Journal: Cell reports

Article Title: HCMV vCXCL1 Binds Several Chemokine Receptors and Preferentially Attracts Neutrophils over NK Cells by Interacting with CXCR2.

doi: 10.1016/j.celrep.2016.04.042

Figure Lengend Snippet: Figure 3. RvCXCL1 Induces Migration of Naive NK Cells (A) Transwell migration assays of freshly isolated NK cells toward increasing concentrations of rvCXCL1 (indicated on the x axis). RhFck and rhIL8 were used as positive controls. Percent of migrating cells was calculated out of total input cells. Data are presented as mean ± SEM (n = 4). (B) Freshly isolated NK cells were incubated for 1 hr with mAb against vCXCL1 or isotype-matched control. RvCXCL1 was placed in the bottom chamber, and migration was performed for 3 hr at 37C. The numbers of migrated cells was determined by FACS following 3 hr of incubation at 37C. The basal migration rate of NK cells toward medium that did not contain rvCXCL1 was set as 1 and the results presented as fold increase (FI). *p < 0.05. **p < 0.005.

Article Snippet: Filters were then plated in bottomwells containing 600 ml migrationmedium (RPMI 1640with 1% fetal calf serum [FCS]) supplemented with either rvCXCL1 (620-CM-025), rhIL8 (208-IL-050), rhFck (365-FR-025), or rhChemerin (2324-CM-025; obtained from R&D Systems), as indicated in each figure.

Techniques: Migration, Isolation, Incubation, Control

Journal: Cell reports

Article Title: HCMV vCXCL1 Binds Several Chemokine Receptors and Preferentially Attracts Neutrophils over NK Cells by Interacting with CXCR2.

doi: 10.1016/j.celrep.2016.04.042

Figure Lengend Snippet: Figure 4. RvCXCL1 Induces Naive NK Cell Migration via CX3CR1 and CXCR1 (A) Freshly isolated NK cells were incubated with and without 0.1 mg and 1 mg (indicated on the left side of the figure) of rhIL8 (red histograms, left), rhFck (blue histograms, middle), or rvCXCL1 (green histograms, right), at 37C for 1 hr. Next, cells were stained with anti-CX3CR1 (two upper rows) or with anti-CXCR1 (two lower rows). Open gray histograms show the staining of the chemokine receptors on untreated cells. Filled gray histograms show staining of the untreated NK cells with an isotype control. The backgrounds of the treated cells were similar to the untreated cells and are not shown in the figure. See also Table S2. (B) Binding of vCXCL1-Ig to 293T-CX3CR1 transfectant (black open histogram) or to the 293T parental cells (gray open histogram). Filled gray histogram is the staining of the control fusion protein (control-Ig) on the 293T-CX3CR1-transfected cells. Staining of the parental 293T cells with control-Ig was similar to the 293T- CX3CR1-transfected cells and is not shown in the figure. (C) Freshly isolated NK cells were incubated for 1 hr with and without the proteins indicated on the x axis. RvCXCL1 was placed in the bottom chamber, and migration was performed for 3 hr at 37C. Migrating cells were counted by FACS. NK cell migration toward rvCXCL1 without pre-blocking was set as 100%, and the results are presented as % of migration. *p < 0.05. NS, not significant. Figure shows one representative experiment out of three performed.

Article Snippet: Filters were then plated in bottomwells containing 600 ml migrationmedium (RPMI 1640with 1% fetal calf serum [FCS]) supplemented with either rvCXCL1 (620-CM-025), rhIL8 (208-IL-050), rhFck (365-FR-025), or rhChemerin (2324-CM-025; obtained from R&D Systems), as indicated in each figure.

Techniques: Migration, Isolation, Incubation, Staining, Control, Binding Assay, Transfection, Blocking Assay

Journal: Cell reports

Article Title: HCMV vCXCL1 Binds Several Chemokine Receptors and Preferentially Attracts Neutrophils over NK Cells by Interacting with CXCR2.

doi: 10.1016/j.celrep.2016.04.042

Figure Lengend Snippet: Figure 5. RvCXCL1 Induces Neutrophil Migration via CXCR1 and CXCR2 (A) Freshly isolated neutrophils were stained with mAb against CD16 and CEACAM1 (left dot plot). The double positive fraction (red square) was stained with specific antibodies against the chemokine receptors CXCR1, CXCR2, and CX3CR1. (B) Transwell migration assays were performed using freshly isolated neutrophils toward the recombinant proteins indicated on the x axis (rhFck, rhIL8, or rvCXCL1). The number of migrating cells was quantified by FACS, following a 30-min incubation period, at 37C. Percent of migrating cells out of total input cells was calculated. Data are presented as mean ± SEM (n = 6). (C) Freshly isolated neutrophils were incubated with and without 0.1 mg and 1 mg (indicated in the left of the figure) of rhIL8 (red histograms, left) or rvCXCL1 (green histograms, right) for 10 min at 37C, followed by staining with anti-CXCR1 (two upper rows) or anti-CXCR2 (two lower rows). Open gray histograms show chemokine receptors staining of the untreated cells. Filled gray histograms represent staining of the untreated cells with an isotype control. The backgrounds of the treated cells were similar to the untreated cells and are not shown in the figure. (D) Freshly isolated neutrophils were incubated at 37C for 10 min with or without the proteins indicated on the x axis. RvCXCL1 was placed in the bottom chamber, and the migrating neutrophils were counted using FACS following 30 min incubation at 37C. Neutrophil migration toward rvCXCL1 after pre-blocking with rhFck was set as 100%, and the results are presented as % of migration. *p < 0.05. **p < 0.005. Figure shows one representative experiment out of three performed. See also Table S3.

Article Snippet: Filters were then plated in bottomwells containing 600 ml migrationmedium (RPMI 1640with 1% fetal calf serum [FCS]) supplemented with either rvCXCL1 (620-CM-025), rhIL8 (208-IL-050), rhFck (365-FR-025), or rhChemerin (2324-CM-025; obtained from R&D Systems), as indicated in each figure.

Techniques: Migration, Isolation, Staining, Recombinant, Incubation, Control, Blocking Assay

Journal: Cell reports

Article Title: HCMV vCXCL1 Binds Several Chemokine Receptors and Preferentially Attracts Neutrophils over NK Cells by Interacting with CXCR2.

doi: 10.1016/j.celrep.2016.04.042

Figure Lengend Snippet: Figure 6. Neutrophils Migrate Faster and More Efficiently Than NK Cells in Response to rvCXCL1 (A) A transwell migration assay was performed using rvCXCL1, rhIL8, or rhFck as the chemoattractant with either freshly isolated neutrophils (gray triangles) or NK cells (black squares) placed in the upper chamber for 10, 20, and 30 min at 37C. Migration of untreated neutrophils and NK cells at the beginning of the experiment was set as 1, and the results are presented as FI. Figure shows one representative experiment out of two performed. (B) Diagram that describes competitive transwell migration assay in which NK cells and neutrophils were incubated together at the upper chamber and their ability to migrate toward rvCXCL1 (lower chamber) is determined. (C) Competitive transwell migration assays described in (B) was performed for 30 min (left panel) and 3 hr (right panel). Percent of migrating cells out of total input cells was calculated separately for neutrophils (gray) and NK cells (black). Data are presented as mean ± SEM (n = 3). *p < 0.05. **p < 0.005. ***p < 0.0005.

Article Snippet: Filters were then plated in bottomwells containing 600 ml migrationmedium (RPMI 1640with 1% fetal calf serum [FCS]) supplemented with either rvCXCL1 (620-CM-025), rhIL8 (208-IL-050), rhFck (365-FR-025), or rhChemerin (2324-CM-025; obtained from R&D Systems), as indicated in each figure.

Techniques: Transwell Migration Assay, Isolation, Migration, Incubation

Journal: Cell reports

Article Title: HCMV vCXCL1 Binds Several Chemokine Receptors and Preferentially Attracts Neutrophils over NK Cells by Interacting with CXCR2.

doi: 10.1016/j.celrep.2016.04.042

Figure Lengend Snippet: Figure 7. Reduced Neutrophil and NK Cell Migration in the Absence of UL146 during HCMV Infection (A) Freshly isolated neutrophils were incubated with increasing concentrations (indicated in the x axis) of rvCXCL1 (gray circles) for 30 min at 37C, followed by staining with anti-CXCR2. MFI of CXCR2 expression without blocking was set on 100%, and the residual CXCR2 expression was calculated. Estimated levels of vCXCL1 following infection of HFF cells with WT HCMV are shown as blue square on the graph. (B) HFFs were infected (MOI of 0.5) with WT HCMV (blue circles) or DUL146 (red circles), and supernatants containing progeny viruses were harvested at the indicated hours postinfection (x axis). The plaque-forming units (PFU) were determined using a standard plaque assay on HFF monolayers. (C and D) HFF cells were infected with WT HCMV or with HCMV DUL146 at a MOI of 1. Three days postinfection, cell supernatants were collected and used for transwell migration assays with either neutrophils (C) or NK cells (D). Neutrophils and NK cell migration toward supernatants from mock-infected HFF was set as 1, and the results are presented as FI. (E and F) Freshly isolated neutrophils were incubated at 37C for 30 min with or without the proteins indicated on the x axis. Transwell migration assays were performed with neutrophils (E) or NK cells (F) toward supernatant from mock-infected or WT-HCMV-infected HFFs. Neutrophils and NK cell migration toward supernatants from mock-infected cells was set as 1, and the results are presented as FI. *p < 0.05. **p < 0.005. ***p < 0.0005. (G) HCMV-infected endothelial cells secrete vCXCL1 (1) and recruit both neutrophils and NK cells to the infection site (2). Migration of NK cells is dependent on CXCR1 and CX3CR1 receptors, whereas neutrophil migration is dependent on CXCR2 and CXCR1. Neutrophils migrate faster and more efficiently in comparison to NK cells due to their CXCR2 receptor (3). Therefore, neutrophils reach the infection site early and can disseminate the virus while proceeding to travel throughout the body (4). This enables the virus to maintain a pool of HCMV-infected cells. NK cells that migrate toward vCXCL1 will get to the infection site at a later time point (5) and will be subverted by HCMV-immune evasion tactics.

Article Snippet: Filters were then plated in bottomwells containing 600 ml migrationmedium (RPMI 1640with 1% fetal calf serum [FCS]) supplemented with either rvCXCL1 (620-CM-025), rhIL8 (208-IL-050), rhFck (365-FR-025), or rhChemerin (2324-CM-025; obtained from R&D Systems), as indicated in each figure.

Techniques: Migration, Infection, Isolation, Incubation, Staining, Expressing, Blocking Assay, Plaque Assay, Comparison, Virus

Journal: Viruses

Article Title: Characterization of Human Cytomegalovirus (HCMV) Long Non-Coding RNA1.2 During Lytic Replication.

doi: 10.3390/v17020149

Figure Lengend Snippet: Figure 2. Viral gene expression and protein analysis TB40E WT, TB40E ∆RNA1.2 half, and TB40E ∆RNA1.2. (A) HF cells were infected with TB40E WT, ∆RNA1.2 half, and ∆RNA1.2 (MOI = 1). Cell lysate was harvested at 4 hpi, 24 hpi, 48 hpi, and 72 hpi. RNA was isolated, and transcript expression was measured by qPCR analysis using primers and probes specific to RNA1.2, RNA4.9, RNA B2.7, IE2, UL54, UL86 and 7SK (cellular). Fold change of each transcript was calculated using ∆∆CT, normalized with cellular 7SK and compared to the 4 hpi time point. Error bars indicate S.D. from independent experiments (N = 3). Statistical analysis was performed using two-way ANOVA, ****, t-test; n.s., not significant. (B) For protein analysis, cells were infected as described above. The mock and infected cell lysate was harvested at 24 hpi and 72 hpi. Cell lysate was denatured, and proteins were separated by SDS PAGE and transferred to PVDF membrane for Western blot. Proteins were identified using antibodies specific for labeling IE2, IE1, pp65, UL44, and H3.

Article Snippet: Membrane with samples from 72 hpi was probed with antibodies to detect UL44 1:1000 (Fitzgerald, 10-C50I), pp65 1:150 (Abcam, ab49214), and H3.

Techniques: Gene Expression, Infection, Isolation, Expressing, SDS Page, Membrane, Western Blot, Labeling

Journal: Viruses

Article Title: Characterization of Human Cytomegalovirus (HCMV) Long Non-Coding RNA1.2 During Lytic Replication.

doi: 10.3390/v17020149

Figure Lengend Snippet: Figure 3. Viral protein localization is comparable between TB40E WT, TB40E ∆RNA1.2 half, and TB40E ∆RNA1.2. HF cells were infected with TB40E WT, TB40E ∆RNA1.2 half, and TB40E ∆RNA1.2 (MOI = 1). After 72 hpi, mock and infected cells were fixed and permeabilized. Cells were labeled with antibodies to detect viral proteins UL44, IE2, UL57, and UL84 (Red). Nuclei were stained with DAPI (blue). GFP expression can be detected in infected cells (green). Cells were mounted and imaged at 63x. Zoom panels are indicated on the right to show viral protein localization patterns.

Article Snippet: Membrane with samples from 72 hpi was probed with antibodies to detect UL44 1:1000 (Fitzgerald, 10-C50I), pp65 1:150 (Abcam, ab49214), and H3.

Techniques: Infection, Labeling, Staining, Expressing

Journal: Viruses

Article Title: Characterization of Human Cytomegalovirus (HCMV) Long Non-Coding RNA1.2 During Lytic Replication.

doi: 10.3390/v17020149

Figure Lengend Snippet: Figure 9. Detection of RNA1.2 interacting with viral and cellular proteins. HF cells were infected with TB40E. After 72 hpi, cell lysate was fixed and sheared. An RNA-IP was performed using antibodies to detect IgG, H2A, RECQ1, and UL44. RNA was isolated, and a qPCR analysis was performed to detect the RNA bound to protein for the IPs. In the qPCR analysis, the primers and probes used to detect transcripts were RNA1.2 and GAPDH. Percent input was calculated and graphed (N = 3). Error bars indicate the standard deviation from the mean. Statistical analysis was performed using Student’s t-test with multiple comparisons *, p < 0.05.

Article Snippet: Membrane with samples from 72 hpi was probed with antibodies to detect UL44 1:1000 (Fitzgerald, 10-C50I), pp65 1:150 (Abcam, ab49214), and H3.

Techniques: Infection, Isolation, Standard Deviation

Journal: Cancer cell

Article Title: Bi-allelic loss of CDKN2A initiates melanoma invasion via BRN2 activation

doi: 10.1016/j.ccell.2018.05.014

Figure Lengend Snippet: (A) Strategy for introducing focal knock-in of CMV-EGFP to replace exon 2 of CDKN2A.

Article Snippet: MISSION pLKO.1-puro Non-Mammalian shRNA Control Plasmid (SHC002) and shPOU3F2 MISSION shRNA Bacterial Glycerol Stock (TRCN0000218221) were purchased from Sigma. pHIV-INK4A-zsGreen (Addgene #110728), pHIV-ARF-zsGreen (Addgene #110729), pHIV-ARF-mOrange2 (Addgene #110731), pHIV-INK4A-mOrange2 (Addgene #110730), and pHIV-BRAF V600E -mOrange2 (Addgene #110732) were all generated by inserting the indicated coding sequence into the Ecorl and Xbal sites of the indicated parental pHIV constructs. pSicoR-Efla-mCh-Puro was a gift from Bruce Conklin (Addgene plasmid # 31845)( Salomonis et al., 2010 ). pGL410-BRN2p (Addgene #110733, BRN2 promoter luciferase reporter) was generated by placing 2kb of sequence upstream of the BRN2 transcriptional start site into the Bgl2 and Xhol sites. pHDR-CDKN2A-Ex2-CMV-EGFP (Addgene #110734) was generated by amplifying 2kb left and right arms from human genomic DNA and using Cold Fusion (System Bio) to combine with the CMV promoter, EGFP ORF, and an Amp/Ori cassette. pHDR-CDKN2A-Ex2-EFla-H2B-mOrange2 (Addgene #110735) was constructed similarly but with a EFla promoter and mOrange2 ORF fused to H2B.

Techniques: Knock-In

Journal: Cancer cell

Article Title: Bi-allelic loss of CDKN2A initiates melanoma invasion via BRN2 activation

doi: 10.1016/j.ccell.2018.05.014

Figure Lengend Snippet: (A) Schematic of experimental set-up. For each experiment, NHMs are derived from donated tissue and engineered for CDKN2A loss as in Figure 1. After isolation, CDKN2A null and wild-type sibling cells are monitored via digital holographic cytometry for 72 hr. The rate of cell division, motility, morphology, growth arrest and detachment are quantified. Representative holographic phase shift images (bottom left) show colored comet tails tracking cells.

Article Snippet: MISSION pLKO.1-puro Non-Mammalian shRNA Control Plasmid (SHC002) and shPOU3F2 MISSION shRNA Bacterial Glycerol Stock (TRCN0000218221) were purchased from Sigma. pHIV-INK4A-zsGreen (Addgene #110728), pHIV-ARF-zsGreen (Addgene #110729), pHIV-ARF-mOrange2 (Addgene #110731), pHIV-INK4A-mOrange2 (Addgene #110730), and pHIV-BRAF V600E -mOrange2 (Addgene #110732) were all generated by inserting the indicated coding sequence into the Ecorl and Xbal sites of the indicated parental pHIV constructs. pSicoR-Efla-mCh-Puro was a gift from Bruce Conklin (Addgene plasmid # 31845)( Salomonis et al., 2010 ). pGL410-BRN2p (Addgene #110733, BRN2 promoter luciferase reporter) was generated by placing 2kb of sequence upstream of the BRN2 transcriptional start site into the Bgl2 and Xhol sites. pHDR-CDKN2A-Ex2-CMV-EGFP (Addgene #110734) was generated by amplifying 2kb left and right arms from human genomic DNA and using Cold Fusion (System Bio) to combine with the CMV promoter, EGFP ORF, and an Amp/Ori cassette. pHDR-CDKN2A-Ex2-EFla-H2B-mOrange2 (Addgene #110735) was constructed similarly but with a EFla promoter and mOrange2 ORF fused to H2B.

Techniques: Derivative Assay, Isolation, Cytometry

Journal: Cancer cell

Article Title: Bi-allelic loss of CDKN2A initiates melanoma invasion via BRN2 activation

doi: 10.1016/j.ccell.2018.05.014

Figure Lengend Snippet: (A) Volcano plot comparing transcriptomes of three independently derived pairs of CDKN2A null NHMs and matched wild-type sibling cells. Differentially expressed transcripts with q-values < 0.05 and log2 fold change > 1.7 are highlighted in aqua. The BRN2 transcript is highlighted in red.

Article Snippet: MISSION pLKO.1-puro Non-Mammalian shRNA Control Plasmid (SHC002) and shPOU3F2 MISSION shRNA Bacterial Glycerol Stock (TRCN0000218221) were purchased from Sigma. pHIV-INK4A-zsGreen (Addgene #110728), pHIV-ARF-zsGreen (Addgene #110729), pHIV-ARF-mOrange2 (Addgene #110731), pHIV-INK4A-mOrange2 (Addgene #110730), and pHIV-BRAF V600E -mOrange2 (Addgene #110732) were all generated by inserting the indicated coding sequence into the Ecorl and Xbal sites of the indicated parental pHIV constructs. pSicoR-Efla-mCh-Puro was a gift from Bruce Conklin (Addgene plasmid # 31845)( Salomonis et al., 2010 ). pGL410-BRN2p (Addgene #110733, BRN2 promoter luciferase reporter) was generated by placing 2kb of sequence upstream of the BRN2 transcriptional start site into the Bgl2 and Xhol sites. pHDR-CDKN2A-Ex2-CMV-EGFP (Addgene #110734) was generated by amplifying 2kb left and right arms from human genomic DNA and using Cold Fusion (System Bio) to combine with the CMV promoter, EGFP ORF, and an Amp/Ori cassette. pHDR-CDKN2A-Ex2-EFla-H2B-mOrange2 (Addgene #110735) was constructed similarly but with a EFla promoter and mOrange2 ORF fused to H2B.

Techniques: Derivative Assay

Journal: Cancer cell

Article Title: Bi-allelic loss of CDKN2A initiates melanoma invasion via BRN2 activation

doi: 10.1016/j.ccell.2018.05.014

Figure Lengend Snippet: (A) Fraction of nevi or melanomas with mono- or bi-allelic CDKN2A disruption. Melanomas are sub-categorized as melanoma in situ (MIS), thin invasive melanoma (Stage T1), thick invasive melanoma (Stage T2+) and distal metastatic melanoma (obtained from the TCGA public database).

Article Snippet: MISSION pLKO.1-puro Non-Mammalian shRNA Control Plasmid (SHC002) and shPOU3F2 MISSION shRNA Bacterial Glycerol Stock (TRCN0000218221) were purchased from Sigma. pHIV-INK4A-zsGreen (Addgene #110728), pHIV-ARF-zsGreen (Addgene #110729), pHIV-ARF-mOrange2 (Addgene #110731), pHIV-INK4A-mOrange2 (Addgene #110730), and pHIV-BRAF V600E -mOrange2 (Addgene #110732) were all generated by inserting the indicated coding sequence into the Ecorl and Xbal sites of the indicated parental pHIV constructs. pSicoR-Efla-mCh-Puro was a gift from Bruce Conklin (Addgene plasmid # 31845)( Salomonis et al., 2010 ). pGL410-BRN2p (Addgene #110733, BRN2 promoter luciferase reporter) was generated by placing 2kb of sequence upstream of the BRN2 transcriptional start site into the Bgl2 and Xhol sites. pHDR-CDKN2A-Ex2-CMV-EGFP (Addgene #110734) was generated by amplifying 2kb left and right arms from human genomic DNA and using Cold Fusion (System Bio) to combine with the CMV promoter, EGFP ORF, and an Amp/Ori cassette. pHDR-CDKN2A-Ex2-EFla-H2B-mOrange2 (Addgene #110735) was constructed similarly but with a EFla promoter and mOrange2 ORF fused to H2B.

Techniques: In Situ

Journal: Cancer cell

Article Title: Bi-allelic loss of CDKN2A initiates melanoma invasion via BRN2 activation

doi: 10.1016/j.ccell.2018.05.014

Figure Lengend Snippet: (A) Gene set enrichment analysis of three CDKN2A null NHM lines compared to three CDKN2A wild-type NHM lines (Engineered NHMs) or CDKN2A wild-type regions compared to CDKN2A null regions of the clinical cohort (Transition case cohort). # indicates both the NOM p value and FDR q-values for the enrichment score < 0.0005.

Article Snippet: MISSION pLKO.1-puro Non-Mammalian shRNA Control Plasmid (SHC002) and shPOU3F2 MISSION shRNA Bacterial Glycerol Stock (TRCN0000218221) were purchased from Sigma. pHIV-INK4A-zsGreen (Addgene #110728), pHIV-ARF-zsGreen (Addgene #110729), pHIV-ARF-mOrange2 (Addgene #110731), pHIV-INK4A-mOrange2 (Addgene #110730), and pHIV-BRAF V600E -mOrange2 (Addgene #110732) were all generated by inserting the indicated coding sequence into the Ecorl and Xbal sites of the indicated parental pHIV constructs. pSicoR-Efla-mCh-Puro was a gift from Bruce Conklin (Addgene plasmid # 31845)( Salomonis et al., 2010 ). pGL410-BRN2p (Addgene #110733, BRN2 promoter luciferase reporter) was generated by placing 2kb of sequence upstream of the BRN2 transcriptional start site into the Bgl2 and Xhol sites. pHDR-CDKN2A-Ex2-CMV-EGFP (Addgene #110734) was generated by amplifying 2kb left and right arms from human genomic DNA and using Cold Fusion (System Bio) to combine with the CMV promoter, EGFP ORF, and an Amp/Ori cassette. pHDR-CDKN2A-Ex2-EFla-H2B-mOrange2 (Addgene #110735) was constructed similarly but with a EFla promoter and mOrange2 ORF fused to H2B.

Techniques: