Extended Data Fig. 4: Role of histone H3.3 in endothelial cells and PKN1-mediated H3.3 phosphorylation.
a, HUAECs were transfected with control siRNA or different siRNA against PKN1 and were exposed to disturbed flow (DF) for 30 min. Histone H3.3 phosphorylation, H3K27 acetylation or H3K36 trimethylation was determined by immunoblotting. b-d, HUAECs were transfected with control siRNA (Con.) or siRNA directed against histone H3.3, and cells were infected with control (non-transducing) lentivirus (Con.) or lentivirus transducing wild-type (WT) H3.3 or the indicated mutants of H3.3. Shown is the relative H3.3 mRNA expression (b), (n = 5 independent experiments), the protein level determined by immunoblotting using an antibody directed against histone H3.3 (c) (n = 3 independent experiments) and the analysis of phosphorylation of histone H3 at the indicated phosphorylation sites using phosphosite-specific antibodies after exposure of HUAECs to disturbed flow for 60 min (d) (n = 3 independent experiments). e, Wild-type (WT) human H3.3 or the indicated mutants of H3.3 were expressed by lentiviral transduction after siRNA-mediated H3.3 knock-down in HUAECs, and cells were kept under static conditions or were exposed to disturbed flow (DF) for 3 h. Thereafter, ICAM1 and CCL2 expression was determined by qRT-PCR (n = 5 independent experiments). f, HUAECs were exposed to disturbed flow (4 dynes/cm2) for 24 hours, and phosphorylation of histone H3.3 at serine 31 was determined by immunoblotting (n = 4 independent experiments). The control immunoblot for GAPDH is from the same experiment as the one shown in Extended Data Fig. 6a. g, Recombinant histone H3.3 or H4 were incubated in kinase buffer without or with recombinant PKN1 and 50 µM [γ-32P]-ATP for 30 min at 30 °C. Thereafter, samples were separated by SDS-PAGE and analyzed by autoradiography and Ponceau staining. h, HUAECs were transfected with control siRNA or siRNA directed against RNAs encoding the kinases CHEK or IKKα (CHEK1 or CHUK, respectively). Thereafter, cells were kept under static conditions (-) or were exposed to disturbed flow (DF) for 60 min. Phosphorylation level of H3.3S31 in lysates was determined by immunoblotting (n = 3 independent experiments). The bar diagrams show the statistical evaluation as well as the knock-down efficiency. Data are shown as mean ± s.e.m.; the p-values are given in the Fig. (1-way ANOVA with Tukey’s post hoc test (b, e), Mann-Whitney two-sided test (f), Kruskal-Wallis test (h)).
