100 | Chapter 4 Figure 6. Rag1 mRNA expression is regulated by RAG-dependent DNA breaks. (A) Realtime quantitative RT-PCR analysis of Rag1 mRNA expression in A70 (WT, black bars) and R2K3 (Rag2-/-, grey bars) mouse v-Abl cells treated for 72h with 5 mM STI571 and 5 mM KU55933 or vehicle (DMSO), Rag1 mRNA expression relative the housekeeping gene 18S rRNA is shown. Means ± SEM are shown (n=3, * p<0.05, one-way ANOVA). (B) Erag-Rag1-driven luciferase activity was measured by transient transfection of the Erag-Rag1 luciferase construct in A70 (WT, black bars) and R2K3 (Rag2-/-, grey bars) mouse v-Abl pre-B cells treated for 72h with 5 mM STI571 or vehicle (DMSO). Relative light units (RLU) normalized to the TK Renilla luciferase reporter vector are shown. Following transfection, cells were treated for 24h with 5 mM KU55933. Means ± SEM are shown (n=3, * p<0.05, two-way ANOVA). (C) Realtime quantitative RT-PCR analysis Rag1 mRNA expression in R2K3 (Rag2-/-) treated overnight with 5 mM STI571. DNA damage induction by 2h treatment with 50 ng/mL NCS resulted in a rapid down-regulation of Rag1 mRNA expression in Rag2-deficient cells, which was prevented by 2h pretreatment with 5 mM KU55933. Means ± SEM are shown (n=3, ** p=0.004). (D) Realtime quantitative RT-PCR analysis of Rag1 mRNA expression in Artemis-/- (black bars) and Artemis-/- Rag2-/- (grey bars) mouse v-Abl cells treated for 72h with 5 mM STI571 or vehicle (DMSO). Means ± SEM are shown (n=3, ** p<0.01, two-way ANOVA).
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