TRIM68 knockdown results in an increase in IFN-β and TFG levels post TLR3 stimulation.

<p>(A) HEK293T cells were transfected with 40 ng full-length IFN-β promoter and increasing amounts of TFG as indicated. Cells were assayed for relative fold increase of reporter gene activity 18–24 hr post-transfection. (B) HEK293T cells were transfected with 400 ng full-length IFN-β promoter and were co-transfected with 250 ng of Scrambled (Scr) or TFG shRNA as indicated and 24 hr later 50 ng of TRIF. Cells were assayed for relative fold increase of reporter gene activity 18–24 hr post-transfection. Presented data is graphed from one experiment. (C) HeLa cells were transiently transfected with 1 µg of TFG and 18 hr later were stimulated with 20 µg/ml poly(I:C) for 24 hr before supernatants were collected for RANTES ELISA. (D) HEK293T cells were transfected with 40 ng full-length IFN-β promoter and were co-transfected with 50 ng of TFG, TBK1 or dsRNA as well as with empty vector (EV) control and 50 ng of full length TRIM68 or TRIM68 mutant lacking the RING and B-box domain (TRIM PRY/SPRY) as indicated. Cells were assayed for relative fold increase of reporter gene activity 18–24 hr post-transfection and % inhibition on promoter activity was calculated. Presented data is graphed from the average of three separate experiments ± S.E.M. *p<0.05 was considered significant. (E) A schematic of the IFN-β promoter which consists of three principle transcription factor binding sites, the PRD IV region which binds the transcription factor ATF-2/c-Jun, the PRD III–I region which binds the transcription factors IRF3 and IRF7 and the PRD II region which binds NF-κB. (F) HEK293T cells were transfected with 40 ng full-length IFN-β promoter or the PRD segments (PRD II, PRD III-I, PRD IV) and 50 ng of TFG as well as with EV control and increasing amounts of TRIM68 as indicated. Cells were assayed for relative fold increase of reporter gene activity 18–24 hr post-transfection. (G–I) Human monocytes were treated with PLGA microparticles containing either Scrambled (Scr) or TRIM68 shRNA for 48 hr. Real-time PCR was used to measure TRIM68 mRNA levels post treatment (G). After treatment with PLGA microparticles, monocytes were stimulated with poly(I:C) for 24 hr before IFN-β mRNA was measured (H). Human monocytes which have been treated with shRNA containing microparticles as above were immunoblotted for TFG and alpha actinin. Immunoblots shown are from a single experiment and are representative of three independent experiments (I). In all other cases, presented data is graphed from the average of three separate experiments ± S.E.M. *p<0.05 was considered significant.