Fig. 5: PKN1-induced FOS/FOSB induction through phosphorylation of H3.3S31 involves EP300 and SETD2.
a, HUAECs were transfected with a control siRNA or siRNAs directed against PKN1. Thereafter, cells were exposed to disturbed flow (DF) for the indicated time periods. H3K27 acetylation or H3K36 tri-methylation in lysates was determined by immunoblotting (n = 3 independent experiments). The presented immunoblots are from the same experiment as the blots shown in Fig. 4a and include the same H3.3 loading control. b, Wild-type (WT) or mutant (S31A) human H3.3 was expressed by lentiviral transduction after siRNA-mediated H3.3 knockdown in HUAECs. Thereafter, cells were exposed to DF for 3 h or were kept under static conditions. H3K27 acetylation or H3K36 tri-methylation in lysates was determined by immunoblotting (n = 3 independent experiments). c–g, HUAECs were transfected with a control siRNA or siRNAs directed against PKN1 (c,e), SETD2 (e) or EP300 (g) and were then exposed to DF for 90 min. Alternatively, wild-type (WT) or mutant (S31A) human H3.3 was expressed by lentiviral transduction after siRNA-mediated knockdown of H3.3 (d,f) in HUAECs. ChIP assay was performed to detect the enrichment of H3K27 acetylation (c,d,g) or H3K36 tri-methylation (e,f) in the FOS/FOSB promoter (c,d,g) or gene body (e,f) (n = 3 independent experiments). h,i, HUAECs were transfected with control siRNA or siRNAs directed against EP300 or SETD2 and were exposed to DF for 3 h. DF-induced expression of FOS and FOSB (h) or inflammatory genes (i) was analyzed by qRT–PCR (n = 3 independent experiments). j, Schematic representation of the role of PKN1 in mediating DF-induced inflammatory gene expression in endothelial cells. Data are shown as mean ± s.e.m.; the P values are given in the figure (Kruskal–Wallis test (a–i)). NS, not significant.
