Production of dNSurR9-C84A. (A) SDS-PAGE analysis of proteins in bacteria transformed with pGEX-2T/dNSurR9-C84A. Bacteria were lysed before IPTG induction and 3 h after IPTG induction of protein expression. Analysis of the total lysate and insoluble and soluble fractions revealed a major ~42 kDa protein (position is indicated in the right-hand margin) induced by IPTG. Lane MW, molecular weights of protein markers in kDa. (B) Western blot analysis confirms the IPTG-induced ~42 kDa protein is dNSurR9-C84A. The insoluble and soluble fractions in (A) were Western blotted with a rabbit anti-survivin antibody, and immunoreactivity was detected with a peroxidase-conjugated goat anti-rabbit antibody. Lane MW, molecular weights of protein markers in kDa. (C) Purification of GST-tagged dNSurR9-C84A by affinity chromatography on glutathione agarose. The soluble fraction of lysates of IPTG-treated bacteria was chromatographed on glutathione agarose, and the glutathione eluted material subjected to SDS-PAGE and stained with Coomassie blue. (D) Formation of 3D cellular spheres of DU145 and HeLa cancer cells. Phase-contrast light microscopy of cellular spheres formed in semi-solid medium. (E) dNSurR9-C84A is able to penetrate into 3D-cultured DU145 and HeLa cells. Cells were incubated with dNSurR9-C84A for 1 to 5 h and cytoplasmic fractions Western blotted with an anti-GST antibody to detect GST-tagged dNSurR9-C84A. Blots were probed with an anti-actin antibody as a control.