4 DNA damage response regulates RAG1/2 expression through ATM-FOXO1 | 87 9 ml using PCR-grade water, to which 1 ml of diluted cDNA was added. The following PCR conditions were used: initial denaturation at 95°C for 5 minutes, followed by 40 cycles of denaturation at 95°C for 15 seconds, annealing at 65°C for 5 seconds, and extension at 72°C for 10 seconds. The fluorescent product was measured by a single acquisition mode at 72°C after each cycle. For distinguishing specific from non-specific products and primer dimers, a melting curve was obtained after amplification by holding the temperature at 65°C for 15 seconds followed by a gradual increase in temperature to 95°C at a rate of 2.5°C s−1, with the signal acquisition mode set to continuous. For each cDNA preparation PCR reactions were performed at least in duplicate, and for each condition at least three independent experiments were performed. Expression of target gene mRNAs were normalized to the housekeeping genes large ribosomal protein PO (RPLPO) for human samples and 18S ribosomal RNA (18S rRNA) for mouse samples. Statistical significance was determined using the one-sample t-test in which the DMSO-treated control samples were set to a value of 1. PCR primers used in this study: human RAG1-forward (5’-GGCAAGGAGAGAGCAGAGGA-3’), human RAG1-reverse (5’-CTCACCCGGAACAGCTTAAA-3’), human CDKN1A/P21-forward (5’-GACTCTCAGGGTCGAAAACG-3’), human CDKN1A/P21-reverse (5’-GGATTAGGGCTTCCTCTTGG-3’), human FOXO1-forward (5’-AAGAGCGTGCCCTACTTCAA-3’), human FOXO1-reverse (5’-TTCCTTCATTCTGCACACGA-3’), human GADD45a-forward (5’-GAGCTCCTGCTCTTGGAGAC-3’), human GADD45a-reverse (5’-GCAGGATCCTTCCATTGAGA-3’), human RPLPO-forward (5’-GCTTCCTGGAGGGTGTCCGC-3’), human RPLPO-reverse (5’-TCCGTCTCCACAGACAAGGCCA-3’), mouse Rag1-forward (5’-AGCTATCACTGGGAGGCAGA-3’), mouse Rag1-reverse (5’-GAGGAGGCAAGTCCAGACAG-3’), mouse Rag2-forward (5’-CTTCCCCAAGTGCTGACAAT-3’), mouse Rag2-reverse (5’-AGTGAGAAGCCTGGCTGAAT-3’), mouse Cdkn1a/ p21-forward (5’-TCCACAGCGATATCCAGACA-3’), mouse Cdkn1a/p21-reverse (5’-AGACAACGGCACACTTTGCT-3’), mouse Foxo1-forward (5’-AAGAGCGTGCCCTACTTCAA-3’), mouse Foxo1-reverse (5’-TGCTGTGAAGGGACAGATTG-3’), mouse Gadd45a-forward (5’-CCGAAAGGATGGACACGGTG-3’), mouse Gadd45a-reverse (5’-TATCGGGGTCTACGTTGAGC-3’), mouse 18S rRNA-forward (5’-TGGTGGAGGGATTTGTCTGG-3’), mouse 18S rRNA-reverse (5’-TCAATCTCGGGTGGCTGAAC-3’). Lentiviral and retroviral transductions Lentiviral constructs for overexpression of WT and mutant kinase-dead AKT (K179M/ T308A/S473A) were generated by cloning the BamH1/EcoR1 fragments from Addgene plasmids 9011 and 901323 into the leGO-iG2 backbone. The leGO-iG2 was a gift from Boris Fehse (Addgene plasmid #27341)24. Lentiviral particles incorporating measles virus glycoproteins H and F were generated by cotransfection of HEK293T cells with the D8.91 (gag/ pol) and Hdel24 and Fdel30 plasmids25 using GENIUS DNA transfection reagent (Westburg, Leusden, the Netherlands). In brief, virus supernatants were harvested 48h after transfection, and concentrated by overnight centrifugation at 3000xg at 4°C. BV173 cells
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