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. Author manuscript; available in PMC: 2015 Jun 19.
Published in final edited form as: Immunity. 2014 Jun 12;40(6):936–948. doi: 10.1016/j.immuni.2014.05.007

Fig. 5. Loss of human OASL and mouse Oasl2 reduces RIG-I signaling and enhances virus replication.

Fig. 5

(A–C) Inhibition of RIG-I signaling in HCT-116 cells following OASL silencing. HCT-116 cells stably expressing OASL shRNA (two different shRNA, sh1-OASL and sh2-OASL targeting two different regions of OASL mRNA) or control vector stimulated with SeV as indicated for 24h followed by IB with indicated antibodies (A). Indicated cells were stimulated with SeV as in (A), and the cellular RNA were analyzed for IFNα mRNA by qRT-PCR (B). Same HCT-116 OASL shRNA and control cells were transfected with LMW for 24 h followed by IB with the indicated antibodies (C). (D–E) Reduction of RIG-I-mediated ISG induction in HCT-116-OASL−/− cells. OASL−/− HCT-116 and wild type HCT-116 cells were either infected with SeV (D) or transfected with LMW (E) as indicated, followed by IB for ISG60, OASL and actin antibodies respectively. (F) ISG induction in OASL and RIG-I targeted 293T cells. Wild type 293T, 293T-OASL−/− and 293T-DDX58−/− cells were transfected with LMW for 24 h, followed by IB for the indicated proteins. (G) Effect of OASL loss on RIG-I-mediated induction of NF-κB target genes. Cells were treated similarly as in (F) followed by qRT-PCR analysis of indicated NF-κB-regulated genes. (H–I) OASL silencing reduces ISG60 induction by RIG-I signaling in primary human keratinocytes. Primary keratinocytes were transfected either with OASL or control siRNA for 48 h, followed by either transfection with LMW or infection with SeV at the indicated doses for another 24 h and analysis by IB. (J–K) Downregulation of RIG-I-mediated Ifnb1 (IFNβ) and Ifit1 (ISG56) mRNA induction in Oasl2−/− macrophages. BMDM from wild type (Wt) or Oasl2−/− mice were infected with SeV for 8 h followed by detection of Ifnb1 (J) and Ifit1 (K) mRNA by qRT-PCR. (L) Secreted IFNβ was measured in the supernatants from SeV infected (8h) Wt (●) and Oasl2−/− (○) BMDM using the mouse IFNβ ELISA kit. (M) Increased VSV replication in BMDM from Oasl2−/− mice. BMDM from Wt or Oasl2−/− mice were infected with VSV for 24 h followed by quantitation of VSV RNA by qRT-PCR.