Fig. 4.

HSP90–IP6K2 interaction regulates drug-induced cell death. (a) Decreased cell survival elicited by HSP90 depletion (filled inverted triangles) is reversed by codepletion of IP6K2 (filled squares). Cotansfection of control siRNA with HSP90 siRNA has no effect (open squares). MTT assay was done after each time period of various siRNA treatments of HEK293 cells. (b) Cell death elicited by HSP90 depletion is further enhanced by IP6K2 overexpression. Cells were either transfected with HSP90 siRNA or cotransfected with Myc-IP6K2 for 48 h, and cell death was monitored by apoptotic nuclei-detection assay. (c) Cell survival (MTT) assay of WT and IP6K2-R136A-transfected HEK293 cells. Cells were transfected with various Myc-IP6K2 constructs; after each interval, the percentage survival was calculated. IP6K2-R136A mutant (filled inverted triangles) causes a significant decrease in cell survival than IP6K2-WT (open circles). (d) Increased cell death elicited by overexpression of IP6K2-R136A that does not bind to HSP90. Cells were transfected by different constructs for 72 h. Cell death caused by IP6K2-WT is reversed by HSP90 overexpression, whereas there is no effect of HSP90 on IP6K2-R136A. The catalytically impaired IP6K2-W131A does not induce cell death. (e) Caspase-3 activity is increased in cells transfected with IP6K2-R136A, which does not bind HSP90. Activity was measured after 72 h of transfection. The magnitude of increase was determined by considering OD₄₀₅ of control samples as unity. (f) Death of cells overexpressing IP6K2-WT after CP treatment is reversed by HSP90 in cells overexpressing IP6K2-WT, but not IP6K2-R136A, which cannot bind HSP90. Cells overexpressing IP6K2-W131A show diminished cell death relative to WT, which is further reduced by HSP90 coexpression. (g) Cell death assay as described for panel f, after NB treatment. (h) CP- and NB-induced cell death in HEK293 cells requires IP6K2. Endogenous IP6K2 was depleted by using siRNA. CP and NB were added after 36 h of transfection for 36 and 24 h, respectively. Cell death in drug-treated cells is diminished by IP6K2 depletion.