In vitro activity: Apoptozole induces caspase dependent apoptosis by blocking interaction of HSP70 with APAF-1, without affecting interactions of HSP70 with ASK1, JNK, BAX, and AIF. However, apoptozole may form aggregates under aqueous conditions that could interact with HSP70 proteins in a non-specific manner, potentially leading to false positives and inconsistent data

Kinase Assay: Stock solutions of malachite green (0.081% w/v), polyvinyl alcohol (2.3% w/v), and ammonium heptamolybdate tetrahydrate (5.7% w/v in 6 M HCl) are prepared and stored at 4°C. Three solutions are mixed with water in the ratio of 2 : 1 : 1 : 2 to prepare the malachite green reagent. For the determination of the ATPase activity of Hsc70, a master mixture of an ATPase domain of Hsc70 is prepared in assay buffer (100 mM Tris-HCl, 20 mM KCl, and 6 mM MgCl2, pH 7.4) as the final concentration of 1 mM. An aliquot (10 mL) of this mixture is added into each well of a 96-well plate. To this solution is added each compound (including Apoptozole) in assay buffer, and the plate is incubated for 30 min at room temperature. To start the reaction, 1 mL of 4 mM ATP is added to the solution. The final concentrations are 1 mM protein and 200 mM ATP in 20 mL of assay buffer. After 3 h incubation at 37°C, 80 mL of the malachite green reagent is added into each well. The samples are mixed thoroughly and incubated at 37°C for 15 min, and 10 mL of 34% sodium citrate is added to stop the nonenzymatic hydrolysis of ATP. The absorbance is determined at 620 nm on a SpectraMax 340 PC 384.

Cell Assay:Several cancer cell lines (A549, lung adenocarcinoma epithelial cells; HeLa, cervical cancer cells; MDA-MB-231, breast cancer cells; HepG2, liver cancer cells) are treated with various concentrations (0-15 μM) of Az or compound 7 as a negative control for 18 hr. Cell viabilities are then determined using an MTT assay.