Journal: PLoS Pathogens

Article Title: Interleukin 16 contributes to gammaherpesvirus pathogenesis by inhibiting viral reactivation

doi: 10.1371/journal.ppat.1008701

Figure Lengend Snippet: (A) 1×10 6 SL-1 and SL-3 cells were cultured for 24 hr, whole-cell lysate (WCL) and supernatant (SUP) were prepared and subjected to cytokine array analyses. Each cytokine has one pair of duplicate spots. A1-2, A23-24, and F1-2 are experimental positive control, and F23-24 is an experimental negative control. (B) C57BL/6 mice were intranasally inoculated with 5×10 4 PFU of MHV68-H2bYFP or mock inoculated with PBS. At day 16 post-infection, splenocytes were isolated and subjected to flow cytometry, the flow plot represented the strategy gating YFP+ MHV68 infected cells (left panel); serum was prepared from 10 virus-infected mice or mock-infected mice, followed by IL16 ELISA assay (right panel). Histograms represented mean ±SD of 10 individual mice (two experiments, n = 5 for each experiment). p value was determined by two-tailed unpaired t-test. (C) WT MEFs were infected with MHV68 at an MOI of 1, total RNA was isolated from infected cells harvested at the indicated time points and subjected to qRT-PCR analyses with specific primers corresponding to IL16 and MHV68 ORF50 gene. The relative RNA amount was normalized to GAPDH in each sample. Histograms represented the mean of three independent biological replicates ±SD, p value was determined by two-tailed unpaired t-test, p ≤ 0.05 represents significance.

Article Snippet: Mouse IL16 Antibody (842373) was from R&D Systems.

Techniques: Cell Culture, Positive Control, Negative Control, Infection, Isolation, Flow Cytometry, Virus, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Quantitative RT-PCR

Journal: PLoS Pathogens

Article Title: Interleukin 16 contributes to gammaherpesvirus pathogenesis by inhibiting viral reactivation

doi: 10.1371/journal.ppat.1008701

Figure Lengend Snippet: (A) Intracellular staining of IL16 in splenocytes isolated from IL16+/+, IL16+/-, and IL16-/- mice. (B) Immunoblot detection of IL16 expression in splenocytes isolated from IL16+/+, IL16+/-, and IL16-/- mice. (C) Representative flow plots showed flow cytometric analyses of splenocytes from IL16+/+ (WT) and IL16-/- (KO) mice. (D) The statistic analyses of CD4+ T, CD8+ T, and B cells (Left panel); follicular (FC) B, marginal zone (MZ) B, and mature B cells (Right panel) in splenocytes from WT and IL16 KO mice. Histograms represented mean ±SD of 8 individual mice (two experiments, n = 4 for each experiment). ns = not significant.

Article Snippet: Mouse IL16 Antibody (842373) was from R&D Systems.

Techniques: Staining, Isolation, Western Blot, Expressing

Journal: PLoS Pathogens

Article Title: Interleukin 16 contributes to gammaherpesvirus pathogenesis by inhibiting viral reactivation

doi: 10.1371/journal.ppat.1008701

Figure Lengend Snippet: (A) WT and IL16 KO MEFs were infected with MHV68 at an MOI of 1 or 0.05. The infected cells were harvested at the indicated time points and immunoblot analyses were performed with specific antibodies as indicated. Actin was used as a loading control. (B) WT and IL16 KO MEFs were infected with MHV68 at an MOI of 5 or 0.05. The supernatant was harvested at the indicated times and viral titers were determined by TCID50 assay. Results are means from triplicate samples. Error bars represented standard deviations. ns = not significant. (C) WT and IL16 KO mice were intranasally infected with 5×10 4 PFU of MHV68. Lungs of infected mice were collected at day 4 and 7 post-infection. Virus titers were determined by TCID50 assay. Data represented one of two independent experiments with 5 or 7 mice per group. ns = not significant. Each symbol represented an individual mouse. The horizon line indicated geometric mean titer. (D) Vector or IL16-expressing plasmids with Flag tag were transfected into BHK21 cells for 24 hr, followed by MHV68 infection at an MOI of 5 or 0.05. The infected cells were harvested at the indicated time points and immunoblot analyses were performed with specific antibodies as indicated.–and + represents the cells transfected with vector and IL16-expressing plasmids with Flag tag, respectively. (E) Supernatant was harvested at the indicated times and viral titers were determined by TCID50 assay. Results were means from triplicate samples. Error bars represented standard deviations. ns = not significant.

Article Snippet: Mouse IL16 Antibody (842373) was from R&D Systems.

Techniques: Infection, Western Blot, Control, TCID50 Assay, Virus, Plasmid Preparation, Expressing, FLAG-tag, Transfection

Journal: PLoS Pathogens

Article Title: Interleukin 16 contributes to gammaherpesvirus pathogenesis by inhibiting viral reactivation

doi: 10.1371/journal.ppat.1008701

Figure Lengend Snippet: (A) WT and three IL16 KO single clones were stimulated with (+) or without (-) anti-mouse Ig(G+M) (5 μg/mL) for 48 hr, respectively. Immunoblot analyses were performed with the indicated antibodies. GAPDH was used as a loading control. (B) WT and IL16 KO cells (clone E6) were stimulated with (+) or without (-) anti-mouse Ig(G+M) (5 μg/mL) for 48 hr. Immunoblot analyses were performed with the indicated antibodies. GAPDH was used as a loading control. MHV68 viral genome was determined by qPCR with the primers specific to the MHV68 ORF50 coding region. The relative copy of the MHV68 viral genome was normalized to GAPDH in each sample. (C) The mRNA expression of MHV68 viral gene ORF73, ORF50, ORF59, and ORF25 was determined by qRT-PCR. The relative RNA amount was normalized to GAPDH in each sample. Histograms represented the mean of three independent biological replicates ±SD, p value was determined by two-tailed unpaired t-test, p ≤ 0.05 represents significance.

Article Snippet: Mouse IL16 Antibody (842373) was from R&D Systems.

Techniques: Clone Assay, Western Blot, Control, Expressing, Quantitative RT-PCR, Two Tailed Test

Journal: PLoS Pathogens

Article Title: Interleukin 16 contributes to gammaherpesvirus pathogenesis by inhibiting viral reactivation

doi: 10.1371/journal.ppat.1008701

Figure Lengend Snippet: (A) The diagram showed the potential cleavage sites of IL16. (B) 293T cells were transfected with IL16 or mutants with Flag tag. At 48 hr post-transfection, the supernatant was collected and subjected to IL16 ELISA assay; whole-cell lysates (WCL) and supernatant (SUP) were prepared and subjected to immunoblot analyses with the indicated antibodies. (C) IL16 KO SL-1 cells were transfected with vector (Vec), IL16-, or IL16(D516A)-expressing plasmid with Flag tag, followed by anti-mouse Ig(G+M) treatment for 48 hr. The whole-cell lysates were prepared and subjected to immunoblot analyses with the indicated antibodies.

Article Snippet: Mouse IL16 Antibody (842373) was from R&D Systems.

Techniques: Transfection, FLAG-tag, Enzyme-linked Immunosorbent Assay, Western Blot, Plasmid Preparation, Expressing

Journal: PLoS Pathogens

Article Title: Interleukin 16 contributes to gammaherpesvirus pathogenesis by inhibiting viral reactivation

doi: 10.1371/journal.ppat.1008701

Figure Lengend Snippet: WT and IL16 KO mice were inoculated intranasally with 5×10 4 PFU of MHV68-H2bYFP. Mice inoculated with 5×10 4 PFU of WT MHV68 were used as a control to gate YFP+ cells. Splenocytes were isolated at day 16 and day 18 post-infection. (A) Representative flow plots showing the identification of MHV68-infected YFP+ cells. (B) Frequency of YFP+ cells at day 16 post-infection. Results were compiled from two independent experiments with 8–9 mice per group. Each symbol represented an individual mouse, and the horizon lines represented the mean frequency of infected cells. ns = not significant. (C) Frequency of splenocytes capable of reactivating virus by ex-vivo assay at day 16 post-infection. Serial dilutions of splenocytes were plated on MEFs and the presence of reactivating virus was determined by the presence of cytopathic effect (CPE). Representative results were from two independent experiments with 8–9 mice per group. (D) Frequency of YFP+ cells at day 18 post-infection. Results were compiled from two independent experiments with 10–12 mice per group. Each symbol represented an individual mouse, and the horizon lines represented the mean frequency of infected cells. ns = not significant. (E) Frequency of splenocytes capable of reactivating virus by ex-vivo assay at day 18 post-infection. Data were generated from two independent experiments, 5 to 6 mice per experiment per group.

Article Snippet: Mouse IL16 Antibody (842373) was from R&D Systems.

Techniques: Control, Isolation, Infection, Virus, Ex Vivo, Generated

Journal: PLoS Pathogens

Article Title: Interleukin 16 contributes to gammaherpesvirus pathogenesis by inhibiting viral reactivation

doi: 10.1371/journal.ppat.1008701

Figure Lengend Snippet: WT and IL16 KO mice were intranasally inoculated with 5×10 4 PFU of MHV68-H2bYFP and splenocytes were harvested at day 16 post-infection. (A) Representative flow plots showing the identification of MHV68-infected YFP+ cells. (B) Representative flow plots of YFP+ germinal center B cells (CD19 + CD95 + GL-7 + YFP + ). (C) Quantitation of the percentage of YFP+ germinal center B cells. Each symbol represented an individual mouse, and the horizon lines represented the mean frequency. (D) Representative flow plots of YFP+ plasma cells (CD3 - YFP + B220 low CD138 + ). (E) Quantitation of the percentage of YFP+ plasma cells. Each symbol represented an individual mouse, and the horizon lines represented the mean frequency. (F) Quantitation of the percentage of total germinal center B cells (CD19 + CD95 + GL-7 + ). (G) Quantitation of the percentage of total plasma cells (CD3 - B220 low CD138 + ). ns = not significant. p value was determined by two-tailed unpaired t-test, p ≤ 0.05 represents significance.

Article Snippet: Mouse IL16 Antibody (842373) was from R&D Systems.

Techniques: Infection, Quantitation Assay, Clinical Proteomics, Two Tailed Test

Journal: PLoS Pathogens

Article Title: Interleukin 16 contributes to gammaherpesvirus pathogenesis by inhibiting viral reactivation

doi: 10.1371/journal.ppat.1008701

Figure Lengend Snippet: WT and IL16 KO mice were inoculated intranasally with 5×10 4 PFU of MHV68-H2bYFP. Splenocytes were isolated at day 16 post-infection and subjected to flow cytometry analyses. (A) Representative flow plots of CD4+ and CD8+ T cells from infected WT and IL16 KO mice. (B) Quantitation of the percentage of CD4+ and CD8+ T cells. Each symbol represented an individual mouse, and the horizon lines represented the mean frequency. (C) Representative flow plots of IFN-γ+CD4+ T cells from infected WT and IL16 KO mice. (D) Quantitation of the percentage of IFN-γ+CD4+ and CD44+CD4+ T cells. Each symbol represented an individual mouse, and the horizon lines represented the mean frequency. (E) Quantitation of the percentage of IL4+CD4+ and IL2+CD4+ T cells. Each symbol represented an individual mouse, and the horizon lines represented the mean frequency. (F) Representative flow plots of IFN-γ+ CD8+ T cells from infected WT and IL16 KO mice. (G) Quantitation of the percentage of IFN-γ+CD8+ and TNF-α+CD8+ T cells. Each symbol represented an individual mouse, and the horizon lines represented the mean frequency. ns = not significant. p value was determined by two-tailed unpaired t-test, p ≤ 0.05 represents significance.

Article Snippet: Mouse IL16 Antibody (842373) was from R&D Systems.

Techniques: Isolation, Infection, Flow Cytometry, Quantitation Assay, Two Tailed Test

Journal: PLoS Pathogens

Article Title: Interleukin 16 contributes to gammaherpesvirus pathogenesis by inhibiting viral reactivation

doi: 10.1371/journal.ppat.1008701

Figure Lengend Snippet: (A) The murine M12 B lymphoma cells were transfected with renilla reporter, a luciferase reporter (pGL2) driven by RTA proximal promoter (RTAp), together with IL16-expressing plasmid with Flag tag or vector alone (Vec). Luciferase activity was normalized to renilla activity and Luciferase value was reported as fold increase in luciferase activity over basal promoter activity. Each sample was done in triplicate (two independent experiments). IL16 expression was detected by immunoblot with a Flag antibody. (B) M12 cells were transfected with RTAp together with vector (Vec), IL16-, IL16(D506A)- or IL16(D516A)-expressing plasmid with Flag tag. At 24 hr post-transfection, transfected cells were treated with (+) or without (-) anti-mouse Ig(G+M) (5 μg/mL) for 12 hr. IL16, IL16 (D506A), and IL16 (D516A) expression was detected by immunoblot with Flag antibody. Luciferase value was reported as fold increase in luciferase activity over basal promoter activity. Each sample was done in triplicate (two independent experiments).

Article Snippet: Mouse IL16 Antibody (842373) was from R&D Systems.

Techniques: Transfection, Luciferase, Expressing, Plasmid Preparation, FLAG-tag, Activity Assay, Western Blot

Journal: PLoS Pathogens

Article Title: Interleukin 16 contributes to gammaherpesvirus pathogenesis by inhibiting viral reactivation

doi: 10.1371/journal.ppat.1008701

Figure Lengend Snippet: (A) WT and IL16 KO SL-1 cells were treated with (+) or without (-) 20 μM JAKs inhibitor AG490 for 1 hr, followed by stimulation with (+) or without (-) anti-mouse Ig(G+M) (5 μg/mL) for 48 hr. Immunoblot analyses were performed with specific antibodies as indicated. GAPDH was used as a loading control. (B) WT and IL16 KO SL-1 cells were treated with (+) or without (-) anti-mouse Ig(G+M) (5 μg/mL) for 48 hr. Immunoblot analyses were performed with specific antibodies as indicated. (C) Quantitation of phosphorylated STAT relative to total STAT based on immunoblot detection in C using the ImageJ image analysis software.

Article Snippet: Mouse IL16 Antibody (842373) was from R&D Systems.

Techniques: Western Blot, Control, Quantitation Assay, Software

Journal: PLoS Pathogens

Article Title: Interleukin 16 contributes to gammaherpesvirus pathogenesis by inhibiting viral reactivation

doi: 10.1371/journal.ppat.1008701

Figure Lengend Snippet: (A) WT and IL16 KO SL-1 cells were pretreated with DMSO, 40 μM, or 80 μM STAT5 inhibitor (STAT5-I) for 1 hr, followed by stimulation with anti-mouse Ig(G+M) (5 μg/mL) for 48 hr. Immunoblot analyses were performed with specific antibodies as indicated. (B) SL-1 cells were transfected with vector, STAT3-expressing plasmid, or STAT3C-expressing plasmid with Flag tag, followed by anti-mouse Ig(G+M) (5 μg/mL) treatment for 48 hr. Immunoblot analyses were performed with the indicated antibodies. (C) WT and IL16 KO SL-1 cells were treated with (+) or without (-) anti-mouse Ig(G+M) (5 μg/mL) for 48 hr. Immunoblot analyses were performed with the specific antibodies as indicated. (D) Quantitation of phosphorylated STAT3(Y705) relative to total STAT3 and p21 relative to GAPDH based on immunoblot detection in C using the ImageJ image analysis software. (E) WT and IL16 KO SL-1 cells were treated with DMSO or Orthovanadate (50 μM) in the presence or absence of anti-mouse Ig(G+M) (5 μg/mL) for 48 hr. Immunoblot analyses were performed with the specific antibodies as indicated.

Article Snippet: Mouse IL16 Antibody (842373) was from R&D Systems.

Techniques: Western Blot, Transfection, Plasmid Preparation, Expressing, FLAG-tag, Quantitation Assay, Software

Journal: PLoS Pathogens

Article Title: Interleukin 16 contributes to gammaherpesvirus pathogenesis by inhibiting viral reactivation

doi: 10.1371/journal.ppat.1008701

Figure Lengend Snippet: MHV68 infection induces IL16 production, which, in turn, increases STAT3(Y705) phosphorylation, subsequently reduces p21 expression, and inhibits MHV68 reactivation. Meanwhile , IL16 partially inhibits RTA promoter activity and STAT3(S727) phosphorylation, contributing to the inhibition of MHV68 reactivation. Ultimately, MHV68-induced IL16 helps to maintain MHV68 latency.

Article Snippet: Mouse IL16 Antibody (842373) was from R&D Systems.

Techniques: Infection, Phospho-proteomics, Expressing, Activity Assay, Inhibition

Journal: bioRxiv

Article Title: Epigenetic regulation of p63 blocks squamous-to-neuroendocrine transdifferentiation in esophageal development and malignancy

doi: 10.1101/2023.09.09.556982

Figure Lengend Snippet: (A) p63 and INSM1 staining in human esophageal small cell carcinoma specimens. Scale bars: 20 µm. (B) Schematics showing that TYUC-1 esophageal neuroendocrine cancer cells were treated with inhibitors of epigenetic regulators and gene expression was analyzed by qRT-PCR. (C) The transcript levels of TAp63 and ΔNp63 in TYUC-1 esophageal neuroendocrine cancer cells treated with various epigenetic inhibitors. Note that the EZH2 inhibitor EPZ-6438 increased the expression of TAp63 and ΔNp63 to the highest levels compared to other inhibitors. (D) Transcript levels of ΔNp63 in TYUC-1 cells treated with EPZ-6438 alone or in combination with JQ1 or A485. (E-F) Immunofluorescence staining and quantification of p63 cells in EPZ-6438 treated TYUC-1 cells. Note that p63 is expressed in the EPZ-6438-treated group but not the DMSO-treated group. Scale bars: 20 µm. (G) Genome browser view at the TP63 locus showing esophagus epithelium H3K27ac ChIP-seq (ENCODE SRX3205374) and TYUC-1 cell H3K27me3, H3K27ac, and BRD4 CUT&Tag upon treatment with EPZ-6438 for 3 and 6 days. Noted are putative enhancer elements near the ΔNp63 isoform TSS where H3K27 methylation is lost, and acetylation is restored.

Article Snippet: For H3K27me3 and H3K27ac, 2 μl of SNAP-ChIP K-MetStat panel and K-AcylStat panel nucleosomes (EpiCypher) respectively, were added as spike-in control at the primary antibody incubation step.

Techniques: Staining, Gene Expression, Quantitative RT-PCR, Expressing, Immunofluorescence, ChIP-sequencing, Methylation