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. Author manuscript; available in PMC: 2009 Jul 10.
Published in final edited form as: Exp Hematol. 2009 Jan;37(1):122–134. doi: 10.1016/j.exphem.2008.09.008

Figure 5.

Figure 5

Regulation of diacylglycerol (DAG) analogue-induced apoptosis in Toledo B-cell non-Hodgkins lymphoma (B-NHL) cells by Bcl-2 family members. (A) Toledo B-NHL cells expressing either the control vector, siLuc, or the siBim vector were examined for Bim expression levels by immunoblotting. Erk was examined as a loading control. Results are representative of two independent experiments. (B) Toledo cells expressing the siLuc and siBim downregulation vectors were compared by tetramethylrhodamine ethyl ester (TMRE) staining after 20 hours (experiment #1) or 26 hours (experiment #2) of 10 nM pico treatment. Results of the two experiments were normalized by expressing the percent viable cells compared to that seen with untreated cells (**p < 0.03). (C) Similarly, siLuc and siBim were compared after 28 hours of 10 nM pico treatment in two separate experiments using the TUNEL assay for nuclear DNA fragmentation (***p < 0.005). (D) Toledo B-NHL cells expressing empty vector (BMG) and overexpressing Bcl-2 were compared, with and without CdCl2 promoter induction, by anti–Bcl-2 immunoblotting. Erk was examined as a loading control. (E) Bcl-2–engineered and empty vector Toledo B-NHL cells were compared in terms of their TMRE staining after 24 hours treatment with 10 nM pico. The results were duplicated in an independent experiment. (F) Similarly, the effects of Bcl-2 overexpression on apoptosis after 48 hours treatment with 10 nM pico was assessed with the TUNEL assay. (G) The pico-induced, Mek-dependent formation of Bak–Bax physical complexes in Toledo B-NHL cells was detected by the presence of Bak in anti-Bax immune- precipitates. Pico treatment was for 24 hours. L = lysate. Similar results were obtained in two additional experiments.