Fig 1: Delivery of pSur/AS-Sur induces both caspase-dependent and caspase-independent apoptosis in cancer cells.Notes: (A) PANC-1 cells were transfected with either scramble siRNA or survivin siRNA for the indicated durations. Caspase-9 and caspase-3 cleavages were determined by Western blotting. The numbers under each blot are the intensities of the blot relative to that of the control (scramble). (B) Cancer cells were transfected with either empty plasmid or pSur/AS-Sur for the indicated durations. The expression of the cleaved caspase-9, caspase-3, and PARP was determined by Western blotting. Equal protein loading was verified by actin. The numbers under each blot are the intensities of the blot relative to that of the control (empty plasmid). (C) MDA-MB-231 cells were transfected with pSur/AS-Sur for 48 hours, and AIF (green fluorescent) translocation was determined by immunofluorescence microscopy. Nuclei were countered stained blue with DAPI. The localization of AIF in the nucleus of confocal images was pixel-by-pixel analyzed by the SigmaPlot software (version 10, Systat Software Inc., CA, USA). “*” denotes statistical significance with a P-value <0.05 between the testing groups. “**” denotes statistical significance of P-value <0.01. (D) Nucleic proteins and cytoplasmic proteins were isolated using the cells fractionation assay. Expression of AIF was analyzed by Western blotting. Equal protein loading was verified by either lamin A/C or GAPDH. The numbers under each blot are the intensities of the blot relative to that of the control (empty plasmid). (E) Cells were transfected with either empty plasmid or pSur/AS-Sur for 48–72 hours. DNA damage was detected using the comet assay. A statistically significant difference in the relative tail moment of cells transfected with empty plasmid versus pSur/AS-Sur is denoted by either “*” or “**”. *P<0.05 and **P<0.01.Abbreviations: siRNA, small interfering RNA; DAPI, 4′,6-diamidino-2-phenylindole; h, hours; PAR, Poly (ADP-ribose) polymerase; AIF, apoptosis-inducing factor; pSur/AS-Sur, survivin promoter-driven antisense survivin.