The formation of ATG16L1-homodimers and ATG16L1-ATG5-heterodimers is not affected by the *300A/T polymorphism.

<p>(A) HEK293T cells were co-transfected with FLAG and MYC-tagged ATG16L1*300A or *300T, as indicated. FLAG-tagged proteins were immunoprecipitated using anti-FLAG antibody. Both the inputs of MYC-ATG16L1 and co-precipitated MYC-ATG16L1 are shown. (B) HEK293T cells were co-transfected with FLAG-tagged ATG16L1*300A or *300T and HA-tagged ATG5, respectively, as indicated. Shown are inputs, as well as immunoprecipitated FLAG-ATG16L1 and co-precipitated monomeric HA-ATG5. Under the conditions used, only the monomeric (not conjugated to ATG12) form of ATG5 was expressed in significant amounts.</p

Dose-dependent HSV-GFP infectivity in untreated and IFNγ-treated RAW 264.7 cells.

<p><b>(A)</b> RAW 264.7 cells were seeded in 96-well glass-bottom plates at a density of 0.3×10⁵ cells/well and either left untreated or treated with IFN-γ (10 ng/ml) overnight before HSV-GFP infection at the indicated dilution of viral stock. Note that when the viral stock was diluted 1 to 10, the MOI was 0.5. The average percentage of GFP-positive cells was calculated from six individual images per treatment condition. Data shown are representative of 3 independent experiments. ***<i>P</i><0.001; **<i>P</i><0.01; *<i>P</i><0.05 by two-way ANOVA with Bonferroni post-test. <b>(B)</b> Representative images of HSV-GFP infection at 1∶25 dilution of the viral stock. Nuclei stained with Hoechst are shown in blue and HSV-GFP infected cells are shown in green.</p

IL-27 functions cooperatively with IFN-γ to suppress the infection of HSV-GFP.

<p><b>(A)</b> Cells were seeded in 96-well glass-bottom plates and either left untreated or treated with recombinant IL-27 alone (50 ng/ml), IFN-γ alone (10 ng/ml) or IL-27 + IFN-γ for 16 h. After infecting cells with HSV-GFP overnight, cells were fixed, stained with nuclear acid stain and subjected to imaging. Percentage of GFP-positive cells in each treatment group was calculated and graphed. Data shown are results from 4 independent experiments. <b>(B)</b> NF-κB/IRF and IRF reporter cells were seeded and treated as in (A). After stimulating with IL-27 and/or IFN-γ for 16 h, cells were lysed and luciferase activities measured. The reporter luciferase activity in untreated cells is normalized to 1. Data shown are results from 3 independent experiments. <b>(C)</b> Cells were seeded and treated as in (A). After infecting cells with HSV-GFP for 6 h, RNA was isolated and qRT-PCR was performed to examine the relative expression levels of downstream effector genes. All expression levels are relative to the level of untreated control and <i>Gapdh</i> was used as internal control for normalization. Data shown are results from 3 independent experiments. ns, not significant; ***<i>P</i><0.001; *<i>P</i><0.05 by one-way ANOVA with Bonferroni post-test.</p

Induction of candidate gene expression by IFN-γ and HSV-GFP infection.

<p>Cells were seeded and either left untreated or treated with IFN-γ (10 ng/ml) for 16 h. HSV-GFP (MOI = 0.5) was used to infect the cells as indicated above. qRT-PCR was performed to measure the relative expression of all candidate genes relative to the untreated control. <i>Gapdh</i> was used as internal control for normalization. Data shown are results from 3 independent experiments. ***<i>P</i><0.001 compared to untreated control by one-way ANOVA with Dunnett's post-test.</p

The relative expression level of candidate genes is correlated with observed antiviral phenotype.

<p>Upper panels: mRNA expression of <i>Sult1a1</i><b>(A)</b> and <i>Il27</i><b>(B)</b> in irrelevant shRNA-transduced cells compared to shRNAs against the indicated gene measured by qRT-PCR. <i>Gapdh</i> was used as internal control for normalization. The results shown are the average from two independent experiments. Lower panels: percentages of GFP-positive cells after HSV-GFP infection in cells previously transduced with irrelevant shRNA (lacZ and luciferase) or targeting shRNA for <i>Sult1a1</i><b>(A)</b> or <i>Il27</i><b>(B)</b>. Data shown are representative of 3 independent experiments. ns, not significant; ***<i>P</i><0.001 compared to negative control by one-way ANOVA with Dunnett's post-test.</p

A Common Mechanism Links Activities of Butyrate in the Colon

Two biological activities of butyrate in the colon (suppression of proliferation of colonic epithelial stem cells and inflammation) correlate with inhibition of the activity of histone deacetylases. Cellular and biochemical studies of molecules similar in structure to butyrate, but different in molecular details (functional groups, chain-length, deuteration, oxidation level, fluorination, or degree of unsaturation), demonstrated that these activities were sensitive to molecular structure, and were compatible with the hypothesis that butyrate acts by binding to the Zn²⁺ in the catalytic site of histone deacetylases. Structure–activity relationships drawn from a set of 36 compounds offer a starting point for the design of new compounds targeting the inhibition of histone deacetylases. The observation that butyrate was more potent than other short-chain fatty acids is compatible with the hypothesis that crypts evolved (at least in part), to separate stem cells at the base of crypts from butyrate produced by commensal bacteria

Tagap deficiency results in increased susceptibility to HSV-GFP infection in macrophages.

<p>(<b>A and B</b>) BMDMs derived from WT or <i>Tagap^−/−</i> mice were infected with HSV-GFP at a MOI of 0.2 (HSV_low) or 0.5 (HSV_high). After 12 h, cells were harvested and stained for GFP. Experiments were performed with a total of 4 mice per genotype. Representative flow plots are shown in (B). ns, not significant; *<i>P</i> = .0463 for HSV_low; *<i>P</i> = 0.0370 for HSV_high. <b>(C)</b> BMDMs were infected with HSV-GFP at a MOI of 0.5. After 10 h, relative mRNA levels of three viral genes (<i>ICP4</i>, <i>ICP27</i>, and <i>UL39</i>) were measured in WT versus <i>Tagap^−/−</i> BMDMs. <i>Gapdh</i> was used as internal control for normalization. n = 2 with a total of 4 mice per genotype. **<i>P</i> = 0.0032 for ICP4; **<i>P</i> = 0.0013 for ICP27; **<i>P</i> = 0.0014 for UL39. <b>(D)</b> BMDMs were infected with HSV-GFP at a MOI of 0.5. After 16 h, DNA was isolated and the relative DNA content of ICP27 in WT versus <i>Tagap^−/−</i> BMDMs was measured. Rplp0 DNA content was used as internal control for normalization. n = 2 with a total of 4 mice per genotype. ***<i>P</i><0.0001. <b>(E)</b> BMDMs were infected with HSV-GFP at a MOI of 0.5. After 24 h, Western blotting was performed using antibodies specific to viral proteins ICP4 and ICP27. β-actin was used as loading control. Representative image from two independent experiments is shown. <b>(F)</b> BMDMs were infected as in C. After 3 or 6 h, RNA was isolated to measure the relative expression of pro-inflammatory cytokines or chemokines, respectively. n = 2 with a total of 4 mice per genotype. ***<i>P</i> = 0.0006 for Ifna; **<i>P</i> = 0.0052 for Ifnb1; **<i>P</i> = 0.0063 for Tnfa; *<i>P</i> = 0.0123 for Il6; **<i>P</i> = 0.0014 for Ccl3; **<i>P</i> = 0.0011 for Ccl5; **<i>P</i> = 0.0052 for Cxcl10. <b>(G)</b> BMDMs were infected as in (A). After 12 h, culture supernatant was collected and ELISA was performed to measure the amount of secreted IFN-β. n = 3 with a total of 6 mice per genotype. ns, not significant; **<i>P</i> = 0.0094 for HSV_low; ***<i>P</i> = 0.0003 for HSV_high. All statistical comparisons were performed by unpaired two-tailed t-tests.</p