Fig. 7. HBO1 stabilized by LPS regulated inflammatory gene transcription.

(A, B) Scrambled-DsiRNA or HBO1-DsiRNA were introduced into THP-1 cells for 72 h. The cells were treated with LPS (125 ng/mL) for 6 h. Efficacy of HBO1 knockdown was determined using immunoblotting (A). Total RNA was extracted and the mRNA levels of IL-1β, IL-6 and IL-10 were determined (B). (C, D) Scrambled-DsiRNA or USP25-DsiRNA were introduced into THP-1 cells for 72 h. The cells were treated with LPS (125 ng/mL) for 6 h. Efficacy of USP25 knockdown was determined using immunoblotting (C). Total RNA was extracted and the mRNA levels of IL-1β, IL-6 and IL-10 were determined (D). (E, F) Scrambled-DsiRNA, pCMV6-XL5/HBO1 plasmid or USP25-DsiRNA were delivered into THP-1 cells using electroporation as indicated. After 72 h, the cells were treated with LPS (125 ng/mL) for 6 h. Efficacy of ectopic HBO1 expression and USP25 knockdown were analyzed using immunoblotting (E). Total RNA was extracted and the mRNA levels of IL-1β, IL-6 and IL-10 were determined (F). (G) Scrambled-DsiRNA or USP25-DsiRNA were introduced into THP-1 cells for 72 h. The cells were treated with LPS (125 ng/mL) for 6 h. Cell lysates were subjected to USP25, TLR4, NF-κB, MyD88, and β-actin immunoblotting. The densitometry results were plotted in right panels. (H) THP-1 cells were introduced with scrambled-DsiRNA, HBO1-DsiRNA or USP25-DsiRNA for 72h. Cell lysates were subjected to USP25, HBO1, β-actin, acH3K14, histone H3 immunoblotting. The densitometry results were plotted in right panel. (I) THP-1 cells were treated with LPS (125 ng/mL) for 6 h. The cells were subjected to ChIP-qPCR, qPCR signals were normalized to that of IgG. Data are representative of three independent experiments. Graphs show mean ± SD, and “*” denotes p<0.05.