Indigenous miR-421 expression was up-regulated in the LA-N-1 cells. We also examined miR-421 expression in five neuroblastoma cell lines. The miR-421 were significantly h Forth in the four cell lines NMYC-verst RKT. We found that miR-421-90-h level CHLA Ago than in the other two N-Mycnonamplified cell lines CHLA-15 and CHLA-255 was. This k Nnte by low-level MPC-3100 expression of N-Myc caused in CHLA-90. An expression profile Similar to the miR-374b was observed in these neuroblastoma cell lines and supports a model that the cluster is entered miR-421 and miR-374b Born by the same promoter. iii) The treatment of LA-N-1 cells with AMO-ATM, the complement to r miR-421 binding sites is � �U TR 3 ATM, and with anti-miR-421 inhibitor, which is complementary r miR-421 led to an increase inATMexpression.
As expected, had AMO ATM treatment does not affect the expression level of miR 421, w During anti-miR-421 inhibitor downregulation of miR-421 expression, suggesting two different mechanisms for AMO-ATM and anti-miR -421 on the cancellation of miR-421-mediated down-regulation of ATM expression. Close Lich was CONFIRMS the obtained Hte expression of ATM by AMO ATM by flow cytometry MLN8054 of phospho-SMC1 test, the recently developed in order best to measure the ATM picture. 5th N-Myc negatively regulated by miR-421 ATM in neuroblastoma cells. Immunoblot of ATM in the expression of N-Myc-verst Markets or unverst Markets neuroblastoma cells. We observed N-myc expression in CHLA-90, even if it is a cell line N-myc-amplified, ATM expression was relatively low in this cell line compared to 15 or CHLA-ABSC -255.
Lymphoblasto cell of cells and lymphoblasto be controlled by WT the negative and positive respectively for ATM expression. A total of 100 g of total protein μ for neuroblastoma cells and 25 g of total protein μ for AT-LCL and LCL-WT were loaded and SMC1 served as a control for loading. Chip-PCR detects the in vivo binding of N-Myc protein to the DNA of miR-421 promoter. A PCR fragment of the expected size E was zipitiert in N-Myc-amplified LA-N-1 cells with specific anti-N-Myc antibody Body immunpr, But not without Antik Body or with unspecific mouse- see IgG. No signal was in the N-Mycnonamplified CHLA-255 cells with no antique Body, non-specific mouse IgG or anti-Myc antibody Body immunpr Zipitiert seen. Used PCR with input DNA was controlled like Positive.
Real-time PCR of endogenous miR-421 in N-Myc-amplified LA-N-1 cells and CHLA-255 cells N-Myc-amplified. RNU66 was used as contr The house. Immunoblot of ATM expression in CHLA-255 and LA-N-1 cells with AMO AMO Schram or ATM for 5 days or 1-L-transfected cells treated with anti-miR-CTL or anti-miR-421 inhibitor 96 h hours change in ATM expression is shown below. Real-time PCR of miR-421 expression in LA-N-1 cells with AMO AMO Schram ATM or treated for 5 days or transfected with anti-miR-CTL or anti-miR-421 inhibitor for 4 days . RNU66 was used as contr The house. FC-IR-induced SMC1 detection of ATM-dependent- Independent Phosphorylation of SMC1 in neuroblastoma cells treated AMO. LA-N-1 and CHLA-255 cells were transfected with AMO AMO Schram or ATM treated for 5 days and a 10 Gy-IR.
PSMC1 level is indicated by the fluorescence intensity t. The filled peaks represent the cells without IR peaks and give unfilled post-IR cells. This plate is repr Sentative for three independent Independent experiments. Linear signal path, in the N-Myc-up regulates the expression of miR-421 and miR-421, which in turn regulates the expression of negative targeting its 3 ATM � �U TR. Hu et al. PNAS | 26 January 2010 | vol. 107 | no. 4 | 1509 GENETIC kinase activity in patients t Tr ger. As shown in Fig. 5F, the treatment of LA-N-1 cells with AMO-ATM resulted in a po