Figure 2

FW-04-806 inhibits Hsp90/Cdc37 chaperone/co-chaperone interactions. (A) FW-04-806 (10, 20, 40 μM) was added into recombinant NBD Hsp90 protein did not affect ATP binding capacity of Hsp90, while 17AAG (0.5, 1 μM) decreased ATP binding. Western blot was analyzed with His-probe antibody. (B) FW-04-806 had little effect on Hsp90 ATPase activity as determined by the malachite green reagent. The assay used 0.5 μM Hsp90 protein, 1 mM ATP, and FW-04-806 or 17AAG at 25, 50, 100, or 200 μM, or vehicle (DMSO) at 620 nm. Results are presented as means ± SD of three independent experiments. *p < 0.05: significant difference from control by analysis of variance; **p < 0.01: very significant difference from control by analysis of variance. (C) FW-04-806 directly affect Hsp90-Cdc37 interaction in Pull-down assay. 400 ug of purified Hsp90 (His-tag)protein was bound with Ni-NTA resin and divided into four groups evenly with the addition of recombinant Cdc37 protein 50 μg each and FW-04-806 0, 10, 20, 40 μM, respectively. After incubation and wash, The resins were boiled with loading buffer and analyzed by western blotting, His-probe antibody and Cdc37 antibody was used respectively. (D) SKBR3 cells were treated with FW-04-806 or DMSO for 24 h. Cdc37 or Hsp90 were immunoprecipitated from whole-cell lysates (500 μg each) with an anti-Cdc37 or anti-Hsp90 antibody respectively, then analyzed by immunoblotting with antibody against Hsp90, Cdc37 and HER2 (E) SKBR3 cells were treated with FW-04-806 at 10, 20, 40 μM for 24 h; 17AAG was used as a positive control at 1 and 2 μM. Hsp70, Hsp90, and Cdc37 protein level were analyzed with western blotting using relevant antibodies. (F) SKBR3 cells were transiently transfected with control siRNA or Hsp90 siRNA for 48 h. Whole-cell lysates were analyzed with western blotting against HER2, Hsp90, and β-actin.