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Millipore lipoxin a4
Interaction between SMARCB1 and LANA-1 in KSHV-infected cells. (A) Western blot for SMARCB1 and LANA-1 in BCBL-1 cells treated with solvent (ethanol) or <t>LXA4</t> (100 nM for 48 h). β-Actin was used as the loading control. The fold change in expression was calculated by considering the level of protein in the solvent-treated sample as 1. (B) Proximity ligation assay (PLA) to visualize the interaction between SMARCB1 and LANA-1. (i) BCBL-1 cells treated with solvent or LXA4 (100 nM) for 48 h were processed as described in Materials and Methods and reacted with the indicated pairs of primary antibodies, followed by PLA to assess the interactions between SMARCB1 and LANA-1. Nuclei were stained with DAPI and viewed at a ×40 magnification. The PLA reaction was detected using the Duolink red detection agent. Red PLA spots in the nucleus indicate a positive PLA signal, suggesting interactions between the two proteins. (ii) Quantitative analysis of the average number of SMARCB1 plus LANA-1 interaction PLA spots per cell is presented in the histogram. *, P
Lipoxin A4, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "Concurrent Control of the Kaposi's Sarcoma-Associated Herpesvirus Life Cycle through Chromatin Modulation and Host Hedgehog Signaling: a New Prospect for the Therapeutic Potential of Lipoxin A4"

Article Title: Concurrent Control of the Kaposi's Sarcoma-Associated Herpesvirus Life Cycle through Chromatin Modulation and Host Hedgehog Signaling: a New Prospect for the Therapeutic Potential of Lipoxin A4

Journal: Journal of Virology

doi: 10.1128/JVI.02177-19

Interaction between SMARCB1 and LANA-1 in KSHV-infected cells. (A) Western blot for SMARCB1 and LANA-1 in BCBL-1 cells treated with solvent (ethanol) or LXA4 (100 nM for 48 h). β-Actin was used as the loading control. The fold change in expression was calculated by considering the level of protein in the solvent-treated sample as 1. (B) Proximity ligation assay (PLA) to visualize the interaction between SMARCB1 and LANA-1. (i) BCBL-1 cells treated with solvent or LXA4 (100 nM) for 48 h were processed as described in Materials and Methods and reacted with the indicated pairs of primary antibodies, followed by PLA to assess the interactions between SMARCB1 and LANA-1. Nuclei were stained with DAPI and viewed at a ×40 magnification. The PLA reaction was detected using the Duolink red detection agent. Red PLA spots in the nucleus indicate a positive PLA signal, suggesting interactions between the two proteins. (ii) Quantitative analysis of the average number of SMARCB1 plus LANA-1 interaction PLA spots per cell is presented in the histogram. *, P
Figure Legend Snippet: Interaction between SMARCB1 and LANA-1 in KSHV-infected cells. (A) Western blot for SMARCB1 and LANA-1 in BCBL-1 cells treated with solvent (ethanol) or LXA4 (100 nM for 48 h). β-Actin was used as the loading control. The fold change in expression was calculated by considering the level of protein in the solvent-treated sample as 1. (B) Proximity ligation assay (PLA) to visualize the interaction between SMARCB1 and LANA-1. (i) BCBL-1 cells treated with solvent or LXA4 (100 nM) for 48 h were processed as described in Materials and Methods and reacted with the indicated pairs of primary antibodies, followed by PLA to assess the interactions between SMARCB1 and LANA-1. Nuclei were stained with DAPI and viewed at a ×40 magnification. The PLA reaction was detected using the Duolink red detection agent. Red PLA spots in the nucleus indicate a positive PLA signal, suggesting interactions between the two proteins. (ii) Quantitative analysis of the average number of SMARCB1 plus LANA-1 interaction PLA spots per cell is presented in the histogram. *, P

Techniques Used: Infection, Western Blot, Expressing, Proximity Ligation Assay, Staining

SMARCB1 and KSHV genome interaction. (A) HMVEC-d cells were infected with KSHV, washed, and incubated with solvent or LXA4 for 48 h. The cells were fixed, permeabilized, and immunostained with an anti-rabbit SMARCB1 antibody followed by donkey anti-rabbit immunoglobulin conjugated to Alexa Fluor 594 secondary antibodies. The cells were then subjected to in situ hybridization with a spectrum green-labeled whole-KSHV-genome probe and viewed under a microscope at a ×40 magnification. (B) ChIP analysis of SMARCB1 in BCBL-1 and BC-3 cells treated with solvent or LXA4 for 48 h. SMARCB1 was immunoprecipitated from nuclei isolated from BCBL-1 and BC-3 cells that had been treated with solvent or LXA4 for 48 h, and bound DNA was analyzed by real-time RT-PCR with primers specific to regions (100 to 200 bp) flanking the transcription start sites of the indicated genes (ORF73 promoter and ORF50 promoter). Promoter occupancy relative to that obtained by IgG immunoprecipitation is presented for pORF50. The results are presented as the mean ± SD of data from three independent experiments. **, P
Figure Legend Snippet: SMARCB1 and KSHV genome interaction. (A) HMVEC-d cells were infected with KSHV, washed, and incubated with solvent or LXA4 for 48 h. The cells were fixed, permeabilized, and immunostained with an anti-rabbit SMARCB1 antibody followed by donkey anti-rabbit immunoglobulin conjugated to Alexa Fluor 594 secondary antibodies. The cells were then subjected to in situ hybridization with a spectrum green-labeled whole-KSHV-genome probe and viewed under a microscope at a ×40 magnification. (B) ChIP analysis of SMARCB1 in BCBL-1 and BC-3 cells treated with solvent or LXA4 for 48 h. SMARCB1 was immunoprecipitated from nuclei isolated from BCBL-1 and BC-3 cells that had been treated with solvent or LXA4 for 48 h, and bound DNA was analyzed by real-time RT-PCR with primers specific to regions (100 to 200 bp) flanking the transcription start sites of the indicated genes (ORF73 promoter and ORF50 promoter). Promoter occupancy relative to that obtained by IgG immunoprecipitation is presented for pORF50. The results are presented as the mean ± SD of data from three independent experiments. **, P

Techniques Used: Infection, Incubation, In Situ Hybridization, Labeling, Microscopy, Chromatin Immunoprecipitation, Immunoprecipitation, Isolation, Quantitative RT-PCR

Noncanonical hedgehog signaling in LXA4-treated BCBL-1 cells. (A) cDNA from LXA4-treated and untreated BCBL-1 cells was harvested, and gene expression for SMARCB1 (i) and Gli-1 (ii) was examined. Each bar represents the fold change in gene expression ± standard deviation (SD) from three independent experiments. These fold changes were calculated after normalization to the expression of the GAPDH gene. (B) (i) Schematic showing the steps of canonical hedgehog signaling. In step 1, the shh ligand protein binds to the Ptch receptor. In step 2, in the absence of the ligand, Ptch inhibits SMO, a downstream protein in the pathway. In step 3, the binding of shh relieves SMO inhibition, leading to activation of the GLI transcription factors: the activators GLI1 and GLI2 and the repressor GLI3. In step 4, activated GLI accumulates in the nucleus, and in step 5, activated GLI controls the transcription of hh target genes. (ii) SMARCB1 directly prevents the transcription of the glioma-associated oncogene homologue (GLI), thus resulting in reduced downstream hedgehog (hh) pathway target genes. (C) Lysates were extracted from LXA4-treated and solvent-treated BCBL-1 cells, and the blots were probed for Gli-1, Ptch-1, and shh. There was no change in the expression of the shh ligand (both the precursor and the 19-kDa active forms), but a significant decrease in the expression of Gli-1 and Ptch-1 was observed. (D) Downregulation of the LXA4-mediated Akt/mTOR pathway. Lysates obtained from latently infected and LXA4-treated BCBL-1 cells were immunoblotted with primary antibody for AKT, phosphorylated AKT (p-AKT), mTOR, phosphorylated mTOR (p-mTOR), S6 kinase, and phosphorylated S6 kinase (pS6kinase). A representative image of the blots with the fold change in the phosphorylation of AKT, mTOR, and S6 kinase is illustrated. (E) Phosphorylation of Gli-1 (p-Gil-1 Thr 1074) inactivates Gli-1, possibly in an AMPK-dependent manner, as represented by Western blotting. (F) iSLK and induced iSLK cells were mock treated or treated with AICAR and/or compound C (i) and 2-DG and/or compound C (ii) and analyzed by Western blotting for AMPK, phospho-AMPK, and phospho-Gli (Thr 1074). (G) Schematic showing the effect of LXA4 treatment on hedgehog signaling in an AMPK-mTOR-S6 kinase-dependent manner. KSHV infection leads to activation of AKT/mTOR signaling in B cells and endothelial cells, and this pathway is essential for both the lytic and the latent phases of the KSHV life cycle. LXA4 binds to the ALX receptor to reduce the levels of KSHV infection-induced AKT and the ERK pathway. LXA4 activates AMPK (Thr 172) to phosphorylate Gli-1 at Thr 1074, causing Gli-1 degradation, which affects cell proliferation and tumorigenesis. Dashed arrows show the pathway followed in KSHV-infected cells. Solid arrows show the pathway followed by LXA4.
Figure Legend Snippet: Noncanonical hedgehog signaling in LXA4-treated BCBL-1 cells. (A) cDNA from LXA4-treated and untreated BCBL-1 cells was harvested, and gene expression for SMARCB1 (i) and Gli-1 (ii) was examined. Each bar represents the fold change in gene expression ± standard deviation (SD) from three independent experiments. These fold changes were calculated after normalization to the expression of the GAPDH gene. (B) (i) Schematic showing the steps of canonical hedgehog signaling. In step 1, the shh ligand protein binds to the Ptch receptor. In step 2, in the absence of the ligand, Ptch inhibits SMO, a downstream protein in the pathway. In step 3, the binding of shh relieves SMO inhibition, leading to activation of the GLI transcription factors: the activators GLI1 and GLI2 and the repressor GLI3. In step 4, activated GLI accumulates in the nucleus, and in step 5, activated GLI controls the transcription of hh target genes. (ii) SMARCB1 directly prevents the transcription of the glioma-associated oncogene homologue (GLI), thus resulting in reduced downstream hedgehog (hh) pathway target genes. (C) Lysates were extracted from LXA4-treated and solvent-treated BCBL-1 cells, and the blots were probed for Gli-1, Ptch-1, and shh. There was no change in the expression of the shh ligand (both the precursor and the 19-kDa active forms), but a significant decrease in the expression of Gli-1 and Ptch-1 was observed. (D) Downregulation of the LXA4-mediated Akt/mTOR pathway. Lysates obtained from latently infected and LXA4-treated BCBL-1 cells were immunoblotted with primary antibody for AKT, phosphorylated AKT (p-AKT), mTOR, phosphorylated mTOR (p-mTOR), S6 kinase, and phosphorylated S6 kinase (pS6kinase). A representative image of the blots with the fold change in the phosphorylation of AKT, mTOR, and S6 kinase is illustrated. (E) Phosphorylation of Gli-1 (p-Gil-1 Thr 1074) inactivates Gli-1, possibly in an AMPK-dependent manner, as represented by Western blotting. (F) iSLK and induced iSLK cells were mock treated or treated with AICAR and/or compound C (i) and 2-DG and/or compound C (ii) and analyzed by Western blotting for AMPK, phospho-AMPK, and phospho-Gli (Thr 1074). (G) Schematic showing the effect of LXA4 treatment on hedgehog signaling in an AMPK-mTOR-S6 kinase-dependent manner. KSHV infection leads to activation of AKT/mTOR signaling in B cells and endothelial cells, and this pathway is essential for both the lytic and the latent phases of the KSHV life cycle. LXA4 binds to the ALX receptor to reduce the levels of KSHV infection-induced AKT and the ERK pathway. LXA4 activates AMPK (Thr 172) to phosphorylate Gli-1 at Thr 1074, causing Gli-1 degradation, which affects cell proliferation and tumorigenesis. Dashed arrows show the pathway followed in KSHV-infected cells. Solid arrows show the pathway followed by LXA4.

Techniques Used: Expressing, Standard Deviation, Binding Assay, Inhibition, Activation Assay, Infection, Western Blot

LXA4, a potential therapeutic agent against KSHV. (A) The overall anti-inflammatory action of LXA4 in vivo ) has shown that the ALX/FPR receptor is vital for LXA4 signaling and that blocking this receptor elevates the levels of NF-κB, ERK, and COX-2. LXA4 downregulates the AKT pathway in KS-IMM cells and KSHV-infected primary endothelial cells. In the present study, we postulate that LXA4 may be an endogenous ligand that facilitates the translocation of AhR into the nucleus, where it heterodimerizes with the AhR nuclear translocator (ARNT). AhR-ARNT induces the transcription of target genes by the recruitment of various components of the transcriptional machinery, such as ATP-dependent chromatin-remodeling components, such as Brg-1 (the mechanism has been explored in other systems but has yet to be proved in KSHV-infected cells). The proposed mechanism of action of LXA4 is based on our two current findings: (i) the interaction of LXA4 with nucleosome complex components possibly mediated by AhR and (ii) the overall decreased expression of Gli-1 in LXA4-treated PEL cells compared to that in untreated PEL cells. LXA4 inhibits Gli-1 expression noncanonically at the cytoplasmic level through AMPK activation (indicated by “1 Cytoplasm”). The AKT pathway is regulated by AMPK and, in turn, regulates the mTOR/S6 kinase 1 pathway. The phosphorylation of Gli-1 at the Thr 1074 site in LXA4-treated PEL cells suggests the direct inhibition of hedgehog signaling mediated through AMPK. LXA4 treatment decreased PD-L1 expression, which is related to a decrease in AKT activation and the associated inflammation in LXA4-treated BCBL-1 cells. LXA4 also inhibits Gli-1 expression noncanonically at the nuclear level (indicated by “2 Nucleus”). LXA4 treatment of KSHV-infected BCBL-1 cell inhibited HDAC expression and increased SMARCB1 expression. SMARCB1 directly interacts with Gli-1 to repress its transcriptional activity. SMARCB1, importantly, regulates RTA epigenetically to initiate and carry viral lytic gene replication and viral progression.
Figure Legend Snippet: LXA4, a potential therapeutic agent against KSHV. (A) The overall anti-inflammatory action of LXA4 in vivo ) has shown that the ALX/FPR receptor is vital for LXA4 signaling and that blocking this receptor elevates the levels of NF-κB, ERK, and COX-2. LXA4 downregulates the AKT pathway in KS-IMM cells and KSHV-infected primary endothelial cells. In the present study, we postulate that LXA4 may be an endogenous ligand that facilitates the translocation of AhR into the nucleus, where it heterodimerizes with the AhR nuclear translocator (ARNT). AhR-ARNT induces the transcription of target genes by the recruitment of various components of the transcriptional machinery, such as ATP-dependent chromatin-remodeling components, such as Brg-1 (the mechanism has been explored in other systems but has yet to be proved in KSHV-infected cells). The proposed mechanism of action of LXA4 is based on our two current findings: (i) the interaction of LXA4 with nucleosome complex components possibly mediated by AhR and (ii) the overall decreased expression of Gli-1 in LXA4-treated PEL cells compared to that in untreated PEL cells. LXA4 inhibits Gli-1 expression noncanonically at the cytoplasmic level through AMPK activation (indicated by “1 Cytoplasm”). The AKT pathway is regulated by AMPK and, in turn, regulates the mTOR/S6 kinase 1 pathway. The phosphorylation of Gli-1 at the Thr 1074 site in LXA4-treated PEL cells suggests the direct inhibition of hedgehog signaling mediated through AMPK. LXA4 treatment decreased PD-L1 expression, which is related to a decrease in AKT activation and the associated inflammation in LXA4-treated BCBL-1 cells. LXA4 also inhibits Gli-1 expression noncanonically at the nuclear level (indicated by “2 Nucleus”). LXA4 treatment of KSHV-infected BCBL-1 cell inhibited HDAC expression and increased SMARCB1 expression. SMARCB1 directly interacts with Gli-1 to repress its transcriptional activity. SMARCB1, importantly, regulates RTA epigenetically to initiate and carry viral lytic gene replication and viral progression.

Techniques Used: In Vivo, Blocking Assay, Infection, Translocation Assay, Expressing, Activation Assay, Inhibition, Activity Assay

SMARCB1 interacts with Gli-1 in KSHV-infected cells (A) (Left) Immunoprecipitation (IP) assay. BCBL-1 cells treated with solvent or LXA4 (48 h) and BJAB cells (KSHV and EBV negative) were harvested, and the lysates were precipitated with SMARCB1 and analyzed by immunoblotting with Gli-1 and SMARCB1. (Right) The input for the left panel. Representative data from three experiments are shown here. (B) (Left) Immunoprecipitation assay using SLK, iSLK, and induced iSLK cells. The cells were harvested, and the lysates were precipitated with Gli-1 and analyzed by immunoblotting with Gli-1 and SMARCB1. (Right) The input for the left panel. Representative data from three experiments are shown here. (C) Proximity ligation assay (PLA) to detect the interaction between SMARCB1 and Gli-1. BCBL-1 cells treated with solvent or LXA4 were washed, fixed, permeabilized, and reacted with SMARCB1 and Gli-1 primary antibodies, followed by PLA to assess the interactions between SMARCB1 and Gli-1. Nuclei were counterstained with DAPI. The differential interference contras t (DIC) image shown in the merged panels corresponds to the fluorescence image. The PLA reaction was detected using the Duolink red detection agent. Red puncta in the nucleus indicate a positive PLA signal, suggesting interactions between the two proteins. Images were captured at a ×40 magnification.
Figure Legend Snippet: SMARCB1 interacts with Gli-1 in KSHV-infected cells (A) (Left) Immunoprecipitation (IP) assay. BCBL-1 cells treated with solvent or LXA4 (48 h) and BJAB cells (KSHV and EBV negative) were harvested, and the lysates were precipitated with SMARCB1 and analyzed by immunoblotting with Gli-1 and SMARCB1. (Right) The input for the left panel. Representative data from three experiments are shown here. (B) (Left) Immunoprecipitation assay using SLK, iSLK, and induced iSLK cells. The cells were harvested, and the lysates were precipitated with Gli-1 and analyzed by immunoblotting with Gli-1 and SMARCB1. (Right) The input for the left panel. Representative data from three experiments are shown here. (C) Proximity ligation assay (PLA) to detect the interaction between SMARCB1 and Gli-1. BCBL-1 cells treated with solvent or LXA4 were washed, fixed, permeabilized, and reacted with SMARCB1 and Gli-1 primary antibodies, followed by PLA to assess the interactions between SMARCB1 and Gli-1. Nuclei were counterstained with DAPI. The differential interference contras t (DIC) image shown in the merged panels corresponds to the fluorescence image. The PLA reaction was detected using the Duolink red detection agent. Red puncta in the nucleus indicate a positive PLA signal, suggesting interactions between the two proteins. Images were captured at a ×40 magnification.

Techniques Used: Infection, Immunoprecipitation, Proximity Ligation Assay, Fluorescence

LXA4 treatment regulates the nuclear translocation of Gli-1 and the SMARCB1–Gli-1 interaction in the nuclei of KSHV-infected cells. (A) The nuclear (Nuc) and cytoplasmic (Cyto) fractions were isolated from BCBL-1 cells and LXA4-treated BCBL-1 using a nuclear isolation kit and immunoblotted with anti-SMARCB1 and anti-Gli-1 antibodies. These membranes were stripped and immunoblotted with anti-GAPDH and anti-TATA-binding protein (anti-TBP) antibodies to check the purity of the cytoplasmic and nuclear lysates, respectively, and to confirm equal loading. (B) Shuttling of Gli-1. Immunoblotting showed the total nuclear translocation of the Gli-1 protein in induced iSLK cells compared with that in iSLK cells. (C) Gli-1 expression in lysates obtained from the nuclear and cytoplasmic fractions of SLK cells, latently infected SLK cells (iSLK), and induced iSLK cells was estimated using an ELISA kit. The experiments were repeated three times, and the results are expressed as the mean ± SD. **, P
Figure Legend Snippet: LXA4 treatment regulates the nuclear translocation of Gli-1 and the SMARCB1–Gli-1 interaction in the nuclei of KSHV-infected cells. (A) The nuclear (Nuc) and cytoplasmic (Cyto) fractions were isolated from BCBL-1 cells and LXA4-treated BCBL-1 using a nuclear isolation kit and immunoblotted with anti-SMARCB1 and anti-Gli-1 antibodies. These membranes were stripped and immunoblotted with anti-GAPDH and anti-TATA-binding protein (anti-TBP) antibodies to check the purity of the cytoplasmic and nuclear lysates, respectively, and to confirm equal loading. (B) Shuttling of Gli-1. Immunoblotting showed the total nuclear translocation of the Gli-1 protein in induced iSLK cells compared with that in iSLK cells. (C) Gli-1 expression in lysates obtained from the nuclear and cytoplasmic fractions of SLK cells, latently infected SLK cells (iSLK), and induced iSLK cells was estimated using an ELISA kit. The experiments were repeated three times, and the results are expressed as the mean ± SD. **, P

Techniques Used: Translocation Assay, Infection, Isolation, Binding Assay, Expressing, Enzyme-linked Immunosorbent Assay

(A and B) Lysates from HMVEC-d cells infected with KSHV (30 DNA copies/cell) for the indicated times (0, 8, and 24 h) (A) and BCBL-1 cells (B) were Western blotted for PD-L1, stripped, and immunoblotted for tubulin and GAPDH, respectively. Representative data from three experiments are shown here, with the fold change being indicated. (C) In vitro PEL cell models. BCBL-1 cells treated with LXA4 were fixed and stained for PD-L1 using an Alexa Fluor 594-labeled antibody. (D) (i) BCBL-1 cells and LXA4-treated BCBL-1 cells were fixed, probed for surface PD-L1, stained with Alexa Fluor 488-labeled antibody, and analyzed by FACS using a Becton, Dickinson LSR II flow cytometer and FlowJo software. (ii) The hatched black peak represents unstained cells, the black peak represents the IgG control, and the solid green peak and hatched green peak represented stained solvent-treated and LXA4-treated BCBL-1 cells, respectively. ***, P
Figure Legend Snippet: (A and B) Lysates from HMVEC-d cells infected with KSHV (30 DNA copies/cell) for the indicated times (0, 8, and 24 h) (A) and BCBL-1 cells (B) were Western blotted for PD-L1, stripped, and immunoblotted for tubulin and GAPDH, respectively. Representative data from three experiments are shown here, with the fold change being indicated. (C) In vitro PEL cell models. BCBL-1 cells treated with LXA4 were fixed and stained for PD-L1 using an Alexa Fluor 594-labeled antibody. (D) (i) BCBL-1 cells and LXA4-treated BCBL-1 cells were fixed, probed for surface PD-L1, stained with Alexa Fluor 488-labeled antibody, and analyzed by FACS using a Becton, Dickinson LSR II flow cytometer and FlowJo software. (ii) The hatched black peak represents unstained cells, the black peak represents the IgG control, and the solid green peak and hatched green peak represented stained solvent-treated and LXA4-treated BCBL-1 cells, respectively. ***, P

Techniques Used: Infection, Western Blot, In Vitro, Staining, Labeling, FACS, Flow Cytometry, Software

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    Millipore lipoxin a4
    Interaction between SMARCB1 and LANA-1 in KSHV-infected cells. (A) Western blot for SMARCB1 and LANA-1 in BCBL-1 cells treated with solvent (ethanol) or <t>LXA4</t> (100 nM for 48 h). β-Actin was used as the loading control. The fold change in expression was calculated by considering the level of protein in the solvent-treated sample as 1. (B) Proximity ligation assay (PLA) to visualize the interaction between SMARCB1 and LANA-1. (i) BCBL-1 cells treated with solvent or LXA4 (100 nM) for 48 h were processed as described in Materials and Methods and reacted with the indicated pairs of primary antibodies, followed by PLA to assess the interactions between SMARCB1 and LANA-1. Nuclei were stained with DAPI and viewed at a ×40 magnification. The PLA reaction was detected using the Duolink red detection agent. Red PLA spots in the nucleus indicate a positive PLA signal, suggesting interactions between the two proteins. (ii) Quantitative analysis of the average number of SMARCB1 plus LANA-1 interaction PLA spots per cell is presented in the histogram. *, P
    Lipoxin A4, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    KSHV lytic infection inhibits PB formation by expression of viral ORF57. ( A ) GW182 and DCP1A are co-stained in the PB in <t>BCBL-1</t> cells with no KSHV reactivation. BCBL-1 cells with latent KSHV infection were co-stained for GW182 (red) and DCP1A (green) using the corresponding antibodies and imaged by confocal microscopy. The nuclei were counterstained with Hoechst DNA stain. Arrows indicate the PB positively co-stained for both GW182 and DCP1A; bar = 5 μm. ( B and C ) Detection of PB during KSHV latent and lytic infection. (B) KSHV-infected BCBL-1 cells were induced with 1 mM valproic acid (VA, 1 mM) for lytic infection. Twenty-four hours after induction, BCBL-1 cells with (+VA) or without (-VA) virus lytic reactivation were co-immunostained for PB formation using a specific PB marker GW182 and for viral lytic protein ORF57 expression. The nuclei were counterstained with Hoechst dye. Images were captured by confocal microscopy; bar = 10 μm. (C) Number of PB in BCBL-1 cells during latent and lytic KSHV infection in the presence or absence of viral ORF57. Total of 50 cells in each group were counted in each experiment. The error bars represent SD from three independent experiments; ** P
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    KSHV lytic infection inhibits PB formation by expression of viral ORF57. ( A ) GW182 and DCP1A are co-stained in the PB in <t>BCBL-1</t> cells with no KSHV reactivation. BCBL-1 cells with latent KSHV infection were co-stained for GW182 (red) and DCP1A (green) using the corresponding antibodies and imaged by confocal microscopy. The nuclei were counterstained with Hoechst DNA stain. Arrows indicate the PB positively co-stained for both GW182 and DCP1A; bar = 5 μm. ( B and C ) Detection of PB during KSHV latent and lytic infection. (B) KSHV-infected BCBL-1 cells were induced with 1 mM valproic acid (VA, 1 mM) for lytic infection. Twenty-four hours after induction, BCBL-1 cells with (+VA) or without (-VA) virus lytic reactivation were co-immunostained for PB formation using a specific PB marker GW182 and for viral lytic protein ORF57 expression. The nuclei were counterstained with Hoechst dye. Images were captured by confocal microscopy; bar = 10 μm. (C) Number of PB in BCBL-1 cells during latent and lytic KSHV infection in the presence or absence of viral ORF57. Total of 50 cells in each group were counted in each experiment. The error bars represent SD from three independent experiments; ** P
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    Interaction between SMARCB1 and LANA-1 in KSHV-infected cells. (A) Western blot for SMARCB1 and LANA-1 in BCBL-1 cells treated with solvent (ethanol) or LXA4 (100 nM for 48 h). β-Actin was used as the loading control. The fold change in expression was calculated by considering the level of protein in the solvent-treated sample as 1. (B) Proximity ligation assay (PLA) to visualize the interaction between SMARCB1 and LANA-1. (i) BCBL-1 cells treated with solvent or LXA4 (100 nM) for 48 h were processed as described in Materials and Methods and reacted with the indicated pairs of primary antibodies, followed by PLA to assess the interactions between SMARCB1 and LANA-1. Nuclei were stained with DAPI and viewed at a ×40 magnification. The PLA reaction was detected using the Duolink red detection agent. Red PLA spots in the nucleus indicate a positive PLA signal, suggesting interactions between the two proteins. (ii) Quantitative analysis of the average number of SMARCB1 plus LANA-1 interaction PLA spots per cell is presented in the histogram. *, P

    Journal: Journal of Virology

    Article Title: Concurrent Control of the Kaposi's Sarcoma-Associated Herpesvirus Life Cycle through Chromatin Modulation and Host Hedgehog Signaling: a New Prospect for the Therapeutic Potential of Lipoxin A4

    doi: 10.1128/JVI.02177-19

    Figure Lengend Snippet: Interaction between SMARCB1 and LANA-1 in KSHV-infected cells. (A) Western blot for SMARCB1 and LANA-1 in BCBL-1 cells treated with solvent (ethanol) or LXA4 (100 nM for 48 h). β-Actin was used as the loading control. The fold change in expression was calculated by considering the level of protein in the solvent-treated sample as 1. (B) Proximity ligation assay (PLA) to visualize the interaction between SMARCB1 and LANA-1. (i) BCBL-1 cells treated with solvent or LXA4 (100 nM) for 48 h were processed as described in Materials and Methods and reacted with the indicated pairs of primary antibodies, followed by PLA to assess the interactions between SMARCB1 and LANA-1. Nuclei were stained with DAPI and viewed at a ×40 magnification. The PLA reaction was detected using the Duolink red detection agent. Red PLA spots in the nucleus indicate a positive PLA signal, suggesting interactions between the two proteins. (ii) Quantitative analysis of the average number of SMARCB1 plus LANA-1 interaction PLA spots per cell is presented in the histogram. *, P

    Article Snippet: BCBL-1 cells (treated with solvent or LXA4) were cultured, fixed, permeabilized with prechilled acetone, and blocked with Duolink blocking buffer for 30 min at 37°C.

    Techniques: Infection, Western Blot, Expressing, Proximity Ligation Assay, Staining

    SMARCB1 and KSHV genome interaction. (A) HMVEC-d cells were infected with KSHV, washed, and incubated with solvent or LXA4 for 48 h. The cells were fixed, permeabilized, and immunostained with an anti-rabbit SMARCB1 antibody followed by donkey anti-rabbit immunoglobulin conjugated to Alexa Fluor 594 secondary antibodies. The cells were then subjected to in situ hybridization with a spectrum green-labeled whole-KSHV-genome probe and viewed under a microscope at a ×40 magnification. (B) ChIP analysis of SMARCB1 in BCBL-1 and BC-3 cells treated with solvent or LXA4 for 48 h. SMARCB1 was immunoprecipitated from nuclei isolated from BCBL-1 and BC-3 cells that had been treated with solvent or LXA4 for 48 h, and bound DNA was analyzed by real-time RT-PCR with primers specific to regions (100 to 200 bp) flanking the transcription start sites of the indicated genes (ORF73 promoter and ORF50 promoter). Promoter occupancy relative to that obtained by IgG immunoprecipitation is presented for pORF50. The results are presented as the mean ± SD of data from three independent experiments. **, P

    Journal: Journal of Virology

    Article Title: Concurrent Control of the Kaposi's Sarcoma-Associated Herpesvirus Life Cycle through Chromatin Modulation and Host Hedgehog Signaling: a New Prospect for the Therapeutic Potential of Lipoxin A4

    doi: 10.1128/JVI.02177-19

    Figure Lengend Snippet: SMARCB1 and KSHV genome interaction. (A) HMVEC-d cells were infected with KSHV, washed, and incubated with solvent or LXA4 for 48 h. The cells were fixed, permeabilized, and immunostained with an anti-rabbit SMARCB1 antibody followed by donkey anti-rabbit immunoglobulin conjugated to Alexa Fluor 594 secondary antibodies. The cells were then subjected to in situ hybridization with a spectrum green-labeled whole-KSHV-genome probe and viewed under a microscope at a ×40 magnification. (B) ChIP analysis of SMARCB1 in BCBL-1 and BC-3 cells treated with solvent or LXA4 for 48 h. SMARCB1 was immunoprecipitated from nuclei isolated from BCBL-1 and BC-3 cells that had been treated with solvent or LXA4 for 48 h, and bound DNA was analyzed by real-time RT-PCR with primers specific to regions (100 to 200 bp) flanking the transcription start sites of the indicated genes (ORF73 promoter and ORF50 promoter). Promoter occupancy relative to that obtained by IgG immunoprecipitation is presented for pORF50. The results are presented as the mean ± SD of data from three independent experiments. **, P

    Article Snippet: BCBL-1 cells (treated with solvent or LXA4) were cultured, fixed, permeabilized with prechilled acetone, and blocked with Duolink blocking buffer for 30 min at 37°C.

    Techniques: Infection, Incubation, In Situ Hybridization, Labeling, Microscopy, Chromatin Immunoprecipitation, Immunoprecipitation, Isolation, Quantitative RT-PCR

    Noncanonical hedgehog signaling in LXA4-treated BCBL-1 cells. (A) cDNA from LXA4-treated and untreated BCBL-1 cells was harvested, and gene expression for SMARCB1 (i) and Gli-1 (ii) was examined. Each bar represents the fold change in gene expression ± standard deviation (SD) from three independent experiments. These fold changes were calculated after normalization to the expression of the GAPDH gene. (B) (i) Schematic showing the steps of canonical hedgehog signaling. In step 1, the shh ligand protein binds to the Ptch receptor. In step 2, in the absence of the ligand, Ptch inhibits SMO, a downstream protein in the pathway. In step 3, the binding of shh relieves SMO inhibition, leading to activation of the GLI transcription factors: the activators GLI1 and GLI2 and the repressor GLI3. In step 4, activated GLI accumulates in the nucleus, and in step 5, activated GLI controls the transcription of hh target genes. (ii) SMARCB1 directly prevents the transcription of the glioma-associated oncogene homologue (GLI), thus resulting in reduced downstream hedgehog (hh) pathway target genes. (C) Lysates were extracted from LXA4-treated and solvent-treated BCBL-1 cells, and the blots were probed for Gli-1, Ptch-1, and shh. There was no change in the expression of the shh ligand (both the precursor and the 19-kDa active forms), but a significant decrease in the expression of Gli-1 and Ptch-1 was observed. (D) Downregulation of the LXA4-mediated Akt/mTOR pathway. Lysates obtained from latently infected and LXA4-treated BCBL-1 cells were immunoblotted with primary antibody for AKT, phosphorylated AKT (p-AKT), mTOR, phosphorylated mTOR (p-mTOR), S6 kinase, and phosphorylated S6 kinase (pS6kinase). A representative image of the blots with the fold change in the phosphorylation of AKT, mTOR, and S6 kinase is illustrated. (E) Phosphorylation of Gli-1 (p-Gil-1 Thr 1074) inactivates Gli-1, possibly in an AMPK-dependent manner, as represented by Western blotting. (F) iSLK and induced iSLK cells were mock treated or treated with AICAR and/or compound C (i) and 2-DG and/or compound C (ii) and analyzed by Western blotting for AMPK, phospho-AMPK, and phospho-Gli (Thr 1074). (G) Schematic showing the effect of LXA4 treatment on hedgehog signaling in an AMPK-mTOR-S6 kinase-dependent manner. KSHV infection leads to activation of AKT/mTOR signaling in B cells and endothelial cells, and this pathway is essential for both the lytic and the latent phases of the KSHV life cycle. LXA4 binds to the ALX receptor to reduce the levels of KSHV infection-induced AKT and the ERK pathway. LXA4 activates AMPK (Thr 172) to phosphorylate Gli-1 at Thr 1074, causing Gli-1 degradation, which affects cell proliferation and tumorigenesis. Dashed arrows show the pathway followed in KSHV-infected cells. Solid arrows show the pathway followed by LXA4.

    Journal: Journal of Virology

    Article Title: Concurrent Control of the Kaposi's Sarcoma-Associated Herpesvirus Life Cycle through Chromatin Modulation and Host Hedgehog Signaling: a New Prospect for the Therapeutic Potential of Lipoxin A4

    doi: 10.1128/JVI.02177-19

    Figure Lengend Snippet: Noncanonical hedgehog signaling in LXA4-treated BCBL-1 cells. (A) cDNA from LXA4-treated and untreated BCBL-1 cells was harvested, and gene expression for SMARCB1 (i) and Gli-1 (ii) was examined. Each bar represents the fold change in gene expression ± standard deviation (SD) from three independent experiments. These fold changes were calculated after normalization to the expression of the GAPDH gene. (B) (i) Schematic showing the steps of canonical hedgehog signaling. In step 1, the shh ligand protein binds to the Ptch receptor. In step 2, in the absence of the ligand, Ptch inhibits SMO, a downstream protein in the pathway. In step 3, the binding of shh relieves SMO inhibition, leading to activation of the GLI transcription factors: the activators GLI1 and GLI2 and the repressor GLI3. In step 4, activated GLI accumulates in the nucleus, and in step 5, activated GLI controls the transcription of hh target genes. (ii) SMARCB1 directly prevents the transcription of the glioma-associated oncogene homologue (GLI), thus resulting in reduced downstream hedgehog (hh) pathway target genes. (C) Lysates were extracted from LXA4-treated and solvent-treated BCBL-1 cells, and the blots were probed for Gli-1, Ptch-1, and shh. There was no change in the expression of the shh ligand (both the precursor and the 19-kDa active forms), but a significant decrease in the expression of Gli-1 and Ptch-1 was observed. (D) Downregulation of the LXA4-mediated Akt/mTOR pathway. Lysates obtained from latently infected and LXA4-treated BCBL-1 cells were immunoblotted with primary antibody for AKT, phosphorylated AKT (p-AKT), mTOR, phosphorylated mTOR (p-mTOR), S6 kinase, and phosphorylated S6 kinase (pS6kinase). A representative image of the blots with the fold change in the phosphorylation of AKT, mTOR, and S6 kinase is illustrated. (E) Phosphorylation of Gli-1 (p-Gil-1 Thr 1074) inactivates Gli-1, possibly in an AMPK-dependent manner, as represented by Western blotting. (F) iSLK and induced iSLK cells were mock treated or treated with AICAR and/or compound C (i) and 2-DG and/or compound C (ii) and analyzed by Western blotting for AMPK, phospho-AMPK, and phospho-Gli (Thr 1074). (G) Schematic showing the effect of LXA4 treatment on hedgehog signaling in an AMPK-mTOR-S6 kinase-dependent manner. KSHV infection leads to activation of AKT/mTOR signaling in B cells and endothelial cells, and this pathway is essential for both the lytic and the latent phases of the KSHV life cycle. LXA4 binds to the ALX receptor to reduce the levels of KSHV infection-induced AKT and the ERK pathway. LXA4 activates AMPK (Thr 172) to phosphorylate Gli-1 at Thr 1074, causing Gli-1 degradation, which affects cell proliferation and tumorigenesis. Dashed arrows show the pathway followed in KSHV-infected cells. Solid arrows show the pathway followed by LXA4.

    Article Snippet: BCBL-1 cells (treated with solvent or LXA4) were cultured, fixed, permeabilized with prechilled acetone, and blocked with Duolink blocking buffer for 30 min at 37°C.

    Techniques: Expressing, Standard Deviation, Binding Assay, Inhibition, Activation Assay, Infection, Western Blot

    LXA4, a potential therapeutic agent against KSHV. (A) The overall anti-inflammatory action of LXA4 in vivo ) has shown that the ALX/FPR receptor is vital for LXA4 signaling and that blocking this receptor elevates the levels of NF-κB, ERK, and COX-2. LXA4 downregulates the AKT pathway in KS-IMM cells and KSHV-infected primary endothelial cells. In the present study, we postulate that LXA4 may be an endogenous ligand that facilitates the translocation of AhR into the nucleus, where it heterodimerizes with the AhR nuclear translocator (ARNT). AhR-ARNT induces the transcription of target genes by the recruitment of various components of the transcriptional machinery, such as ATP-dependent chromatin-remodeling components, such as Brg-1 (the mechanism has been explored in other systems but has yet to be proved in KSHV-infected cells). The proposed mechanism of action of LXA4 is based on our two current findings: (i) the interaction of LXA4 with nucleosome complex components possibly mediated by AhR and (ii) the overall decreased expression of Gli-1 in LXA4-treated PEL cells compared to that in untreated PEL cells. LXA4 inhibits Gli-1 expression noncanonically at the cytoplasmic level through AMPK activation (indicated by “1 Cytoplasm”). The AKT pathway is regulated by AMPK and, in turn, regulates the mTOR/S6 kinase 1 pathway. The phosphorylation of Gli-1 at the Thr 1074 site in LXA4-treated PEL cells suggests the direct inhibition of hedgehog signaling mediated through AMPK. LXA4 treatment decreased PD-L1 expression, which is related to a decrease in AKT activation and the associated inflammation in LXA4-treated BCBL-1 cells. LXA4 also inhibits Gli-1 expression noncanonically at the nuclear level (indicated by “2 Nucleus”). LXA4 treatment of KSHV-infected BCBL-1 cell inhibited HDAC expression and increased SMARCB1 expression. SMARCB1 directly interacts with Gli-1 to repress its transcriptional activity. SMARCB1, importantly, regulates RTA epigenetically to initiate and carry viral lytic gene replication and viral progression.

    Journal: Journal of Virology

    Article Title: Concurrent Control of the Kaposi's Sarcoma-Associated Herpesvirus Life Cycle through Chromatin Modulation and Host Hedgehog Signaling: a New Prospect for the Therapeutic Potential of Lipoxin A4

    doi: 10.1128/JVI.02177-19

    Figure Lengend Snippet: LXA4, a potential therapeutic agent against KSHV. (A) The overall anti-inflammatory action of LXA4 in vivo ) has shown that the ALX/FPR receptor is vital for LXA4 signaling and that blocking this receptor elevates the levels of NF-κB, ERK, and COX-2. LXA4 downregulates the AKT pathway in KS-IMM cells and KSHV-infected primary endothelial cells. In the present study, we postulate that LXA4 may be an endogenous ligand that facilitates the translocation of AhR into the nucleus, where it heterodimerizes with the AhR nuclear translocator (ARNT). AhR-ARNT induces the transcription of target genes by the recruitment of various components of the transcriptional machinery, such as ATP-dependent chromatin-remodeling components, such as Brg-1 (the mechanism has been explored in other systems but has yet to be proved in KSHV-infected cells). The proposed mechanism of action of LXA4 is based on our two current findings: (i) the interaction of LXA4 with nucleosome complex components possibly mediated by AhR and (ii) the overall decreased expression of Gli-1 in LXA4-treated PEL cells compared to that in untreated PEL cells. LXA4 inhibits Gli-1 expression noncanonically at the cytoplasmic level through AMPK activation (indicated by “1 Cytoplasm”). The AKT pathway is regulated by AMPK and, in turn, regulates the mTOR/S6 kinase 1 pathway. The phosphorylation of Gli-1 at the Thr 1074 site in LXA4-treated PEL cells suggests the direct inhibition of hedgehog signaling mediated through AMPK. LXA4 treatment decreased PD-L1 expression, which is related to a decrease in AKT activation and the associated inflammation in LXA4-treated BCBL-1 cells. LXA4 also inhibits Gli-1 expression noncanonically at the nuclear level (indicated by “2 Nucleus”). LXA4 treatment of KSHV-infected BCBL-1 cell inhibited HDAC expression and increased SMARCB1 expression. SMARCB1 directly interacts with Gli-1 to repress its transcriptional activity. SMARCB1, importantly, regulates RTA epigenetically to initiate and carry viral lytic gene replication and viral progression.

    Article Snippet: BCBL-1 cells (treated with solvent or LXA4) were cultured, fixed, permeabilized with prechilled acetone, and blocked with Duolink blocking buffer for 30 min at 37°C.

    Techniques: In Vivo, Blocking Assay, Infection, Translocation Assay, Expressing, Activation Assay, Inhibition, Activity Assay

    SMARCB1 interacts with Gli-1 in KSHV-infected cells (A) (Left) Immunoprecipitation (IP) assay. BCBL-1 cells treated with solvent or LXA4 (48 h) and BJAB cells (KSHV and EBV negative) were harvested, and the lysates were precipitated with SMARCB1 and analyzed by immunoblotting with Gli-1 and SMARCB1. (Right) The input for the left panel. Representative data from three experiments are shown here. (B) (Left) Immunoprecipitation assay using SLK, iSLK, and induced iSLK cells. The cells were harvested, and the lysates were precipitated with Gli-1 and analyzed by immunoblotting with Gli-1 and SMARCB1. (Right) The input for the left panel. Representative data from three experiments are shown here. (C) Proximity ligation assay (PLA) to detect the interaction between SMARCB1 and Gli-1. BCBL-1 cells treated with solvent or LXA4 were washed, fixed, permeabilized, and reacted with SMARCB1 and Gli-1 primary antibodies, followed by PLA to assess the interactions between SMARCB1 and Gli-1. Nuclei were counterstained with DAPI. The differential interference contras t (DIC) image shown in the merged panels corresponds to the fluorescence image. The PLA reaction was detected using the Duolink red detection agent. Red puncta in the nucleus indicate a positive PLA signal, suggesting interactions between the two proteins. Images were captured at a ×40 magnification.

    Journal: Journal of Virology

    Article Title: Concurrent Control of the Kaposi's Sarcoma-Associated Herpesvirus Life Cycle through Chromatin Modulation and Host Hedgehog Signaling: a New Prospect for the Therapeutic Potential of Lipoxin A4

    doi: 10.1128/JVI.02177-19

    Figure Lengend Snippet: SMARCB1 interacts with Gli-1 in KSHV-infected cells (A) (Left) Immunoprecipitation (IP) assay. BCBL-1 cells treated with solvent or LXA4 (48 h) and BJAB cells (KSHV and EBV negative) were harvested, and the lysates were precipitated with SMARCB1 and analyzed by immunoblotting with Gli-1 and SMARCB1. (Right) The input for the left panel. Representative data from three experiments are shown here. (B) (Left) Immunoprecipitation assay using SLK, iSLK, and induced iSLK cells. The cells were harvested, and the lysates were precipitated with Gli-1 and analyzed by immunoblotting with Gli-1 and SMARCB1. (Right) The input for the left panel. Representative data from three experiments are shown here. (C) Proximity ligation assay (PLA) to detect the interaction between SMARCB1 and Gli-1. BCBL-1 cells treated with solvent or LXA4 were washed, fixed, permeabilized, and reacted with SMARCB1 and Gli-1 primary antibodies, followed by PLA to assess the interactions between SMARCB1 and Gli-1. Nuclei were counterstained with DAPI. The differential interference contras t (DIC) image shown in the merged panels corresponds to the fluorescence image. The PLA reaction was detected using the Duolink red detection agent. Red puncta in the nucleus indicate a positive PLA signal, suggesting interactions between the two proteins. Images were captured at a ×40 magnification.

    Article Snippet: BCBL-1 cells (treated with solvent or LXA4) were cultured, fixed, permeabilized with prechilled acetone, and blocked with Duolink blocking buffer for 30 min at 37°C.

    Techniques: Infection, Immunoprecipitation, Proximity Ligation Assay, Fluorescence

    LXA4 treatment regulates the nuclear translocation of Gli-1 and the SMARCB1–Gli-1 interaction in the nuclei of KSHV-infected cells. (A) The nuclear (Nuc) and cytoplasmic (Cyto) fractions were isolated from BCBL-1 cells and LXA4-treated BCBL-1 using a nuclear isolation kit and immunoblotted with anti-SMARCB1 and anti-Gli-1 antibodies. These membranes were stripped and immunoblotted with anti-GAPDH and anti-TATA-binding protein (anti-TBP) antibodies to check the purity of the cytoplasmic and nuclear lysates, respectively, and to confirm equal loading. (B) Shuttling of Gli-1. Immunoblotting showed the total nuclear translocation of the Gli-1 protein in induced iSLK cells compared with that in iSLK cells. (C) Gli-1 expression in lysates obtained from the nuclear and cytoplasmic fractions of SLK cells, latently infected SLK cells (iSLK), and induced iSLK cells was estimated using an ELISA kit. The experiments were repeated three times, and the results are expressed as the mean ± SD. **, P

    Journal: Journal of Virology

    Article Title: Concurrent Control of the Kaposi's Sarcoma-Associated Herpesvirus Life Cycle through Chromatin Modulation and Host Hedgehog Signaling: a New Prospect for the Therapeutic Potential of Lipoxin A4

    doi: 10.1128/JVI.02177-19

    Figure Lengend Snippet: LXA4 treatment regulates the nuclear translocation of Gli-1 and the SMARCB1–Gli-1 interaction in the nuclei of KSHV-infected cells. (A) The nuclear (Nuc) and cytoplasmic (Cyto) fractions were isolated from BCBL-1 cells and LXA4-treated BCBL-1 using a nuclear isolation kit and immunoblotted with anti-SMARCB1 and anti-Gli-1 antibodies. These membranes were stripped and immunoblotted with anti-GAPDH and anti-TATA-binding protein (anti-TBP) antibodies to check the purity of the cytoplasmic and nuclear lysates, respectively, and to confirm equal loading. (B) Shuttling of Gli-1. Immunoblotting showed the total nuclear translocation of the Gli-1 protein in induced iSLK cells compared with that in iSLK cells. (C) Gli-1 expression in lysates obtained from the nuclear and cytoplasmic fractions of SLK cells, latently infected SLK cells (iSLK), and induced iSLK cells was estimated using an ELISA kit. The experiments were repeated three times, and the results are expressed as the mean ± SD. **, P

    Article Snippet: BCBL-1 cells (treated with solvent or LXA4) were cultured, fixed, permeabilized with prechilled acetone, and blocked with Duolink blocking buffer for 30 min at 37°C.

    Techniques: Translocation Assay, Infection, Isolation, Binding Assay, Expressing, Enzyme-linked Immunosorbent Assay

    (A and B) Lysates from HMVEC-d cells infected with KSHV (30 DNA copies/cell) for the indicated times (0, 8, and 24 h) (A) and BCBL-1 cells (B) were Western blotted for PD-L1, stripped, and immunoblotted for tubulin and GAPDH, respectively. Representative data from three experiments are shown here, with the fold change being indicated. (C) In vitro PEL cell models. BCBL-1 cells treated with LXA4 were fixed and stained for PD-L1 using an Alexa Fluor 594-labeled antibody. (D) (i) BCBL-1 cells and LXA4-treated BCBL-1 cells were fixed, probed for surface PD-L1, stained with Alexa Fluor 488-labeled antibody, and analyzed by FACS using a Becton, Dickinson LSR II flow cytometer and FlowJo software. (ii) The hatched black peak represents unstained cells, the black peak represents the IgG control, and the solid green peak and hatched green peak represented stained solvent-treated and LXA4-treated BCBL-1 cells, respectively. ***, P

    Journal: Journal of Virology

    Article Title: Concurrent Control of the Kaposi's Sarcoma-Associated Herpesvirus Life Cycle through Chromatin Modulation and Host Hedgehog Signaling: a New Prospect for the Therapeutic Potential of Lipoxin A4

    doi: 10.1128/JVI.02177-19

    Figure Lengend Snippet: (A and B) Lysates from HMVEC-d cells infected with KSHV (30 DNA copies/cell) for the indicated times (0, 8, and 24 h) (A) and BCBL-1 cells (B) were Western blotted for PD-L1, stripped, and immunoblotted for tubulin and GAPDH, respectively. Representative data from three experiments are shown here, with the fold change being indicated. (C) In vitro PEL cell models. BCBL-1 cells treated with LXA4 were fixed and stained for PD-L1 using an Alexa Fluor 594-labeled antibody. (D) (i) BCBL-1 cells and LXA4-treated BCBL-1 cells were fixed, probed for surface PD-L1, stained with Alexa Fluor 488-labeled antibody, and analyzed by FACS using a Becton, Dickinson LSR II flow cytometer and FlowJo software. (ii) The hatched black peak represents unstained cells, the black peak represents the IgG control, and the solid green peak and hatched green peak represented stained solvent-treated and LXA4-treated BCBL-1 cells, respectively. ***, P

    Article Snippet: BCBL-1 cells (treated with solvent or LXA4) were cultured, fixed, permeabilized with prechilled acetone, and blocked with Duolink blocking buffer for 30 min at 37°C.

    Techniques: Infection, Western Blot, In Vitro, Staining, Labeling, FACS, Flow Cytometry, Software

    KSHV lytic infection inhibits PB formation by expression of viral ORF57. ( A ) GW182 and DCP1A are co-stained in the PB in BCBL-1 cells with no KSHV reactivation. BCBL-1 cells with latent KSHV infection were co-stained for GW182 (red) and DCP1A (green) using the corresponding antibodies and imaged by confocal microscopy. The nuclei were counterstained with Hoechst DNA stain. Arrows indicate the PB positively co-stained for both GW182 and DCP1A; bar = 5 μm. ( B and C ) Detection of PB during KSHV latent and lytic infection. (B) KSHV-infected BCBL-1 cells were induced with 1 mM valproic acid (VA, 1 mM) for lytic infection. Twenty-four hours after induction, BCBL-1 cells with (+VA) or without (-VA) virus lytic reactivation were co-immunostained for PB formation using a specific PB marker GW182 and for viral lytic protein ORF57 expression. The nuclei were counterstained with Hoechst dye. Images were captured by confocal microscopy; bar = 10 μm. (C) Number of PB in BCBL-1 cells during latent and lytic KSHV infection in the presence or absence of viral ORF57. Total of 50 cells in each group were counted in each experiment. The error bars represent SD from three independent experiments; ** P

    Journal: Nucleic Acids Research

    Article Title: KSHV RNA-binding protein ORF57 inhibits P-body formation to promote viral multiplication by interaction with Ago2 and GW182

    doi: 10.1093/nar/gkz683

    Figure Lengend Snippet: KSHV lytic infection inhibits PB formation by expression of viral ORF57. ( A ) GW182 and DCP1A are co-stained in the PB in BCBL-1 cells with no KSHV reactivation. BCBL-1 cells with latent KSHV infection were co-stained for GW182 (red) and DCP1A (green) using the corresponding antibodies and imaged by confocal microscopy. The nuclei were counterstained with Hoechst DNA stain. Arrows indicate the PB positively co-stained for both GW182 and DCP1A; bar = 5 μm. ( B and C ) Detection of PB during KSHV latent and lytic infection. (B) KSHV-infected BCBL-1 cells were induced with 1 mM valproic acid (VA, 1 mM) for lytic infection. Twenty-four hours after induction, BCBL-1 cells with (+VA) or without (-VA) virus lytic reactivation were co-immunostained for PB formation using a specific PB marker GW182 and for viral lytic protein ORF57 expression. The nuclei were counterstained with Hoechst dye. Images were captured by confocal microscopy; bar = 10 μm. (C) Number of PB in BCBL-1 cells during latent and lytic KSHV infection in the presence or absence of viral ORF57. Total of 50 cells in each group were counted in each experiment. The error bars represent SD from three independent experiments; ** P

    Article Snippet: KSHV lytic infection in BCBL-1 cells or JSC-1 cells and Bac36 cells was reactivated by 1 mM sodium valproate (VA, cat. # P4543, Millipore Sigma) or 3 mM sodium butyrate (Bu, cat. # B5887, Millipore Sigma) treatment, respectively, for 24 h. KSHV lytic replication in iSLK/Bac16 was induced by simultaneous treatment with 1 mM sodium butyrate and 1 μg/ml doxycycline (DOX, cat. # NC0424034, Fisher Scientific).

    Techniques: Infection, Expressing, Staining, Confocal Microscopy, Marker