Journal: Nature Communications

Article Title: Akt-p53-miR-365-cyclin D1/cdc25A axis contributes to gastric tumorigenesis induced by PTEN deficiency

doi: 10.1038/ncomms3544

Figure Lengend Snippet: ( a ) The protein level of p53 was measured using western blot analysis in BGC-823 cells treated with FBS, IGF-1 or LY294002 ( n= 3). ( b ) The protein level of p53 was measured using western blot in the PTEN -mutant and control epithelium at P20 ( n= 5). ( c , d ) The relative level of miR-365 was measured using qRT–PCR in BGC-823 cells upon the change of p53 expression ( n= 4). p53 abundance was verified using western blot from the same cell lysates. ( c ) Cells were transfected with pCMV-Neo-Bam-p53 vector for 24 h or treated with doxorubicin (1 μM) for 12 h ( n= 6). ( d ) Cells were transfected with p53 or control siRNA for 48 h ( n= 3). ( e ) The relative level of miR-365 was measured using qRT–PCR in p53 mutant and control epithelium at P20 ( n= 4). ( f ) ChIP assay detecting the binding of p53 to the miR-193b-365 promoter ( n= 3). BGC-823 cells were transfected with pCMV-Neo-Bam-p53 plasmid for 24 h. The p21 promoter was the positive control. A schematic diagram (upper) shows the promoter region of miR-193b-365 cluster in the genomic region and the luciferase vector containing the 4-kb region, in which the transcriptional start site (TSS) is represented as a black box and the p53-binding site (BS) as a pink box. ( g ) The luciferase reporter assay of the miR-193b-365 promoter constructs ( n= 5). BGC-823 cells were cotransfected with wild-type miR-193b-365 promoter construct (BS-WT) or p53-binding-site-mutant construct (BS-mut) with pCMV-Neo-Bam-p53. ( h ) The relative level of miR-365 was measured using qRT–PCR ( n= 4). BGC-823 cells were transfected with p53 siRNA for 36 h, followed by LY294002 addition for another 12 h. ( i ) Flow cytometry analysis detecting the distribution of the population to the different cell cycle stages ( n= 5). BGC-823 cells were cotransfected with miR-365 (365) or scrambled (Scr) mimic and p53 or control siRNA for 48 h. Values represent the mean±s.d. * P <0.05 and ** P <0.01 were calculated using Student’s t -test.

Article Snippet: The Akt siRNA (CST; no. 6211), p53 siRNA (CST; no. 6512), cyclin D1 siRNA (Santa Cruz; SC-29286) and cdc25A siRNA (Santa Cruz; SC-29254) were transfected with a final concentration of 100 nM.

Techniques: Western Blot, Mutagenesis, Quantitative RT-PCR, Expressing, Transfection, Plasmid Preparation, Binding Assay, Positive Control, Luciferase, Reporter Assay, Construct, Flow Cytometry