Fig. 7: HSF1 phosphorylation supports tumor formation.

a Equal amounts of total cell extracts from melanoma cell lines, HeLa cells (control and heat-shocked), and OUMS-36T-3F cells were subjected to HSF1 immunoprecipitation and immunoblotting. Relative levels of HSF1-S419 phosphorylation normalized by HSF1 protein levels are shown. b Phosphorylation of HSF1-S419 is induced by overexpression of PLK1 in OUMS-36T-3F cells. HSF1 immunoprecipitation and immunoblotting were performed as described in Fig. 5a. c, d Endogenous HSF1 was replaced with GFP, hHSF1-HA, S419A-HA, S419G-HA (c, d), or S326A/S419A-HA (d) in melanoma and OUMS-36T-3F cells. Cell extracts were subjected to immunoblotting. e MeWo and OUMS-36T-3F cells were treated as described in d. HSP72 mRNA levels were quantified and shown. f, g Tumor sizes and masses of melanoma cells expressing hHSF1 phosphorylation site mutants in athymic nude mice. The sizes (f) and masses (g) of tumors at indicated time points after injection were calculated until 22 days. Bars in g indicate mean values (n = 8). h Schematic model for establishing an active chromatin state in HSP72 promoter. PLK1 phosphorylates HSF1-S419 (1), which recruits the TRRAP-TIP60 complex. TIP60 and p300 are responsible for H3K18ac and H3K23ac, respectively (2, 3). TRIM33 and TRIM24, recruited to the promoter by interactions with HSF1 and the histone acetylation marks, are required for H2BK120ub (4). Norminal p values were determined by one-way ANOVA, followed by Tukey-Kramer test in a and e or by two-way ANOVA in c, d and f. Error bars indicate SEM (n = 3) in a and e, (n = 4) in c and d, or (n = 8) in f. Experiments were repeated two times for b.