6 DNA damage-induced p53 downregulates expression of RAG1 | 151 Figure 1. (A) Rag1 mRNA expression in primary murine WT bone marrow pre-B cells measured by quantitative RT-PCR; the cells were first cultured 7 days on the OP9 feeder layer cell line with IL7 and Flt3L: conditions that promote expansion of small pre-B cells that have not yet undergone the Igk recombination because of the low RAG1/2 expression. To induce the RAG1/2 expression, IL7 and Flt3L were withdrawn from the media for 24h. After that, the cells were treated either with DMSO, subjected to 5 Gy g-irradiation with subsequent 2h recovery time, pre-treated with ATM inhibitor KU55933 for 2h prior to g-irradiation, or treated for 4h with KU55933 with no irradiation (n=2, mean±SD, **p<0.05). (B) Rag1 and (C) Rag2 mRNA expression in primary WT and Atm-/- bone marrow pre-B cells first cultured 7 days on OP9 cells with addition of IL7 and Flt3L, 24h after the withdrawal of Flt3L and IL7 the cells were treated either with either DMSO or subjected to 5 Gy g-irradiation with subsequent 2h recovery time (n=2, mean±SD, **p<0.05). (D) and (E) Rag1 and Rag2 mRNA expression in primary WT and Tp53-/- bone marrow pre-B cells treated, 24h after the withdrawal of Flt3L and IL7, with either DMSO or 5 Gy g-irradiation with subsequent 2h recovery time (n=2, mean±SD, fold difference as compared to DMSO, *p<0.1). (F) Representative FACS staining of WT primary bone marrow cells and cells derived from Tp53-/- mice. The bone marrow cells were first cultured 7 days on OP9, Flt3L, and IL7, then, the amounts of B220+IgM+ pre-B cells were measured 48h following the withdrawal of Flt3L and IL7 from the medium. (G) Foxp1 mRNA expression in primary WT and Tp53-/- bone marrow pre-B cells treated, 24h after the withdrawal of Flt3L and IL7, with either DMSO or 5 Gy g-irradiation with subsequent 2h recovery time (n=2, mean±SD, fold difference as compared to DMSO, *p<0.1). (H) Western blotting analysis of RAG1, TP53 phosphorylated at Ser15 and FOXP1 protein levels in BV173 cells. The cells were treated with v-Abl kinase inhibitor STI571 for 72h to induce RAG1/2 expression and Igk recombination, after which the cells were exposed to 5Gy g-irradiation with subsequent 2h recovery time or pre-treated with ATM inhibitor KU55933 for 2hours prior to the irradiation with subsequent 2h recovery time or treated with p53 stabilizer Nutlin-3 for 4 hours. KU70 was used as a control. (I) Rag2 and (J) Foxp1 mRNA expression in primary WT and Rag1-/- bone marrow pre-B cells. After 7 days of culture, the Flt3L and IL7 were withdrawn from the media and the Rag2 and Foxp1 mRNA expression were measured on the day of the withdrawal and 1, 3, 4, and 5 days following the withdrawal (n=2, mean±SD, **p<0.05).
RkJQdWJsaXNoZXIy MTk4NDMw