Figure 4.

Changes in cell cycle phase distribution and apoptogenic/anti-apoptotic regulatory proteins in control and I'm-Yunity™ (PSP)-treated HL-60 cells. Panel A. Cellular DNA content frequency histograms showing the cell cycle phase distribution changes of U-937 and HL-60 cells following 3 day treatment with 1.0 mg/ml doses of I'm-Yunity™ (PSP). Flow cytometric analysis was performed as described in Materials and Methods. Induction of apoptosis in treated cells is illustrated by cells showing fractional DNA content. Panels B-C, changes in apoptogenic/anti-apoptotic proteins in control and 3 day I'm-Yunity™ (PSP)-treated HL-60 cells. Expression of bax, cytochrome c, bcl-2, survivin, and PARP, identified by their respective molecular weights, was determined by immunoblot analysis, then adjusted for protein loading using cytochrome c oxidase in panel B, or actin in panel C, and presented as fold differences, with the control value showing as 1. Panel D, changes in bax, bcl-2 and cytochrome c in the cytosolic and mitochondria fractions separated using the procedure detailed in Materials and Methods. Expression of the various proteins was adjusted for loading using either cytochrome c oxidase (for mitochondria fraction) or actin (for cytosolic fraction) and presented as fold differences, with the control value showing as 1. The adjusted bax and bcl-2 values were used to calculate the ratio of bax/bcl-2 expression.