Fig 1: DYRK2 phosphorylates HSF1.A 293T cells were transiently transfected to express GFP-tagged DYRK2 wild-type (WT) or a kinase dead (KD) version. After 48 h, cells were lysed and the levels of endogenous HSF1 and phospho-HSF1 were analysed as indicated. B 293T cells were transiently transfected with a GFP-tagged DYRK2 analogue sensitive (AS) version. After 48 h, cells were treated for a further 3 h with increasing concentrations of three different PP1 inhibitors as indicated. Cells were lysed and the levels of endogenous HSF1 and phospho-HSF1 were analysed by western blot. C Upper panel, purified recombinant His-HSF1 (1 µg) was incubated in kinase buffer with increasing concentrations of recombinant GST-DYRK2 or GST-DYRK2 kinase dead (KD) at 30 °C for 30 min. Lower panel, purified recombinant His-HSF1 (1 µg) was incubated with either 20 ng of GST-DYRK2 WT or KD at 30 °C for various times as indicated. The reactions were terminated by the addition of SDS gel loading buffer, the proteins were resolved by SDS-PAGE, and the levels of phosphorylated HSF1 were analysed. D MDA-MB-468 cells were treated with either DMSO, the p38 inhibitor SB202190 (10 µM), the mTOR inhibitor rapamycin (30 nM) or harmine (10 µM). After 1 h, cells were incubated at 42 °C. After 1 h, cells were lysed in SDS buffer and the levels of the indicated proteins were analysed. Upper panel is a representative western blot and the bottom panel shows the quantification of the ratio between the phospho-HSF1 and total HSF1 levels. Data represent means ± SD (n = 3) and are expressed relative to the DMSO treated samples. See also Fig. S1.
Fig 2: Ex vivo heat shock response in middle age compared to old Ames dwarf mice. a A representative EMSA of HSF1–HSE binding activity is shown for middle aged (12M) and old aged (24M) mouse liver that were either extracted freshly or cultivated ex vivo at 37 °C or 43 °C for 60 min. A bar graph shows HSF1–HSE binding levels from 12M and 24M liver samples taken from freshly sacrificed mice (fresh) and ex vivo liver samples incubated at 37 °C and 43 °C in oxygenated normal saline. Both 12M and 24M ex vivo liver samples display diminished HSF1–HSE binding at the recovery temperature of 37 °C when compared to freshly isolated liver, whereas heat-shocked ex vivo samples show mildly inducible HSF1–DNA binding only in 12M but not 24M samples. The heat inducible HSF1-HSE binding in ex vivo 12M samples was lower than freshly isolated liver tissue from both 12M and 24M old mice. Statistical analysis showed 12M fresh versus 24M freshly isolate liver with a p value = 0.9999; 12M freshly isolate liver versus 12M 37 °C ex vivo samples with a p value = 0.1889; 12M freshly isolated liver versus 24M ex vivo liver at 37 °C with a p value = 0.8276; 24M freshly isolated liver versus ex vivo liver at 43 °C with a p value = 0.4034. Thus, a 1-h “recovery period” of liver samples at 37 °C only partially attenuated HSF1 into its non-DNA binding state, and there was nominally inducible HSF1 DNA binding activity in the 12M but not 24M samples. b Western blot and densitometry graph analyses of HSF1 protein levels from 12M and 24M old dwarf mouse liver compares freshly isolated liver with ex vivo liver samples held at 37 °C or 43 °C for 1 h. A bar graph of the western blot analyses of HSF1 protein levels from ex vivo liver samples normalized against GADPH protein levels demonstrates a 2.5-fold increase in HSF1 protein levels when compared to freshly isolated liver samples. No age-dependent changes are noted in the ex vivo accumulation of HSF1 protein levels. All p values are < .05 with p = 0.0274 for 12M and p = 0.0045 for 24M when ex vivo liver samples are compared to freshly isolated liver samples.
Fig 3: TRRAP-TIP60 complex is responsible for histone acetylation at specific residues.a Occupancy of pan-acetylated histone H3 and H4 in HSP72 promoter. ChIP assay was performed using HeLa cells treated as described in Fig. 2f. b Components of the TRRAP-TIP60 complex identified dominantly in heat-shocked cells. Nuclear extracts were prepared from cells overexpressing hTRRAP-3 × FLAG, and proteins co-immunoprecipitated with anti-FLAG were identified by MS. c Interaction between HSF1 and TIP60. Complexes co-immunoprecipitated using anti-HSF1 in nuclear extracts of heat-shocked cells were subjected to immunoblotting. d Occupancy of TIP60 in cells expressing HSF1-S419 mutants. ChIP assay was performed using cells treated as described in a. e Cells, in which components of HAT complexes were knocked down, were treated with heat shock. Levels of HSP72 mRNA were quantified, and relative levels are shown. f Occupancy of active chromatin marks in HSP72 promoter. ChIP assay was performed using untreated (Cont.) or heat-shocked (HS) cells, in which HSF1, TRRAP, TIP60, or p300 were knocked down. Norminal p-values were determined by one-way ANOVA, followed by Tukey-Kramer test in a and d–f. Error bars indicate SEM (n = 3) in a and d–f. Experiments were repeated two times for c.
Fig 4: Effect of Nar on HG-reduced HSP70 levels. HUVECs were exposed to NG (5.5 mM glucose) or HG (30 mM glucose) media with or without the 50 mM of Nar for 36 h. (a). Effect of Nar on iHSP70 levels and HSF1 phosphorylation. (b) Quantification of iHSP70 levels in (a). (c) Quantification of HSF1 phosphorylation in (a). (d) Effect of Nar on HSP70 concentration in the culture media. (e) Effect Nar on Hspa1a mRNA levels. (f) Effect of Nar on Hspa1a promotor activity. Data are expressed as means ± SD (n = 3). **P < 0.01 and ***P < 0.001 versus control (NG) group. ##P < 0.01 and ###P < 0.001 versus high-glucose (HG) group.
Fig 5: 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.
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