Fig. 2.

Effect of Edwardsiella tarda on RIG-I/MDA5 pathway activation. (A) RAW264.7 cells were infected with or without (control) E. tarda for indicated hours. The cell lysates and supernatants were immunoblotted (IB) with antibodies against the indicated proteins. (B) RAW264.7 cells were treated as above, and the IFN-β mRNA level was determined by qRT-PCR. (C) RAW264.7 cells were treated with live or dead E. tarda for different hours. RIG-I/MDA5 production and FN-β secretion were determined as above. (D) RAW264.7 cells were transfected with different doses of E. tarda RNA for different hours. ISG-Lucia activity was then assessed. (E) RIG-I^-/-, MDA5^-/-, or wild type (WT) RAW264.7 cells were treated with 50 ng E. tarda RNA for 16 h, and ISG-Lucia activity was then determined. (F-G) IFN-β secretion (F) and ISG-Lucia activity (G) in RIG-I^-/-, MDA5^-/-, or wild type (WT) RAW264.7 cells following E. tarda infection were determined. (H) RIG-I^-/-, MDA5^-/-, or WT RAW264.7 cells were incubated with a NO probe for 1 h and then infected with E. tarda. The production of NO was measured at different hours. (I) RIG-I^-/-, MDA5^-/-, or WT RAW264.7 cells were infected with E. tarda for indicated hours, and the intracellular bacterial proliferation rate was determined. For (B, and d-I), values are the means of three experimental replicates and shown as means ± SD. ***p < 0.001; **p < 0.01.