1. DDX6 Represses Aberrant Activation of Interferon-Stimulated Genes
Jennifer H. Lumb, Qin Li, Lauren M. Popov, Siyuan Ding, Marie T. Keith, Bryan D. Merrill, Harry B. Greenberg, Jin Billy Li, Jan E. Carette 
Cell Reports 
Volume 20, Issue 4, Pages (July 2017)
DOI: /j.celrep
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2. Cell Reports 2017 20, 819-831DOI: (10.1016/j.celrep.2017.06.085)
Copyright © 2017 The Author(s) Terms and Conditions

3. Figure 1
Unbiased Haploid Screen Identifies Genes that Prevent Aberrant Activation of IFN-Stimulated Genes
(A) Schematic of the genome-wide screening approach.
(B) Screen results for genes that prevent activation of ISGs in the absence of IFN. The y axis represents significance of enrichment of gene trap insertions in genes in selected cells compared with unselected HAP1 cells (Fisher’s exact test corrected for false discovery rate). Each circle represents a specific gene. The top 15 significant genes are colored blue. Genes with −log10 p > 1 are shown. Genes in bold were chosen for further study.
(C) IFIT2 qPCR of USP14KO and DDX6KO HAP1 cells. Two independently generated knockout clonal cell lines for each genotype are shown by (1) and (2). Expression is relative to parental HAP1 cells shown as a black dotted line.
Data are mean ± SEM of three separate RNA extractions. NGS, next generation signaling.
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4. Figure 2
DDX6 Regulates ISGs and Adaptive Immune Genes Independently of IFN
(A) Global gene expression in WT and DDX6KO HAP1 cells. Dark gray dots are differentially expressed genes (DEGs). Light gray dots are non-DEGs. The two most significantly enriched GO terms in upregulated DEGs are GO: (orange circles) and GO: (blue circles). Red dot, DDX6.
(B) qPCR analyses of IFIT1, IFITM1, ISG15, and OAS1 in WT and DDX6KO HAP1 cells. Each circle represents a unique clonal cell line as determined by genotyping (DDX6KO1-4; Table 1). Data are mean ± SEM of the clonal cell lines (n = 4).
(C) qPCR analyses of IFITM1 and OAS1 in NHDF cells. Two unique DDX6KO NHDF clones are depicted by (1) and (2). WT is shown as a black dotted line. Data are mean ± SEM of four independent RNA extractions.
(D) OAS1 qPCR in WT, DDX6KO, or DDX6KO complemented with DDX6 (+DDX6) or vector control expressing Cherry (+Cherry) ± doxycycline (Dox) treatment. Minus signs (−) denote uncomplemented DDX6KO cells. Data are mean ± SEM of three separate RNA extractions.
(E) Cytokine secretion in WT (black dotted line) and two independently generated DDX6KO cell lines, DDX6KO(1) and DDX6KO(2). Cytokines with a fold change >2 are displayed except for IFNA, IFNB, and IFNG, which were unchanged between WT and DDX6KO HAP1 cells. Data are mean ± SEM of three independent experiments.
(F) VCAM1 surface expression in WT and DDX6KO(1) and DDX6KO(2) HAP1 clones.
∗p < 0.05, ∗∗p < 0.01, ∗∗∗p <
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5. Figure 3
ISG Activation Occurs Independently of STAT1 and Is Cell Intrinsic
(A) Protein expression of STAT1 and DDX6 in three unique clonal DDX6_STAT1KO cell lines. GAPDH was used as a loading control.
(B) qPCR of ISG15, IFITM1, and VCAM1 in WT, DDX6KO, and DDX6_STAT1KO HAP1 cells. Each circle represents a unique clonal cell line [DDX6_STAT1KO(1-3); Table 1]. Data are mean ± SEM of the clones (n = 3).
(C) Schematic depicting the co-culture of WT and DDX6KO cells. WT+ refers to WT cells that have been grown in the presence of DDX6KO cells.
(D–F) qPCR of IFITM1 (D), OAS1 (E), and VCAM1 (F) in WT, DDX6KO, and WT cells grown in the presence of DDX6KO cells (WT+). Data are mean ± SEM of three independent experiments.
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6. Figure 4
DDX6 Deficiency Primes the IFN System for Enhanced Antiviral Response
(A) Total STAT1 and phosphorylation of STAT1 (P-STAT1 Tyr701) in WT and DDX6KO HAP1 cells ± IFNλ2. GAPDH served as a loading control. Two unique clones were included for WT and DDX6KO cells, denoted by (1) and (2).
(B) VEEV-GFP infection of HuH7 cells. WT, DDX6KO, and DDX6KO complemented with DDX6 or a vector control expressing Cherry were treated with the indicated amounts of IFN overnight and then infected with VEEV-GFP.
(C) DENV-GFP infection of WT or DDX6KO HuH7 cells pre-treated with IFN overnight.
(D) VSV-GFP infection of WT or DDX6KO HuH7 cells pre-treated with IFN overnight.
Data are mean ± SEM of three independent infections.
Cell Reports  , DOI: ( /j.celrep )
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7. Figure 5
VCAM1 Upregulation Is Dependent on MDA5 and Can Be Uncoupled from ISG Upregulation
(A) OAS1 qPCR in WT, DDX6KO, and DDX6KO with an additional knockout of MAVS (DDX6_MAVSKO), MDA5 (DDX6_MDA5KO), RIG-I (DDX6_RIG-IKO), and PKR (DDX6_PKRKO) HAP1 cells. Two unique clones for each genotype are denoted by (1) and (2). Data are mean ± SEM of three independent RNA extractions.
(B) qPCR of VCAM1 in WT, DDX6KO and DDX6_MAVSKO, MDA5KO, DDX6_MDA5KO, DDX6_RIG-IKO, and DDX6_PKRKO HAP1 cells. Independent knockouts are denoted by (1) and (2). Data are mean ± SEM of three independent RNA extractions.
(C) VCAM1 surface expression in WT, DDX6KO, MDA5KO, and two independent DDX6_MDA5KO HAP1 cells.
(D) qPCR of OAS1 in WT, MAVSKO, MDA5KO, and RIG-IKO HAP1 cells infected with influenza virus (INFVPR8) and Sendai virus (SENV). Data are mean ± SEM of three independent RNA extractions. The experiment was performed three times and a representative is shown.
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8. Figure 6
P-Body Disruption Does Not Cause ISG Upregulation, and DDX6 Represses ISG Activation via LSM1
(A) Localization of LSM1 and DCP1A in WT, DDX6KO and LSM1KO HAP1 cells. Scale bar, 5 μm.
(B) qPCR of OAS1 in WT, DDX6KO, and LSM1KO. Two unique clones for each genotype are shown by (1) and (2). Data are mean ± SEM of three independent RNA extractions.
(C) Protein expression of DDX6 and LSM1 in WT, DDX6KO, and LSM1KO cells. For each genotype, two unique clonal cell lines are shown by (1) and (2).
(D) Localization of DDX6 in WT or LSM1KO HAP1 cells. Scale bar, 10 μm.
(E) qPCR analysis of OAS1 in WT cells and DDX6KO cells complemented with WT DDX6 (+DDX6WT), ATPase-deficient DDX6 (+DDX6E247Q), or a vector control expressing Cherry (+Cherry). Minus signs (−) denote uncomplemented DDX6KO cells. Data are mean ± SEM of three independent RNA extractions.
(F) qPCR of OAS1 in DDX6KO and two unique DDX6 LSM1 double-knockout (DDX6_LSM1KO) clones denoted by (1) and (2) complemented with LSM1 or vector control expressing RFP. Data are mean ± SEM of n = 18 (DDX6KO) or n = 9 (all other cell lines) from three independent experiments.
(G) Summary of genetic relationships discovered in this study.
Dox, doxycycline.
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