In vitro activity: KRIBB11 inhibits HSF1 activity in a concentration-dependent manner. It down-regulates HSP70 and HSP27. KRIBB11 can inhibit cancer cell proliferation, arrests the cell cycle at G2/M phase and induces apoptosis. But it does not inhibit heat shock-induced recruitment of HSF1 to the hsp70 promoter or phosphorylation of HSF1 Ser-230. KRIBB11 inhibits heat shock-induced recruitment of pTEFb to the hsp70 promoter and p-TEFb-dependent Phosphorylation of polⅡ CTD Ser-2

Kinase Assay: HCT-116 cells are washed with PBS and then homogenized with a 27-gauge syringe in binding buffer (10 mm Tris-HCl (pH 7.4), 50 mm KCl, 5 mm MgCl2, 1 mm EDTA, and 0.1 mm Na3VO4). The cell lysate is centrifuged at 13,000 rpm for 30 min at 4°C, and the supernatant is collected. The HCT-116 cell lysate supernatant is precleared by incubating with Dynabeads M-280 streptavidin for 30 min at 4°C and captured by magnet separation. The cleared supernatants are incubated with biotinyl-KRIBB11 compound. After overnight incubation at 4°C, proteins associated with the biotinyl-KRIBB11 compound are precipitated with Dynabeads M-280 streptavidin. Precipitated samples are separated by a magnet. Samples are washed with 1 mL of ishing buffer containing 50 mm HEPES (pH 7.5), 50 mm NaCl, 1 mm EDTA, 1 mm EGTA, 0.1% Tween 20, 10% (v/v) glycerol, 1 mm NaF, 0.1 mm Na3VO4, and protease inhibitor mixture tablets (1 tablet/10 mL). Samples are boiled in SDS-PAGE sample buffer, separated by 10% polyacrylamide gel, and immunoblotted with antibodies against HSF1, HSF2, HSP90, or CDK9.

Cell Assay:HCT-116 cells are treated with KRIBB11 at various concentrations for 48 h. Cells are then harvested by trypsinization, fixed with 70% chilled ethanol, and preserved at –20 °C before FACS analysis. Fixed cells are washed twice with phosphate-buffered saline (PBS) solution before being suspended in 500 μl of PBS and treated with 100 mg/ml RNase A at 37 °C for 30 min. Propidium iodide is then added to a final concentration of 50 mg/ml for DNA staining, and 20,000 fixed cells are analyzed on a FACSCalibur system. Cell cycle distribution is analyzed using the ModFit program.