110 | Chapter 4 Supplement Supplemental Figure 1. Short term exposure to neocarzinostatin does not result in appreciable cell death, and DNA damage regulates RAG1 expression in SUP-B15 pre-B cells. (A) Overnight STI571-treated (STI) BV173, SUPB15 and A70 pre-B cells were treated for 2.5hrs with 200 ng/mL neocarzinostatin (NCS). STI, NCS and KU55933 treatment did not result in increased cell death as judged by propidium-iodide (PI) staining and flow-cytometry analysis. (n=3, independent replicate experiments). (B) Western blot analysis of SUP-B15 pre-B cells. DNA damage resulted in the ATM-kinase dependent down-regulation of RAG1 protein levels. The β-actin blot was used to control for loading. (C) Realtime quantitative RT-PCR analysis of RAG1 mRNA expression in SUP-B15 pre-B cells treated overnight with 5 mM STI571. DNA damage induction by 2h treatment with 50 ng/mL NCS resulted in the down-regulation of RAG1 mRNA expression, which was prevented by 2h pretreatment with 5 mM KU55933. Means +/- SEM are shown (n=3, ** p<0.01, one-sample t test).
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