Journal: Molecular Therapy Oncolytics

Article Title: Systemic immunity upon local oncolytic virotherapy armed with immunostimulatory genes may be supported by tumor-derived exosomes

doi: 10.1016/j.omto.2021.02.007

Figure Lengend Snippet: Expression of CD40L and 4-1BBL on Mel526 cells Mel526 cells infected with LOAd700 or LOAd703 express CD40L (upper histograms), while cells infected with LOAd703 also present 4-1BBL (lower histograms), as shown with flow cytometry at day 2 post-infection. Gray and black histograms represent isotype control and transgene staining, respectively.

Article Snippet: Exosomes were then incubated with a primary antibody, either CD40L (AF617, R&D Systems) or 4-1BBL (ab126274, Abcam), followed with goat anti-mouse or goat anti-rabbit as a secondary antibody conjugated with 6-nm gold particles (Jackson ImmunoResearch) in a ratio of 1:80 (40 min incubation each).

Techniques: Expressing, Infection, Flow Cytometry, Control, Staining

Journal: Molecular Therapy Oncolytics

Article Title: Systemic immunity upon local oncolytic virotherapy armed with immunostimulatory genes may be supported by tumor-derived exosomes

doi: 10.1016/j.omto.2021.02.007

Figure Lengend Snippet: Characterization of exosomes (A) Nanoparticle tracking analysis of exosomes isolated from Mel 626 cells using NanoSight NS500. Captures 1–5 represent five captured videos for calculation of size and concentration of exosomes. (B) Exosomal markers (CD63 and CD81) were evaluated on cells (either infected or uninfected) and their exosomes using western blot. (C) Electron micrographs of isolated exosomes stained for CD40L or 4-1BBL. Arrows indicate positive staining. Cells exo, exosomes from uninfected cells; LOAd cells, cells infected with LOAd viruses; LOAd exo, exosomes released by LOAd-infected cells.

Article Snippet: Exosomes were then incubated with a primary antibody, either CD40L (AF617, R&D Systems) or 4-1BBL (ab126274, Abcam), followed with goat anti-mouse or goat anti-rabbit as a secondary antibody conjugated with 6-nm gold particles (Jackson ImmunoResearch) in a ratio of 1:80 (40 min incubation each).

Techniques: Isolation, Concentration Assay, Infection, Western Blot, Staining

Journal: Molecular Therapy Oncolytics

Article Title: Systemic immunity upon local oncolytic virotherapy armed with immunostimulatory genes may be supported by tumor-derived exosomes

doi: 10.1016/j.omto.2021.02.007

Figure Lengend Snippet: CD40L and 4-1BBL protein content of cells and exosomes (A and B) Exosomes derived from Mel526 cells infected with LOAd viruses or left uninfected were investigated for the presence of CD40L (A) and 4-1BBL (B) with ELISA. Each group was tested in duplicate. Error bars represent standard error and median values are displayed in the graphs.

Article Snippet: Exosomes were then incubated with a primary antibody, either CD40L (AF617, R&D Systems) or 4-1BBL (ab126274, Abcam), followed with goat anti-mouse or goat anti-rabbit as a secondary antibody conjugated with 6-nm gold particles (Jackson ImmunoResearch) in a ratio of 1:80 (40 min incubation each).

Techniques: Derivative Assay, Infection, Enzyme-linked Immunosorbent Assay

Journal: Molecular Therapy Oncolytics

Article Title: Systemic immunity upon local oncolytic virotherapy armed with immunostimulatory genes may be supported by tumor-derived exosomes

doi: 10.1016/j.omto.2021.02.007

Figure Lengend Snippet: Surface expression of CD40L and 4-1BBL on exosomes from LOAd-infected cells using flow cytometry (A) Total percentage of CD40L + and 4-1BBL + exosomes. (B) Mean fluorescence intensity (MFI) of CD40L and 4-1BBL expression on CD63 + bead-coupled exosomes. ∗p ≤ 0.05, n = 3. Error bars represent standard error and median values are displayed in the graphs.

Article Snippet: Exosomes were then incubated with a primary antibody, either CD40L (AF617, R&D Systems) or 4-1BBL (ab126274, Abcam), followed with goat anti-mouse or goat anti-rabbit as a secondary antibody conjugated with 6-nm gold particles (Jackson ImmunoResearch) in a ratio of 1:80 (40 min incubation each).

Techniques: Expressing, Infection, Flow Cytometry, Fluorescence

Journal: Molecular Therapy Oncolytics

Article Title: Systemic immunity upon local oncolytic virotherapy armed with immunostimulatory genes may be supported by tumor-derived exosomes

doi: 10.1016/j.omto.2021.02.007

Figure Lengend Snippet: TMZ-CD40L and 4-1BBL mRNA in infected cells and exosomes (copy number per picogram) (A and B) LOAd-infected or uninfected Mel526 cells and cell-derived exosomes were evaluated for their TMZ-CD40L (A) or 4-1BBL (B) mRNA content using quantitative real-time PCR. Samples were analyzed in triplicates (cells) or duplicates (exosomes). Statistical analyses were done using a Kruskal-Wallis test for non-parametric samples with a Dunn’s multi-comparison test. A p value of ≤0.05 was considered significant. n = 3.Error bars represent standard error and median values are displayed in the graphs.

Article Snippet: Exosomes were then incubated with a primary antibody, either CD40L (AF617, R&D Systems) or 4-1BBL (ab126274, Abcam), followed with goat anti-mouse or goat anti-rabbit as a secondary antibody conjugated with 6-nm gold particles (Jackson ImmunoResearch) in a ratio of 1:80 (40 min incubation each).

Techniques: Infection, Derivative Assay, Real-time Polymerase Chain Reaction, Comparison

Journal: Mammalian genome : official journal of the International Mammalian Genome Society

Article Title: Derivation of stable embryonic stem cell-like, but transcriptionally heterogenous, induced pluripotent stem cells from non-permissive mouse strains

doi: 10.1007/s00335-020-09849-x

Figure Lengend Snippet: Cell surface marker expression in under different culture conditions. A, B Control NOD/ShiLtJ ESC and NOD/EiJ EpiSC grown in serum-free media on a mitotically inactive MEF feeder layer. NOD/ShiLtJ ESC form three-dimensional dome shaped colonies and NOD/ShiLtJ EpiSC formed broad, flattened colonies. C NOD/ShiLtJ ESC label positive for the ESC surface marker PECAM1 and do not label for the EpiSC surface marker CD40. D NOD/ShiLtJ EpiSC label positive for the EpiSC surface marker CD40 and do not label for the PECAM1 embryonic stem cell surface marker. E NOD/ShiLtJ and WSB/EiJ iPSC derived with shortened 2iS culture and returned to 2iS culture after 16 passages. F,G In 2iS free conditions, these cells label heterogeneously for both PECAM1 and CD40. H,I When these colonies are grown in 2iS media again after 16 passages they label homogenously for PECAM1 expression. J NOD/ShiLtJ and WSB/EiJ iPSC.2iS colonies that were derived and maintained in 2iS for 16 passages, were then removed from 2iS culture. K,L In 2iS conditions cells label homogenously for PECAM1. M,N In 2iS free conditions the cells transition to a heterogenous expression pf PECAM1 and CD40.

Article Snippet: Primary antibodies were as follows: PECAM1 monoclonal primary antibody (BD Biosciences 553370) at 1:400 and CD40 polyclonal primary antibody (R&D Systems AF440) diluted to 1:500.

Techniques: Marker, Expressing, Control, Derivative Assay

Journal: Mammalian genome : official journal of the International Mammalian Genome Society

Article Title: Derivation of stable embryonic stem cell-like, but transcriptionally heterogenous, induced pluripotent stem cells from non-permissive mouse strains

doi: 10.1007/s00335-020-09849-x

Figure Lengend Snippet: Differential gene expression in cells grown under different condition. A PCA plot of subpopulation relationships. PECAM1+ cells from both the flow sorted heterogeneous populations cluster together separately from flow sorted CD40+ or dual expressing cells from the same source population. There is less variance between PECAM1+ cells and dual expressing cells than between either and CD40+ cells. B There are fewer differentially expressed genes between PECAM1+ cells and the ESC state than PECAM1+ cells and the EpiSC state. The same is true for flow-sorted dual expressing cells. However, CD40+ cells are equally dissimilar to both the ESC state and the EpiSC state as judged by a roughly equal number of differentially expressed genes in each comparison. C1-C4 Culture of PECAM1+ cells flow sorted cells separately yields a re-emergence of all three subpopulations. C1-C2 single channel images of each marker. C3 Merged image of both markers. C1-C3 scale bar 200 μm. C4 enlarged region of C3 panel, scale bar 30 μm. D1-D4 Culture of CD40+ flow sorted cells separately yields a re-emergence of all three subpopulations. These images are representative of the population three to four days after separate growth. The cells have not been passaged.

Article Snippet: Primary antibodies were as follows: PECAM1 monoclonal primary antibody (BD Biosciences 553370) at 1:400 and CD40 polyclonal primary antibody (R&D Systems AF440) diluted to 1:500.

Techniques: Gene Expression, Expressing, Comparison, Marker

Journal: Mammalian genome : official journal of the International Mammalian Genome Society

Article Title: Derivation of stable embryonic stem cell-like, but transcriptionally heterogenous, induced pluripotent stem cells from non-permissive mouse strains

doi: 10.1007/s00335-020-09849-x

Figure Lengend Snippet: Control iPSC cell surface marker expression under different culture conditions. A 129S1/SvImJ ESC derived in serum/LIF conditions label heterogeneously for PECAM1 and CD40. B 2iS transitions colonies from heterogeneous to homogenous PECAM1 expression. C,D iPSC derived in serum/LIF conditions from permissive C57BL/6J and 129S1/SvImJ strains label homogenously for the PCEAM1. E iPSC from 129S1/SvImJ continue to label homogenously for PECAM1 after being grown in 2iS conditions for eight passages.

Article Snippet: Primary antibodies were as follows: PECAM1 monoclonal primary antibody (BD Biosciences 553370) at 1:400 and CD40 polyclonal primary antibody (R&D Systems AF440) diluted to 1:500.

Techniques: Control, Marker, Expressing, Derivative Assay

Journal: Mammalian genome : official journal of the International Mammalian Genome Society

Article Title: Derivation of stable embryonic stem cell-like, but transcriptionally heterogenous, induced pluripotent stem cells from non-permissive mouse strains

doi: 10.1007/s00335-020-09849-x

Figure Lengend Snippet: Flow cytometry of cells grown under different conditions. A The occurrence of each subpopulation in heterogeneous iPSC populations is about the same regardless of the historical length of 2iS exposure. The majority of live cells are PECAM1 positive. B Repeat of flow sorting experiment. The majority of live cells are positive for PECAM1. There are slightly less dual expressing cells in this second sort. C Flow sorted CD40+ cells cluster separately (highlighted in yellow) from flow sorted PECAM+ cells and flow sorted dual expressing cells. The PECAM1+ cells and dual expressing cells cluster together with captured single cells. The separate cluster of flow sorted CD40+ cells indicate that no CDO40+ cells were captured in our signal cells analysis.

Article Snippet: Primary antibodies were as follows: PECAM1 monoclonal primary antibody (BD Biosciences 553370) at 1:400 and CD40 polyclonal primary antibody (R&D Systems AF440) diluted to 1:500.

Techniques: Flow Cytometry, Expressing

Journal: Mammalian genome : official journal of the International Mammalian Genome Society

Article Title: Derivation of stable embryonic stem cell-like, but transcriptionally heterogenous, induced pluripotent stem cells from non-permissive mouse strains

doi: 10.1007/s00335-020-09849-x

Figure Lengend Snippet: Functional classifications and genes differentially expressed in flow sorted CD40+ cells

Article Snippet: Primary antibodies were as follows: PECAM1 monoclonal primary antibody (BD Biosciences 553370) at 1:400 and CD40 polyclonal primary antibody (R&D Systems AF440) diluted to 1:500.

Techniques: Functional Assay

Journal: Mammalian genome : official journal of the International Mammalian Genome Society

Article Title: Derivation of stable embryonic stem cell-like, but transcriptionally heterogenous, induced pluripotent stem cells from non-permissive mouse strains

doi: 10.1007/s00335-020-09849-x

Figure Lengend Snippet: Transcription factor activity in cells expressing different markers. A FOXO1 production is significantly higher in PECAM1+ cells than in CD40+ cells. MYC production is significantly higher in PECAM1+ cell than in CD40+ cells. It is also significantly higher in PECAM1+ cells than in dual expressing cells. B, C There appears to be little difference in the level of each stem cell transcription factor between PECAM1 and CD40+ cells. D Dual expressing cells from the 2iS culture group have higher production of all stem cell transcription factors than cells not maintained in 2iS.

Article Snippet: Primary antibodies were as follows: PECAM1 monoclonal primary antibody (BD Biosciences 553370) at 1:400 and CD40 polyclonal primary antibody (R&D Systems AF440) diluted to 1:500.

Techniques: Activity Assay, Expressing

Journal: Mammalian genome : official journal of the International Mammalian Genome Society

Article Title: Derivation of stable embryonic stem cell-like, but transcriptionally heterogenous, induced pluripotent stem cells from non-permissive mouse strains

doi: 10.1007/s00335-020-09849-x

Figure Lengend Snippet: Pluripotency potential of stem cells used in this study. At the top of the hill are cells with the most potential. These are transcriptionally homogeneous iPSC colonies. The 2i inhibitors foster a transcriptionally homogeneous PECAM1 positive state, with the exception of iPSC derived from permissive strains such as the 129S1/SvImJ strain that can achieve transcriptional homogeneity in the absence of 2i. ESC from permissive strains and iPSC from non-permissive strains not maintained in 2iS conditions exist at an intermediate level of pluripotency that is both pluripotent and poised for differentiation. This intermediate stage is defined by heterogeneous PECAM1 and CD40 expression. EpiSC are the most poised for differentiation and have the least pluripotency potential of the stem cells studied here. They are the last state of stem cell identity before terminal differentiation. Although CD40 is expressed in EpiSC, it is not unique to the EpiSC state as intermediate ESC also express CD40. A complete loss of pluripotency occurs when cells terminally differentiate.

Article Snippet: Primary antibodies were as follows: PECAM1 monoclonal primary antibody (BD Biosciences 553370) at 1:400 and CD40 polyclonal primary antibody (R&D Systems AF440) diluted to 1:500.

Techniques: Derivative Assay, Expressing

Journal: The Journal of Biological Chemistry

Article Title: The Transitional Endoplasmic Reticulum ATPase p97 Regulates the Alternative Nuclear Factor NF-κB Signaling via Partial Degradation of the NF-κB Subunit p100 *

doi: 10.1074/jbc.M114.630061

Figure Lengend Snippet: p97 regulates the processing of p100 to p52 in a manner dependent on its ATPase activity. A, bar plot for the mRNA levels of NFKB2 and IL6 in the IBMPFD datasets (***, p < 0.001, n.s., no significance, same below). B, box plot for p97 mRNA levels in the lymphoma datasets from the Oncomine database (*, p < 0.05, same below). C, scatter plots between p97 and NFKB2 in the lymphoma datasets. D–E, immunoblotting for p100/p52 in cells overexpressing HA-p100 and V5-NIK with or without IBMPFD-related p97 mutants T262A (D) and R155H (E). F, immunoblotting for p100/p52 in cells overexpressing HA-p100 and V5-NIK with or without sip97 (p97 siRNA, same below). G, immunoblotting for p100/p52 in cells overexpressing HA-p100 and V5-NIK with or without p97 (AA, K251AK524A, same below) mutant. H, immunoblotting for p100/p52 in the lysates of control (scramble, same below) and p97-knockdown (shp97, p97 shRNA, same below) MEF cells after treatment with anti-LTβR for 4 h. I, immunoblotting for p100/p52 in the lysates of control and p97-knockdown Raji cells after stimulation with CD40L for 6 h.

Article Snippet: Recombinant human soluble CD40 ligand (rh-CD40L) was obtained from R&D systems (Minneapolis, MN).

Techniques: Activity Assay, Western Blot, Mutagenesis, Control, Knockdown, shRNA

Journal: The Journal of Biological Chemistry

Article Title: The Transitional Endoplasmic Reticulum ATPase p97 Regulates the Alternative Nuclear Factor NF-κB Signaling via Partial Degradation of the NF-κB Subunit p100 *

doi: 10.1074/jbc.M114.630061

Figure Lengend Snippet: p97 regulates the alternative NF-κB signaling. A–B, transcription of NFKB2 and MCP1, in anti-LTβR-treated MEF cells after transfection with p97 mutant T262A (A) or R155H (B) (**, p < 0.01, same below). C, immunofluorescent staining of p100 and p52 in MEF cells transfected with n.c. (negative control) or sip97 after incubation with anti-LTβR for 12 h. Cells were labeled with DAPI (blue) to visualize the nuclei (left), fluorescent intensities of p100/p52 in cytosol and nuclear were calculated (n = 100, right). D, EMSA assay for detecting the p52 binding to κB promoter by using a 22-bp probe containing κB promoter sequence. E-F, mRNA levels of NFKB2, MCP1, and VCAM-1 in control and p97-knockdown MEF cells after stimulation with anti-LTβR (E), and those in Raji cells after treatment with CD40L (F).

Article Snippet: Recombinant human soluble CD40 ligand (rh-CD40L) was obtained from R&D systems (Minneapolis, MN).

Techniques: Transfection, Mutagenesis, Staining, Negative Control, Incubation, Labeling, Binding Assay, Sequencing, Control, Knockdown

Journal: The Journal of Biological Chemistry

Article Title: The Transitional Endoplasmic Reticulum ATPase p97 Regulates the Alternative Nuclear Factor NF-κB Signaling via Partial Degradation of the NF-κB Subunit p100 *

doi: 10.1074/jbc.M114.630061

Figure Lengend Snippet: The p97-Npl4-Ufd1 complex regulates the p100 processing and the alternative NF-κB signaling. A–B, immunoblotting for p100/p52 in NIK-transfected HEK293T cells (A) or anti-LTβR-incubated MEF cells (B) after transfection with indicated shRNAs. C, transcription of NFKB2, MCP1, RANTES, and BAFF in CD40L-stimulated Raji cells after transfection with indicated shRNAs. D, immunofluorescent staining of p100 and p52 in MEF cells transfected with vector or p97 (3A, F52AD55AY110A) mutant after treatment with CD40L. Cells were labeled with DAPI (blue) to visualize the nuclei (left), fluorescent intensities of p100/p52 in cytosol and nuclear were calculated (n = 100, right). E, mRNA levels of NFKB2, MCP1, RANTES, and BAFF in control and p97 (3A) Raji cells after treatment with CD40L.

Article Snippet: Recombinant human soluble CD40 ligand (rh-CD40L) was obtained from R&D systems (Minneapolis, MN).

Techniques: Western Blot, Transfection, Incubation, Staining, Plasmid Preparation, Mutagenesis, Labeling, Control

Journal: PLoS ONE

Article Title: Soluble CD40 Ligand Stimulates CD40-Dependent Activation of the β2 Integrin Mac-1 and Protein Kinase C Zeda (PKCζ) in Neutrophils: Implications for Neutrophil-Platelet Interactions and Neutrophil Oxidative Burst

doi: 10.1371/journal.pone.0064631

Figure Lengend Snippet: A . Neutrophil firm adhesion to activated surface-adherent platelets. B . Neutrophil transmigration across activated, surface-adherent platelets. Microtiter wells or transwell insert membranes were pre-coated with thrombin-activated platelets adherent to fibrinogen (Fgn) or fibrinogen alone. Washed platelets were prepared from WT or CD40L −/− mice. Neutrophils were prepared from WT or CD40 −/− mice. Cell adhesion and migration were performed as described in Methods . Data obtained from five independent experiments are shown. *P<0.05 versus Bar 1; # P<0.05 versus bar 3. C . Flow cytometry showing the surface expression of CD40L in thrombin-activated WT and CD40 −/− platelets. D . Representative microscopic pictures (×400) showing Giemsa-stained peritoneal neutrophils from WT and CD40 −/− mice. Bar = 10 μm.

Article Snippet: Recombinant mouse CD40 ligand (rm-CD40L) was purchased from R&D Systems (Minneapolis, MN).

Techniques: Transmigration Assay, Migration, Flow Cytometry, Expressing, Staining

Journal: PLoS ONE

Article Title: Soluble CD40 Ligand Stimulates CD40-Dependent Activation of the β2 Integrin Mac-1 and Protein Kinase C Zeda (PKCζ) in Neutrophils: Implications for Neutrophil-Platelet Interactions and Neutrophil Oxidative Burst

doi: 10.1371/journal.pone.0064631

Figure Lengend Snippet: A. Flow cytometric analysis of Mac-1 (CD11b and CD18) expression in WT and CD40 −/− neutrophils stimulated without or with rm-CD40L (100 ng/ml, 60 min). Data obtained from five independent experiments are shown. *P<0.05 versus respective WT control group. B. Immunocytochemical staining showing clustering (a sign of Mac-1 activation) of Mac-1 (CD11b) on the cell surface of neutrophils, with robust clustering seen in WT neutrophils stimulated with rm-CD40L, but with weak signals seen in CD40 −/− neutrophils. Images were obtained by confocal microscopy. Scale bar = 10 μm. Similar results are obtained from five independent experiments.

Article Snippet: Recombinant mouse CD40 ligand (rm-CD40L) was purchased from R&D Systems (Minneapolis, MN).

Techniques: Expressing, Control, Staining, Activation Assay, Confocal Microscopy

Journal: PLoS ONE

Article Title: Soluble CD40 Ligand Stimulates CD40-Dependent Activation of the β2 Integrin Mac-1 and Protein Kinase C Zeda (PKCζ) in Neutrophils: Implications for Neutrophil-Platelet Interactions and Neutrophil Oxidative Burst

doi: 10.1371/journal.pone.0064631

Figure Lengend Snippet: A. Comparison of WT versus Mac-1 −/− neutrophil firm adhesion to activated, surface-adherent platelets. B . Comparison of WT versus Mac-1 −/− neutrophil transmigration across activated, surface-adherent platelets. Neutrophils were stimulated without and with rm-CD40L (100 ng/ml, 60 min). Data obtained from five independent experiments are shown. *P<0.01 versus respective WT control.

Article Snippet: Recombinant mouse CD40 ligand (rm-CD40L) was purchased from R&D Systems (Minneapolis, MN).

Techniques: Comparison, Transmigration Assay, Control

Journal: PLoS ONE

Article Title: Soluble CD40 Ligand Stimulates CD40-Dependent Activation of the β2 Integrin Mac-1 and Protein Kinase C Zeda (PKCζ) in Neutrophils: Implications for Neutrophil-Platelet Interactions and Neutrophil Oxidative Burst

doi: 10.1371/journal.pone.0064631

Figure Lengend Snippet: A. Immunoprecipatation and Western blotting. Neutrophils were stimulated with rm-CD40L (100 ng/ml) for the indicated periods of time. The cell lysates were immunoprecipitated (IP) with anti-PKCζ antibody. Immunoblotting (IB) was carried out using anti-CD11b (Mac-1) antibody. The blots were stripped and reprobed with anti-PKCζ antibody. A set of representative blots from three independent experiemnts is shown. B. Double immunofluoresence staining of PKCζ and Mac-1 (CD11b). Stimulation with rm-CD40L (100 ng/ml, 15 min) significantly increased focal surface clustering of Mac-1 and the colocalization PKCζ and Mac-1 in WT but not in CD40 −/− neutrophils. C. Inhibition of PKCζ activity reduces CD40L-induced neutrophil firm adhesion to activated, surface-adherent platelets. Neutrophils were pretreated (30 min) with a PKC ζ pseudosubstrate (PS, 5 μM) or calphostin C (Cal C, 1 μM), an inhibitor of conventional and novel PKC isoforms. Dimethyl sulfoxide (DMSO) was used as a solvent control. Data obtained from five independent experiments are shown. *P<0.05 versus Bar 1; # P<0.05 versus bar 2.

Article Snippet: Recombinant mouse CD40 ligand (rm-CD40L) was purchased from R&D Systems (Minneapolis, MN).

Techniques: Western Blot, Immunoprecipitation, Staining, Inhibition, Activity Assay, Solvent, Control

Journal: PLoS ONE

Article Title: Soluble CD40 Ligand Stimulates CD40-Dependent Activation of the β2 Integrin Mac-1 and Protein Kinase C Zeda (PKCζ) in Neutrophils: Implications for Neutrophil-Platelet Interactions and Neutrophil Oxidative Burst

doi: 10.1371/journal.pone.0064631

Figure Lengend Snippet: A & B: CD40, but not Mac-1, is required for sCD40L- stimulated neutrophil oxidative burst. A . Representative flow cytometry plots showing ROS generation in WT, CD40 −/− , and Mac-1 −/− neutrophils using CM-H2DCFDA as a fluorescent probe (dashed gray line: without the probe as a control). Neutrophils were stimulated without (green line) or with (red line) rm-CD40L (100 ng/ml) for 30 min. B . Quantitation of ROS generation in the indicated groups of neutrophils. Data obtained from five independent experiments are shown. NS = not significant. C . Effect of the indicated protein kinase inhibitors on sCD40L-stimulated neutrophil oxidative burst. WT neutrophils were stimulated without or with rm-CD40L (100 ng/ml) for 30 min in the presence of PKC ζ pseudosubstrate (PS, 5 μM), PI3-kinase (LY294002,10 µM), or NF-κB (BAY11-7082, 10 µM) inhibitors, or dimethyl sulfoxide (DMSO) as a solvent control. Data obtained from five independent experiments are shown. *P<0.01 versus bar 1, and #P<0.01 versus bar 2.

Article Snippet: Recombinant mouse CD40 ligand (rm-CD40L) was purchased from R&D Systems (Minneapolis, MN).

Techniques: Flow Cytometry, Control, Quantitation Assay, Solvent

Journal: PLoS ONE

Article Title: Soluble CD40 Ligand Stimulates CD40-Dependent Activation of the β2 Integrin Mac-1 and Protein Kinase C Zeda (PKCζ) in Neutrophils: Implications for Neutrophil-Platelet Interactions and Neutrophil Oxidative Burst

doi: 10.1371/journal.pone.0064631

Figure Lengend Snippet: A. Representative images of Immunofluorescence staining showing NF-kB p65 (green) nuclear translocation in WT, CD40 −/− , and Mac-1 −/− neutrophils stimulated without or with rm-CD40L (100 ng/ml, 60 min). Cell nuclei were detected by DAPI (blue). Scale bars: 20 μm. B. Quantitation of NF-kB p65 nuclear translocation in the indicated groups. Results are expressed as the percentage of the p65 nuclei-positively stained cells to the total cells. C. Effect of the indicated protein kinase inhibitors on sCD40L-stimulated NF-kB p65 nuclear translocation in neutrophils. WT neutrophils were stimulated without or with rm-CD40L (100 ng/ml) for 30 min in the presence of PKC ζ pseudosubstrate (PS, 5 μM), PI3-kinase (LY294002,10 µM), or NF-κB (BAY11-7082, 10 µM) inhibitors, or DMSO as a solvent control. Data obtained from five independent experiments are shown. *P<0.01 versus bar 1, and #P<0.01 versus bar 2.

Article Snippet: Recombinant mouse CD40 ligand (rm-CD40L) was purchased from R&D Systems (Minneapolis, MN).

Techniques: Immunofluorescence, Staining, Translocation Assay, Quantitation Assay, Solvent, Control

Journal: Journal of Virology

Article Title: Interplay between the Epigenetic Enzyme Lysine (K)-Specific Demethylase 2B and Epstein-Barr Virus Infection

doi: 10.1128/JVI.00273-19

Figure Lengend Snippet: EBV-dependent silencing of KDM2B expression in vitro. (A) Primary B cells from 3 independent donors were infected with EBV and collected to make dry pellets at the indicated time points. Some of the infected cells were left in culture for 4 weeks (4w) to generate LCL. Cell pellets were processed and analyzed for the expression level of KDM2B mRNA by RT-qPCR. The difference between the levels of KDM2B in primary B cells (time point 0) and in EBV-infected cells was significant (*, P < 0.05; **, P < 0.01). The levels of KDM2B were measured relative to its levels in LCL (which was used as the calibrator, for which the value was 1). (B) KDM2B protein expression levels in primary B cells mock infected (MI) or infected with EBV for 48 h were analyzed by Western blotting (left). The KDM2B protein signal was normalized to the levels of GAPDH. The histogram (right) shows the average from 3 independent experiments. (C to E) Primary B cells from 2 independent healthy donors were activated by treating them for 24 h with CD40L and IL-4, as described in Materials and Methods, and/or infected with EBV for 48 h. Cells were then processed and analyzed for the expression levels of EBNA1 (C), CCL22 (D), and KDM2B (E) mRNA by RT-qPCR. The results shown in the histogram are the average from 2 independent experiments (*, P < 0.05; ns, not significant). EBNA1 mRNA levels were measured relative to its levels in EBV-infected untreated cells. CCL22 and KDM2B mRNA levels were measured relative to their levels in mock-infected untreated cells. (F) Primary B cells were infected with 2 aliquots of the same EBV batch, one of which was UV inactivated before infection. At 48 h after infection, cells were collected and EBNA1 and KDM2B expression levels were assessed by RT-PCR and qPCR, respectively. The levels of KDM2B mRNA were measured relative to its levels in mock-infected cells. (G) Akata2000 and Akata31 cells were collected, processed for DNA/RNA extraction, and analyzed for the presence of the EBV genome by PCR (left) as well as for KDM2B expression levels by RT-qPCR (lower right) or RT-PCR (upper right). Viral DNA (left) indicates the relative amount of the BFRF3 gene present in Akata2000 versus Akata31 cells that was analyzed by real-time PCR and normalized to the amount of GAPDH in the extracted DNA. The levels of KDM2B mRNA were measured relative to its levels in mock-infected cells (*, P < 0.05). (H) Raji cells untreated or treated with TPA (50 ng/ml)-NaB (3 mM) for 48 h were analyzed for expression levels of BZLF1 and KDM2B mRNA by RT-qPCR (*, P < 0.05). (I) RPMI cells untreated or infected with EBV were collected, processed for RNA and protein extraction, and analyzed for the levels of the KDM2B protein (Western blotting, top) and mRNA (RT-qPCR, bottom). The levels of KDM2B mRNA were measured relative to its levels in mock-infected cells.

Article Snippet: To induce their activation, primary B cells were seeded at a density of 0.5 × 10 6 cells in 6-well dishes and treated with 100 ng/ml recombinant human CD40 ligand (hCD40L; catalog number 6245-CL; R&D Systems) and 20 ng/ml of recombinant human IL-4 (R&D Systems) for 48 h. Cells were then collected and processed for further analysis.

Techniques: Expressing, In Vitro, Infection, Quantitative RT-PCR, Western Blot, Reverse Transcription Polymerase Chain Reaction, RNA Extraction, Real-time Polymerase Chain Reaction, Protein Extraction