Structure/function of survivin–Hsp90 interaction. (A) In vitro pull-down. Survivin was incubated with Sepharose-GST, -GST-Hsp90, or the indicated -GST-Hsp90 N, M, or C domains, and protein binding was determined by Western blotting. (B) Immunoprecipitation. HeLa cells transfected with the indicated FLAG-Hsp90 domains were immunoprecipitated with a mAb to FLAG and analyzed by Western blotting. MW, molecular weight. (C) Peptide mapping. The indicated survivin peptides were used to compete the binding between Hsp90 and survivin by ELISA. (D) Folding requirement. Protein binding to immobilized survivin (Upper) or Hsp90 (Lower) was determined by ELISA. (E) In vivo turnover. HeLa cells transfected with GFP-survivin (Left) or GFP-survivin(C84A) (Right) were treated with cycloheximide, and protein turnover at the indicated time intervals was determined by Western blotting with an Ab to GFP.

Ab disruption of survivin–Hsp90 complex induces apoptosis and cell cycle arrest. (A) In vitro targeting. Survivin was incubated with mAbs 58 or 8E2 to survivin (24) or IgG, and binding to GST-Hsp90 or GST-N-Hsp90 (GST-Hsp90 N) was determined by Western blotting. (B) In vivo targeting. HeLa cells were loaded intracellularly with mAb 8E2 or IgG, treated with cycloheximide, and analyzed by Western blotting (Upper). MW, molecular weight. Protein amounts were quantified by densitometry (Lower). (C) Caspase activity. YUSAC-2 cells loaded with mAb 8E2 or IgG were analyzed for caspase-3/7 activity by Asp-Glu-Val-Asp expression and flow cytometry in the absence (Tet–) or presence (Tet+) of Tet. Data are mean ± SD of three independent experiments. (Inset) Proteolytic processing of caspase-9 in HeLa cells transduced with mAbs 8E2 or 58 to survivin. The positions of proform (Casp.9) or cleaved (Cleaved) caspase-9 are indicated. (D) Mitotic defects. HeLa cells loaded with mAbs 8E2 or 58 to survivin or IgG were stained with an Ab to β-tubulin and analyzed by fluorescence microscopy. (Magnification, ×400.) Multinucleated cells in mAb 8E2-transduced cultures are indicated by arrows. (E) Summary of cell division defects. Mitotic or multinucleated cells in Ab-transduced HeLa cells were scored by fluorescence microscopy. Data are the means ± SEM of four independent determinations.

Survivin–Hsp90 interaction. (A) Heat shock. HeLa cells exposed to 42°C for 1 h were harvested at the indicated time intervals and analyzed by Western blotting. (B) Cell cycle analysis. HeLa cells were harvested 8 h after heat shock and analyzed for DNA content by flow cytometry. The percentage of G₂/M cells is indicated. (C)Affinity purification. HeLa cell extracts were fractionated on a survivin-Sepharose column. Eluted proteins (arrows) were identified by Western blotting. (D) Coimmunoprecipitation. Raji cell extracts were immunoprecipitated with IgG or an Ab to survivin, and pellets (P) and supernatants (S) were analyzed by Western blotting. (E) In vivo pull-down. Asynchronous or taxol-treated HeLa cell extracts were incubated with Sepharose (None) or survivin-Sepharose (Survivin), and pellets or supernatants (25% of reaction) were analyzed for coassociated Hsp90 by Western blotting. MW, molecular weight.

Regulation of survivin stability by Hsp90. (A) Requirement for Hsp90 chaperone function. HeLa cells were treated with GA and analyzed after 24 h by Western blotting. MW, molecular weight. (B) Cell cycle analysis. Untreated (Left) or GA-treated (Right) HeLa cells were harvested after 24 h and analyzed for DNA content by flow cytometry. The percentage of cells at each cell cycle transition is indicated. Apoptotic cells were electronically excluded. (C) Proteasome inhibition. GA-treated HeLa cells with or without the proteasome inhibitor lactacystin were analyzed after 30 h by Western blotting. (D) GA-induced apoptosis. GA-treated HeLa cells were analyzed for plasma membrane integrity (red fluorescence) and caspase-3/7 activity (green fluorescence) by multiparametric flow cytometry. The percentage of cells in each quadrant is indicated. (E) Apoptosome requirement. WT or Apaf-1^–/– MEF were treated with GA and analyzed for apoptosis (Upper) or G₂/M arrest (Lower) by DNA content and flow cytometry. (F) Modulation of GA-induced apoptosis. YUSAC-2 cells conditionally expressing survivin after Tet withdrawal (25) were treated with GA with or without Tet and analyzed for apoptosis by DNA content and flow cytometry. Data in E and F are the means ± SD of three independent experiments.