FIG. 8.

Effect of GSTμ on IL-3 withdrawal mediated apoptosis of 32Dcl3 cells. (A) Constitutive expression of GSTμ cDNA in 32Dcl3 cells with the IPTG-inducible pOPRSVI/MCS vector. Total RNA was extracted from mock-transfected and GSTμ-transfected cells and probed with full-length GSTμ cDNA in the absence or presence of IPTG. As a control for RNA loading, filters were stained with ethidium bromide to compare the levels of 28S and 18S RNAs (shown below). (B) Analysis of the viability of GSTμ-transfected cells in the absence of IL-3. Mock-transfected (32D/E) and GSTμ-transfected (32D/GST) 32Dcl3 cells were washed in IL-3-free medium and incubated up to 8 days. At each indicated time point, the cells were analyzed for viability by trypan blue exclusion. The curves represent means of three experiments. (C) Analysis of DNA fragmentation. At the indicated times following IL-3 withdrawal, DNA fragments released from 10⁷ cells from the two 32D cell lines were extracted and separated by electrophoresis and stained with ethidium bromide. (D) Analysis of caspase activity by determining the breakdown of nuclear lamin B. Mock-transfected and GSTμ-transfected cells were incubated in IL-3-free medium, and the cell lysates from the indicated time points were subjected to Western blotting with anti-lamin B antibody. The full-length lamin B is detected as a 69-kDa protein. The caspase activity is marked by lamin B cleavage, detected as a 32-kDa product.