1. Volume 18, Issue 6, Pages 714-722 (December 2015)
microRNA Function Is Limited to Cytokine Control in the Acute Response to Virus Infection 
Lauren C. Aguado, Sonja Schmid, David Sachs, Jaehee V. Shim, Jean K. Lim, Benjamin R. tenOever 
Cell Host & Microbe 
Volume 18, Issue 6, Pages (December 2015)
DOI: /j.chom
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2. Cell Host & Microbe 2015 18, 714-722DOI: (10.1016/j.chom.2015.11.003)
Copyright © 2015 Elsevier Inc. Terms and Conditions

3. Figure 1
VP55-Mediated Degradation of Endogenous miRNAs Modulates Cellular Transcriptome
(A) Small RNA northern blot of 293T cells cotransfected with a plasmid expressing miR-124 and either pEGFP or pEFGP-VP55. Total RNA was collected 24 hr posttransfection.
(B) Gaussia luciferase reporter assay containing two perfect let-7 target sites or a control insert in the 3′UTR cotransfected with either pEGFP or pEGFP-VP55. Targeted constructs were normalized to control in each condition. Average of three independent experiments. Error bars denote ± SD. ∗p < 0.05 from one-tailed Student’s t test.
(C) Small RNA northern blot of total RNA from 293T cells treated with either AdV-GFP or AdV-VP55 for 24 hr.
(D) Small RNA deep sequencing of 293T cells mock, AdV-GFP, or AdV-VP55 treated for 24 hr.
(E) Differentially expressed transcripts from mRNA-Seq of samples in (C) and (D). Graph depicts data from libraries generated from two biological replicates.
Cell Host & Microbe  , DOI: ( /j.chom )
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4. Figure 2
Depletion of miRNAs Does Not Modulate the Intrinsic Response to Virus
(A) qPCR of RNA from BJ cells treated with poly(I:C) for 6 hr. All samples were normalized to tubulin.
(B) Small RNA northern blot of BJ cells treated with AdV-GFP or AdV-VP55 for 24 hr prior to transfection with dsRNA. Total RNA collected 6 hr posttransfection.
(C) Differentially expressed transcripts with a greater than 2-fold induction from mRNA-Seq of samples in (B). Statistical analysis was performed on two biological replicates per condition.
(D) qPCR of RNA from BJ cells treated with IFNβ for 6 hr. All samples were normalized to tubulin.
(E) Small RNA northern blot of BJ cells treated with AdV-GFP or AdV-VP55 for 24 hr alone or prior to treatment with IFNβ for 6 hr.
(F) Differentially expressed transcripts with a greater than 2-fold induction from mRNA-Seq of samples in (E). Analysis was performed on two biological replicates per condition.
(G) qPCR validation of selected genes from (F). All samples were normalized to tubulin. Fold change over samples treated with AdV-GFP alone.
(H) Western blot (left) and quantification (right) of BJ cells treated with AdV-GFP or AdV-VP55 as described in (E). IRF1 levels were normalized to actin. Fold change over AdV-GFP+IFN. Average of two independent experiments.
(I) Gaussia luciferase reporter assay containing either the 3′UTR of IRF1 or the 3′UTR with mutated putative miR-23 target site. NoDice cells were cotransfected with indicated reporter and either a miR-23 or scrambled miRNA mimetic. ∗∗p < from a one-tailed Student’s t test. Representative of three independent experiments. For all graphs, error bars denote ± SD.
Cell Host & Microbe  , DOI: ( /j.chom )
Copyright © 2015 Elsevier Inc. Terms and Conditions

5. Figure 3
Loss of miRNA Silencing Directly Alters Cellular Cytokine Production
(A) Differentially expressed transcripts from mRNA-Seq of BJ cells treated for 24 hr with AdV-GFP or AdV-VP55. Analysis was performed on two biological replicate samples per condition.
(B) Differentially expressed transcripts from mRNA-Seq of BJ cells treated for 9 days with AdV-GFP or AdV-VP55. Analysis was performed on two biological replicate samples per condition.
(C) Heatmap depicting log2 fold induction over AdV-GFP of cytokines differentially expressed in at least one of the indicated time points (in A or B).
(D) qPCR of IL6 expression in BJ fibroblasts treated with AdV-GFP or AdV-VP55 for 24 hr alone or prior to treatment with IFNβ for 6 hr. Graph depicts relative expression over AdV-GFP.
(E) Small RNA northern blot of RNA from 293T cells mock treated or transfected with protected let-7f miRNA mimetic 4 hr prior to treatment with AdV-GFP or AdV-VP55. Total RNA was collected 24 hr posttransfection.
(F) BJ cells transfected with a let-7 mimetic for 1 hr, then infected with indicated AdV. Total RNA was collected 24 hr postinfection and analyzed by qPCR for IL6 expression. All samples were normalized to tubulin. Average of three independent experiments. ∗p < 0.05 from a one-tailed Student’s t test.
(G) Gaussia luciferase reporter assay utilizing either the 3′UTR of IL6 or the 3′UTR with mutated putative let-7 target site. NoDice cells cotransefected with indicated reporter and either a let-7 or scrambled miRNA mimetic. ∗p < 0.05 from a one-tailed Student’s t test. Representative of three independent experiments. For all graphs, error bars denote ± SD.
Cell Host & Microbe  , DOI: ( /j.chom )
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6. Figure 4 Loss of miRNA Silencing Induces Cytokine Expression In Vivo
(A) Mice were mock treated or transduced with 2.5 × 108 pfu of either AdV-GFP or AdV-VP55 for 48 hr. Cytokine concentrations from homogenized whole-lung tissue are indicated. Significance was determined using a one-tailed Student’s t test, ∗p < 0.05 and ∗∗p < n = 5 mice per cohort.
(B) qPCR of RNA from lungs in (A) for indicated genes. All samples normalized to tubulin. Fold change over mock-treated cohort.
(C) Diagram depicting significantly enriched gene ontology categories of the 139 differentially expressed genes from (A) that are induced by 2 or more log2 fold change. Connecting lines denote 25 or more shared genes between ontology categories. Circle size represents the number of genes in each indicated category.
(D) Diagram as described in (C) for the 138 differentially expressed genes decreased by 2 or more log2 fold change.
Cell Host & Microbe  , DOI: ( /j.chom )
Copyright © 2015 Elsevier Inc. Terms and Conditions