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. 2020 Dec 1;34(23-24):1697–1712. doi: 10.1101/gad.340604.120

Figure 1.

Figure 1.

DUSP11 modulates RIG-I signaling sensitivity to liposomal 5′-triphosphate RNAs. (A) Schematic diagram of the 5′-ppp-RNA transfection assay, and immunoblot analysis of A549 parental wild-type (WT) and DUSP11 knockout (KO) cells transduced with pLenti empty vector (vector), DUSP11-3xFLAG (D11), or DUSP11-3xFLAG-catalytic mutant (D11-CM). A549 WT or DUSP11 knockout cells (12-well) were transfected with 5–10 ng of in vitro transcribed 5′-ppp-RNA for 18 h posttransfection. (B) RT-qPCR analysis of IFNB1 and ISG15 mRNA normalized to GAPDH mRNA in 5′-ppp-RNA transfected in WT and DUSP11 KO cells as in (A). Results are presented relative to mock-transfected WT cells. (C) RT-qPCR analysis of ISG15 mRNA normalized to GAPDH mRNA in DUSP11 KO cells stably expressing empty vector, DUSP11, or DUSP11 catalytic mutant as in A, transfected with 5′-ppp-RNA. Results are presented relative to those of DUSP11-expressing cells (+D11). (D) RT-qPCR analysis of IFNB1 and ISG15 mRNA normalized to GAPDH mRNA in DUSP11 knockout cells transfected with 5′-ppp-RNA pretreated with or without calf intestinal phosphatase (CIP) or in vitro-translated DUSP11-core. Results are presented relative to mock-treated DUSP11 knockout cells. (E) RT-qPCR analysis (left) of ISG15 mRNA normalized to GAPDH mRNA in DUSP11 knockout cells transfected with negative control siRNA (siNC) or siRNA targeting RIG-I (siRIG-I) and subsequently transfected with 5′-ppp-RNA, and immunoblot analysis (right) assessing siRIG-I knockdown efficiency of A549 cells transfected with or without 5′ppp-RNA. Data are derived from n = 3 independent replicates in B–E and are presented as mean ± SEM.