Fig. 6. Co-condensation of TRIM25 and G3BP1 activates the RIG-I signaling pathway.
a HEK293T cells were transfected with HA-Ub, Flag-RIG-I plasmids, together with mCherry-TRIM25 (mCh-TRIM25) and its indicated mutant plasmids. After poly(I:C) treatment, anti-Flag immunoprecipitates were analyzed by immunoblot with HA, Flag, and mCherry antibodies. b HEK293T WT or G3BP1 KO cells were transfected with the indicated plasmids and TRIM25 plasmid with and without poly(I:C) treatment. Anti-Flag immunoprecipitates were analyzed by immunoblot. c HEK293T cells were transfected with plasmids encoding TRIM25 WT and its mutants, then treated with poly(I:C) for 12 h. The p-IRF3 was then measured. d HEK293T WT or G3BP1 KO cells were transfected with the indicated vector and TRIM25 plasmids, the levels of p-IRF3 were analyzed by immunoblot. e, f WT or G3BP1 KO HEK293T cells were first transfected with TRIM25 WT or ∆PTFG, then treated with or without poly(I:C). The mRNA levels of IFNβ (e) and ISG56 (f) were determined using qPCR. Poly(I:C) (−) vs. Poly(I:C) (+): p value: <0.001 (e, f), Vector (+) vs. mCh-TRIM25 (+): p value: <0.0001 (e, f), V2 (+) vs. G3BP1 KO (+): p value: <0.0001 (e, f), V2-mCh-TRIM25 (+) vs. G3BP1 KO-mCh-TRIM25 (+): p value: <0.0001 (e, f), G3BP1 KO-V2 (+) vs. G3BP1 KO-mCh-TRIM25 (+): p value: 2.96e−003 (e); 0.4607 (f). g–j HEK293T cells were transfected with TRIM25 WT, TRIM25 PTFGAAAA or ∆PTFG, then treated with poly(I:C) for 12 h. The mRNA levels of IFNα (g), IFNβ (h), IFNγ (i), and ISG56 (j) were determined using qPCR. Poly(I:C) (−) vs. Poly(I:C) (+): p value: 8.57e−004 (g); <0.001 (h, j); 1.02e−003 (i), Vector (+) vs. WT (+): p value: <0.0001 (g–j), WT (+) vs. PTFGAAAA (+): p value: <0.0001 (g–j), WT (+) vs. ∆PTFG (+): p value: <0.0001 (g, h, j); 2.97e−004 (i). All data are representative of at least three independent experiments (a–j). Mean ± s.d., statistical analysis was performed using one-way ANOVA (c, e–j).