Fig 2: Exogenous Introduction of Different Apoptotic Caspases May Result in Unique Apoptotic OutcomesApoptotic pathways are mediated by caspases, calling into question which exogenously introduced caspase will be most effective at inducing cell death. Initiators casp-8 and casp-9 are activated in response to extrinsic and intrinsic stimuli, respectively. After initiator caspase activation, the executioners casp-3, -6, and -7 are activated, resulting in feedback activation of initiator caspases and direct substrate proteolysis, leading to cell death. Caspase activity is inhibited by pro-survival factors which are overexpressed to various extents in various cancerous tissues. Example executioner caspase substrates include poly(ADP-ribose) polymerase 1 (PARP-1), p23, and lamin A, colored to match the caspase that cleaves them (i.e., red for casp-6, green for casp-7, and green/purple for casp-3 and -7). Exogenous introduction of each of the apoptotic caspases, via nanogel intracellular delivery, allowed us to assess which caspase is best at cleaving cells of a given pro-survival profile.

Fig 3: Two-dimensional WB confirmation of ʎ-PPase treatment sensitive proteins CALM, PTGES3, TALDO and CAP1. With IEF, 30 µg of Cy5-labeled DU145 protein lysate were separated on a 7 cm IPG strip, pH 3–10, followed by molecular weight separation by 11.5% SDS-PAGE and blotted onto a PVDF membrane. The 2D-WB membranes were scanned with a laser scanner at 650 nm wavelength and shown as black spots. The 2D-WB images of the Cy5-stained proteomes are stained with antibodies against (A) CALM1, (B) PTGES3, (C) TALDO and (D) CAP1 and the specific signals are visualised by secondary HRP-conjugated antibodies and chemiluminescence (white spots). Overlays indicate the particular Cy5- labelled spots in black spots and specific antibody signals with white spots. Red arrows indicate phosphorylated protein spots, and green arrows indicate non-phosphorylated protein spots. Abbreviations: 2D-DIGE—two-dimensional difference gel electrophoresis; IEF—isoelectric focusing; IPG—immobilised pH gradient; WB—western blot; MW—molecular weight; pI—isoelectric point; ʎ-PPase—lambda phosphatase.