Fig. 1.

Acute treatment with gambogic acid induces a heat shock response (HSR). a Immunoblot analysis of HSF1 and HSP70 expression. U2OS, HeLa, HDF, and RWPE-1 cells were treated with heat shock (HS, 42 °C, 1 h), 1 h HS with 3-h recovery at 37 °C (H+R), and gambogic acid (GB), celastrol (Cel) or 17-AAG (17A) for 4 h. U2OS: 1.25 μM GB and Cel, 0.25 μM 17A; HeLa: 2 μM GB, 1.5 μM 17A; RWPE-1: 1.25 μM GB and Cel, 3 μM 17A; HDF: 2.5 μM GB and Cel, 3 μM 17A. The HSF1 uppershift induced by HS and treatments (labeled with #) correspond to hyperphosphorylated HSF1 forms. β-tubulin was used as a loading control. The amount of relative HSP70 protein related to β-tubulin was quantified with ImageJ (n=2-4). b Oligonucleotide-mediated pulldown of HSF1 and HSF2 in WT U2OS cells, untreated (C), treated with heat shock 42 °C for 1 h (HS) or with 1.25 μM gambogic acid for 4.5 h (GB). Input indicates total cell lysates from treated cells. β-tubulin serves as a loading control. Single asterisk indicates previously blotted HSF2. c Immunofluorescence staining of endogenous HSF1 in nuclear stress bodies in HeLa cells. GB: 4 μM GB, HS: 2 h heat shock (HS, 42 °C) (n=3). Scale bar: 20 μm. Mean ± SEM shown