Journal: Frontiers in Microbiology

Article Title: Metabolic Regulators Nampt and Sirt6 Serially Participate in the Macrophage Interferon Antiviral Cascade

doi: 10.3389/fmicb.2019.00355

Figure Lengend Snippet: Expression of Nampt and Sirt6 is upregulated by MCMV infection. (A) Quantification of relative Nampt and Sirt6 mRNA expression in NIH-3T3 cells, at 6 and 10 h, using RT-qPCR following mCMV infection with mock-infected cells serving as controls ( n = 3). (B) Quantification of relative Nampt and Sirt6 mRNA expression in p53-MEF cells, at 3 and 6 h, using RT-qPCR following mCMV infection ( n = 3). ANOVA with Tukey post-test was used to assess statistical signficance. (C) Normalized temporal Nampt and Sirt6 expression (antilog) in mCMV infected bone marrow derived macrophages (BMDM). The expression was measured over the first 24 h of infection using microarray and compared to timepoint 0. The expression levels between 0 and 10 h were smoothened and fitted to a linear (+), quadatic (#), or cubic ( ∗ ) polynomal on time and the statistical significance ( p -values) was assessed. +/#/∗ p < 0.05, ++/##/∗∗ p < 0.01, and +++/###/∗∗∗ p < 0.001 were considered to be significant (ns, not significant). Bars represent standard error of the mean (SEM). ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001 were considered to be significant (ns, not significant).

Article Snippet: Cells infected with MCMV or treated with poly(I:C) were harvested at 0, 2, 4, 6, 8, 10, and 24 h post-treatment for RNA isolation and microarray analysis (Affymetrix Mouse Gene 1.0ST microarray).

Techniques: Expressing, Infection, Quantitative RT-PCR, Derivative Assay, Microarray

Journal: Frontiers in Microbiology

Article Title: Metabolic Regulators Nampt and Sirt6 Serially Participate in the Macrophage Interferon Antiviral Cascade

doi: 10.3389/fmicb.2019.00355

Figure Lengend Snippet: Expression of Nampt is induced by both type-I and type-II IFN, while respone of Sirt6 is restricted to type-I IFN response. (A) Quantification of Nampt mRNA expression, using microarray, in wild-type (wt) and Tyk2 -/- BMDM following mock- (control) and mCMV-infection. (B) Normalized temporal expression (antilog) of Nampt and Sirt6 in mCMV infected wild-type and Ifnb -/- BMDM. The expression was measured over the first 24 h of infection using microarray and compared to timepoint 0. The expression levels between timepoints 0–10 h post-streatment were smoothened and fitted to a linear (+), quadatic (#), or cubic ( ∗ ) polynomal on time to assess significance ( p -values). +/#/∗ p < 0.05, ++/##/∗∗ p < 0.01, and +++/###/∗∗∗ p < 0.001 were considered to be significant (ns, not significant). (C) Normalized temporal expression (antilog) of Nampt and Sirt6 in poly(I:C) treated wild-type and Ifnb -/- BMDM. The expression was measured over the first 24 h using microarray and compared to timepoint 0. The expression levels between timepoints 0–10 h post-streatment were smoothened and fitted to a linear (+), quadatic (#), or cubic ( ∗ ) polynomal on time to assess significance ( p -values). +/#/∗ p < 0.05, ++/##/∗∗ p < 0.01, and +++/###/∗∗∗ p < 0.001 were considered to be significant (ns, not significant). (D) Quantification of de novo synthesis of Nampt, Sirt6, and Srebf2 mRNA in IFNγ-stimulated BMDMs using qRT-PCR. Expression was measured every 30 min for a total of 8 h. (E) Quantification of relative Nampt mRNA expression in wild-type and Stat1 -/- p53-MEF cells, at 1, 2, 4, 8, and 16 h, using RT-qPCR following IFNγ stimulation ( n = 3). Expression is relative to wild-type untreated (0 h) cells, set as 1 (not shown). One-way ANOVA with a Tukey’s post-test was used to assess statistical signficance to untreated controls. One-way ANOVA with a Sidak’s multiple comparisons test was used to assess statistical signficance between wild-type and Stat1 -/- mutants. Bars represent SEM. Statisical significance between groups (wild-type and Stat1 -/- ) were depicted with ∗ . Statistical significance in relation to untreated controls (wild-type and Stat1 -/- , respectively) were depicted with #. ∗ / # p < 0.05, ∗∗ / ## p < 0.01, ∗∗∗ / ### p < 0.001 were considered to be significant. (F) Quantification of relative Sirt6 mRNA expression in wild-type and Stat1 - / - p53-MEF cells, at 1, 2, 4, 8, and 16 h, using RT-qPCR following IFNγ stimulation ( n = 3). Expression is relative to wild-type untreated (0 h) cells, set as 1 (not shown). One-way ANOVA with a Tukey’s post-test was used to assess statistical signficance to untreated controls. One-way ANOVA with a Sidak’s multiple comparisons test was used to assess statistical signficance between wild-type and Stat1 -/- mutants. Bars represent SEM. Statisical significance between groups (wild-type and Stat1 -/- ) were depicted with ∗ . Statistical significance in relation to untreated controls (wild-type and Stat1 -/- , respectively) were depicted with #. ∗ / # p < 0.05, ∗∗ / ## p < 0.01, ∗∗∗ / ### p < 0.001 were considered to be significant.

Article Snippet: Cells infected with MCMV or treated with poly(I:C) were harvested at 0, 2, 4, 6, 8, 10, and 24 h post-treatment for RNA isolation and microarray analysis (Affymetrix Mouse Gene 1.0ST microarray).

Techniques: Expressing, Microarray, Infection, Quantitative RT-PCR

Journal: Frontiers in Microbiology

Article Title: Metabolic Regulators Nampt and Sirt6 Serially Participate in the Macrophage Interferon Antiviral Cascade

doi: 10.3389/fmicb.2019.00355

Figure Lengend Snippet: Expression of upstream, but not downstream, TLR signaling pathway components is dependent on IFNβ/type-I IFN signaling. (A) Normalized temporal expression (antilog) of Myd88 , p50 ( Nfkb1 ), p65 ( Rela ), Trif ( Ticam1 ), Rig-I ( Ddx58 ), Mda-5 ( Ifih1 ), Ips-1 ( Mavs ), Sting ( Tmem173 ), and cGas ( Mb21d1 ) in mCMV infected wild-type and Ifnb - / - BMDM. The expression was measured over the first 24 h of infection using microarray and compared to timepoint 0. The expression levels between timepoints 0–10 h post-streatment were smoothened and fitted to a linear (+), quadatic (#), or cubic ( ∗ ) polynomal on time to assess significance ( p -values). +/#/∗ p < 0.05, ++/##/∗∗ p < 0.01, and +++/###/∗∗∗ p < 0.001 were considered to be significant (ns, not significant). (B) Normalized temporal expression (antilog) of Myd88 , p50 ( Nfkb1 ), p65 ( Rela ), Trif ( Ticam1 ), Rig-I ( Ddx58 ), Mda-5 ( Ifih1 ), Ips-1 ( Mavs ), Sting ( Tmem173 ), and cGas ( Mb21d1 ) in poly(I:C) treated wild-type and Ifnb - / - BMDM. The expression was measured, as in (A) , over the first 24 h using microarray and compared to timepoint 0. The expression levels between timepoints 0–10 h post-streatment were smoothened and fitted to a linear (+), quadatic (#), or cubic ( ∗ ) polynomal on time to assess significance ( p -values). +/#/∗ p < 0.05, ++/##/∗∗ p < 0.01, and +++/###/∗∗∗ p < 0.001 were considered to be significant (ns, not significant).

Article Snippet: Cells infected with MCMV or treated with poly(I:C) were harvested at 0, 2, 4, 6, 8, 10, and 24 h post-treatment for RNA isolation and microarray analysis (Affymetrix Mouse Gene 1.0ST microarray).

Techniques: Expressing, Infection, Microarray