HSF1A is a cell-permeable activator of HSF1 that protects mammalian cells against stress-induced apoptosis.

HSF1A是一种小分子HSF1激活剂，能保护细胞免受应激诱导的凋亡，抑制TRiC活性，但不扰乱ATP水解。TRiC复合体的遗传失活或耗尽会导致人类HSF1激活，而HSF1A可抑制纯化的TRiC与HSF1之间的直接相互作用。此外，使用与HSF1A偶联的FITC进行的荧光偏振实验显示，HSF1A-FITC以约600 nM的亲和力结合到TRiC的纯化Tcp1亚基。通过将纯化的Tcp1滴定到含有500 nM生物素或HSF1A-生物素的结合反应中，这一点得到定性验证。通过统计含有聚集体的细胞数占总细胞数的比例进行量化，显示在HSF1A浓度低至2 μM时，观察到含聚集体细胞的数量减少。预处理12 μM HSF1A的细胞，约20％显示出通过荧光显微镜可见的聚集体，且随剂量增加细胞中含聚集体的比例持续减少。

HSF1A通过增强HSF1活性、稳定HSF1表达并减轻多柔比星(DOX)引起的心脏损伤效果显著。在多柔比星（累积剂量：30mg/kgw）和多柔比星联合HSF1A（100mg/kgw/天）挑战的WKY大鼠中，补充HSF1A能显著提升心脏功能至对照组水平。研究表明，HSF1A可促进人HSF1向核内转移，提高蛋白质伴侣表达量，改善蛋白质错误折叠及细胞死亡在神经退行性疾病模型中的情况。超声心动图结果显示，HSF1A亦能缓解DOX引起的心脏功能障碍。

Protein extracts are generated from mammalian, yeast and E. coli cultures using biotin-binding buffer (20 mM HEPES, 5 mM MgCl2, 1 mM EDTA, 100 mM KCl, 0.03% NP-40) supplemented with 1% Trition-X100 and protease inhibitors. Approximately 0.5 mg of protein extract is incubated with 100 μM HSF1A-Biotin for 4 h at 4°C and HSF1A-Biotin associated proteins captured by with NeutrAvidin Agarose Resin. After washing in biotin binding buffer proteins are eluted using 50 μL biotin elution buffer (100 mM Tris, 150 mM NaCl, 0.1 mM EDTA, 2 mM D-biotin), resolved on a 4-20% SDS-PAGE, and immunoblotted. For purified TRiC and Hsp70 analyses, 5 nM protein is incubated in biotin-binding buffer+0.5% Triton X-100 with 100 μM biotin or 100 μM HSF1A-Biotin for 4 h at 4°C and captured with NeutrAvidin Resin. For NiNTA purified yeast Tcp1, different concentrations of Tcp1 0.5 μM, 1 mM, 2 mM, 3 mM and 4 mM in 25 mM Hepes pH 7.5, 150 mM NaCl are incubated with 0.5 μM Biotin or HSF1A-Biotin for 4 h at 4°C and captured with NeutrAvidin Resin.

PC12 cells seeded into a 96-well plate (5×104 cells/well) are treated with increasing concentrations of HSF1A (2, 4, 8 and 12 μM) for 15 h, at which time httQ74-GFP expression is stimulated by incubation in the presence of 1 μg/mL Doxycycline for 5 d. Cell viability is assessed via the XTT viability assay.

RatTen-week-old Wistar Kyoto rats (WKY) are used. The rats are housed at a constant temperature (22°C) on a 12-h light/dark cycle with food and tap water. The animals are arranged into three groups: WKY rats (the control group), DOX rats and DOX rats treated with HSF1A. Each group contain five animals. The DOX group is injected with DOX (5?mg/kg) for 6 consecutive weeks intraperitoneal injection to achieve a cumulative dose of 30?mg/kg, which has been well documented to achieve cardiotoxicity. The small molecular HSF1 activator HSF1A (100?mg/kg/day) is injected intraperitoneally.