GPATCH3 deficiency potentiated RLR-mediated signaling.

(A) GPATCH3-/- 293T cells were generated by CRISPR-Cas9 technology. Two GPATCH3 knockout clones and one wild-type clone (1 x 10⁶) were analyzed by immunoblotting with the anti-GAPTCH3 antibody. (B) Wild-type or GPATCH3-/- cells (1 x 10⁶) were left uninfected, infected with SeV, VSV (MOI = 0.1) or transfected with poly(I:C) (1 μg/ml). Twelve hours later, total RNAs were extracted and the mRNA level of IFNB was analyzed by qPCR. (C) Wild-type (WT) or GPATCH3-deficient cells (KO 1#&2#) (4 x 10⁵) were transfected with control or GPATCH3 expression plasmids (1 μg). Twenty-four hours after transfection, cells were left uninfected or infected with SeV. Twelve hours later, total RNAs were extracted and the mRNA level of IFNB was analyzed by qPCR. Expression of transfected GPATCH3 was detected by immunoblotting with anti-GPATCH3 antibody. (D) Wild-type (WT) or GPATCH3-deficient cells (KO 1#) (4 x 10⁵) were transfected with empty vector or GPATCH3 expression plasmids (1 μg). Cells were left uninfected or infected with SeV for the indicated times. Immunoblotting was then performed with the indicated antibodies. Graphs show mean ± SD. n = 3. *P<0.05, **P<0.01 (Student’s t-test).