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. 2021 Aug 23;22(16):9094. doi: 10.3390/ijms22169094

Figure 4.

Figure 4

TRIM25 and DDX3X cooperatively enhance IFNB1 promoter induction. Activity of firefly luciferase expressed under the control of the IFNB1 promoter (pGL3-IFN-β1) measured after 24 h of RLR signalling cascade activation with RIG-I 2CARD (A,C,E,G,I,J,K) or 18 h of poly(I:C) stimulation (B,D,F,H) in HEK293T cells transfected to express recombinant proteins as indicated. (A,B) Increasing amounts of HA-TRIM25 expression vector or control FLAG vector. (C,D) Increasing amounts of FLAG-DDX3X expression vector or control FLAG vector. (E,F) FLAG-DDX3X and HA-TRIM25. (G,H) FLAG-DDX3X and HA-TRIM25 in cells transfected with pSuper-shTRIM25 (TRIM25 KD) or pSuper-shScrambled (WT). (I) FLAG-tagged WT, K55R or K66R DDX3X and HA-TRIM25. (J) FLAG-tagged WT, K55R or K66R DDX3X and HA-tagged WT or ΔRING TRIM25. (K) FLAG-tagged WT, K55R or K66R DDX3X and HA-tagged WT or ΔPRY-SPRY TRIM25. Firefly luciferase results normalised to the activity of Renilla luciferase internal control. The empty vector pcDNA3.1 FLAG was transfected as the “–” for all data. All results are representative of three independent experiments. Representative anti-FLAG, anti-HA and anti-actin immunoblots are shown. Graphs show the mean ± SD of three replicates. Statistical significance was determined using one-way ANOVA with Tukey’s multiple comparisons test and assessed based on the p value: NS p > 0.05, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, and **** p ≤ 0.0001.