were incubated with LIGHT/IFN-g for 20 hours followed by digitonin-based subcellular fractionation to separate the cell lysate into cytosol-enriched (left) and mitochondria-enriched (right) fractions and by Western blot analysis using antigen-specific antibodies. (Each fraction represents 5 106 cells.) Actin and COX IV served as references for the cytosol and mitochondria, respectively, as described in Materials and Methods. B, colocalization of survivin-DEx3 and Smac. Hep3BT2 cells were incubated with LIGHT/IFN-g for 20 hours. Survivin-DEx3 and KSHV-K7 were stained with anti- Myc tag, whereas cytochrome c (Cyt c) and Smac were stained with anti–cytochrome c and anti-Smac antibodies, respectively. Smac and anti-Myc signals were merged. The cell nucleus was stained with Hoechst 33342. C, association of survivin-DEx3 with Smac. Hep3BT2 cells were cotransfected with FLAG-tagged survivin-DEx3, KSHV-K7, or survivin in conjunction with HA-tagged Smac for 18 hours followed by incubation with LIGHT/IFN-g for another 20 hours. Cells were then harvested, and cell lysates were incubated with M2 anti-FLAG antibody for immunoprecipitation before blotting onto nitrocellulose membrane. Blots were probed with anti-FLAG or anti-HA mAb. D, translocation of survivin-DEx3 to mitochondria. Hep3BT2 cells were cotransfected pDsRed2-Mito and either survivin-DEx3 or KSHV-K7 for 18 hours followed by incubation with LIGHT/IFN-g for various time intervals. Survivin-DEx3 and KSHV-K7 were stained with anti-Myc tag antibody; the cell nucleus was stained with Hoechst 33342. Colocalization in the merged images of green and red channels. E, BIR domain is essential for the interaction between survivin-DEx3 and Smac. Hep3BT2 cells (1 106) were cotransfected with FLAG-tagged survivin-DEx3 (2 Ag/mL) or survivin-DEx3 (DBIR; 2 Ag/mL) in conjunction with HA-tagged Smac (2 Ag/mL) for 18 hours followed by incubation with LIGHT/IFN-g for another 20 hours. Cells were then harvested, and cell lysates were incubated with M2 anti-FLAG antibody for immunoprecipitation before blotting onto nitrocellulose membrane. Blots were probed with anti-FLAG or anti-HA mAb. F, colocalization of Smac with survivin-DEx3 but not survivin-DEx3 (DBIR). Hep3BT2 cells (3 105) were cotransfected with pDsRed2-Mito, HA-tagged Smac, in conjunction with Myc-tagged survivin-DEx3 or survivin-DEx3 (DBIR) for 18 hours followed by incubation with LIGHT/IFN-g for 20 hours. Cells were incubated with anti-Myc antibody and FITC-conjugated anti-mouse IgG subsequently to detect survivin-DEx3 and survivin-DEx3 (DBIR) or stained with anti-Smac antibody and Cy5-conjugated goat anti-rabbit antibody subsequently. Colocalization in the merged images of green [FITC for survivin-DEx3 or survivin-DEx3 (DBIR)], red (DsRed for mitochondria), and blue (Cy5 for Smac/DIABLO) channels. Mt, mitochondrial fraction.

Article Snippet: The blots were probed with mAbs against cytochrome c (6H2.B4; Imgenex, San Diego, CA), cytochrome oxidase IV (COX IV; Molecular Probes), actin (Sigma), and Smac/DIABLO (R&D Systems).

Techniques: Integrity Assay, Fractionation, Incubation, Western Blot, Staining, Immunoprecipitation, Membrane, Translocation Assay

Journal: Cancer Research

Article Title: Inhibition of Lymphotoxin-β Receptor–Mediated Cell Death by Survivin-ΔEx3

doi: 10.1158/0008-5472.can-05-2479

Figure Lengend Snippet: Figure 6. Translocation of survivin-DEx3 from nucleus to cytosol and colocalization of survivin-DEx3 with ASK1 after LIGHT/IFN-g treatment. A, translocation of survivin-DEx3 from nucleus to cytosol. Hep3BT2 cells were transfected with Myc-tagged survivin-DEx3 followed by treatment with LIGHT/IFN-g for various time intervals. Localization of survivin-DEx3 was determined by confocal microscopy using anti-HA (for ASK1) and Cy3-conjugated donkey anti-rat secondary antibody (red) subsequently, whereas survivin-DEx3 was detected by anti-Myc antibody and FITC-conjugated goat anti-mouse secondary antibody (green) subsequently. Merge, merged images from survivin-DEx3 and ASK1 staining. The cell nucleus was stained with Hoechst 33342. Bar, 8 Am. B, subcellular fractionation of survivin-DEx3 and ASK1. Hep3BT2 cells were transfected with Myc-tagged survivin-DEx3 and treated with LIGHT/IFN-g. Cytosolic and nuclear fractions were prepared as described in Materials and Methods. Western blots of the two fractions (nuclear and cytosol) were probed with anti-FLAG (for survivin-DEx3) or anti-HA (for ASK1) antibody. Anti-tubulin and anti–poly(ADP-ribose) polymerase (PARP) were used as controls for the cytoplasmic and nuclear fractions, respectively. C, association of survivin-DEx3 with ASK1. Hep3BT2 cells were cotransfected with FALG-survivin-DEx3 and HA-ASK1 for 18 hours and incubated with LIGHT/IFN-g. Following subcellular fractionation, the nuclear and cytoplasmic fractions were immunoprecipitated with antibody and blotted onto nitrocellulose membrane. The blot was probed with anti-FLAG and anti-HA antibody to detect the presence of survivin-DEx3 and ASK1, respectively. D, mapping of interaction domain of survivin-DEx3 with ASK-1. HeLa (1 106) cells were transfected with pCR3.1, pCR3.1-survivin-DEx3, or its deletion mutants for 18 hours followed by immunoprecipitation using anti-HA to pull down survivin-DEx3 and deletion mutants and incubated with anti-ASK-1 mAb (top). Alternatively, cell lysates were precipitated by anti-ASK-1 mAb followed by incubation with anti-HA mAb to detect survivin-DEx3 and its mutants (bottom). E, HeLa cells were transfected with pCR3.1, pCR3.1-survivin-DEx3, or pCR3.1-survivin-DEx3 (DNLS) for 18 hours followed by incubation with LIGHT-R228E (100 ng/mL) for various time intervals, and the ASK1 activities in the immunoprecipitates were determined by in vitro kinase assay. Nu, nuclear fraction.

Article Snippet: The blots were probed with mAbs against cytochrome c (6H2.B4; Imgenex, San Diego, CA), cytochrome oxidase IV (COX IV; Molecular Probes), actin (Sigma), and Smac/DIABLO (R&D Systems).

Techniques: Translocation Assay, Transfection, Confocal Microscopy, Staining, Fractionation, Western Blot, Incubation, Immunoprecipitation, Membrane, In Vitro, Kinase Assay