Skip to main content
. 2011 Jan 11;108(5):1897–1902. doi: 10.1073/pnas.1019059108

Fig. 3.

Fig. 3.

RIG-I induction triggers ISG production and STAT1 activation in U937 cells independent of IPS-1. (A and C) U937/vector and U937/RIG-I cells were incubated in the Tc− medium for the different days as indicated. (A) Relative mRNA levels of RIG-I, STAT1, and other ISGs, including TRAIL, KLF4, OAS1, and PKR, were determined by real-time PCR assays (n = 3, mean ± SD; *P < 0.05). The total and phosphorylated levels of STAT1 (B) or those of STAT3 and STAT5 (C) were examined by Western blotting analyses. (D) GAS- or ISRE-coupled luciferase reporter plasmid was transfected into U937/vector or U937/RIG-I cells at day 6 of culture in Tc− medium. The relative luciferase activities of each group were expressed as mean ± SD (n = 3, mean ± SD; *P < 0.05). U937/vector and U937/RIG-I were measured by real-time PCR assays for IFN-β mRNA levels (E) or by ELISA for IFN-β protein levels in the culture supernatant (F) at day 6 of culture in Tc− medium. IFN-β mRNA or protein induction levels in the 293T cells transfected with 1 μg/mL Poly(I:C)[poly(rI):poly(rC)] were measured as the positive control. (G) Two knockdown plasmids specific for IPS-1 (IPS-1sh1 and IPS-1sh2) and one negative control plasmid (NC) were separately transduced into U937/RIG-I cells at day 2 of tetracycline withdrawal. Cells were cultured in the Tc− medium for another 4 d before protein extracts were collected for Western blotting analysis.