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. 2014 Jul 7;5(15):6113–6129. doi: 10.18632/oncotarget.2176

Figure 6. c-Myc transcriptionally activates rad-51 and survivin expression in LAD cells.

Figure 6

(A) qRT-PCR and Western blotting detection of rad-51 and survivin mRNA and protein expression in sh-c-Myc#3 (or sh-control) -transfected SPC-A1/DTX cells. (B) qRT-PCR and Western blotting detection of rad-51 and survivin mRNA and protein expression in pcDNA/c-Myc (or pcDNA/control)-transfected SPC-A1 cells. (C) Analysis of the reporter activity of survivin and rad-51 promoter (survivin or rad-51 promoter/Luc) in pcDNA/miR-451 (or pcDNA/miR-NC) and sh-c-Myc#3 (or sh-control)-transfected SPC-A1/DTX cells. Each cell type was transiently transfected with survivin or rad-51 promoter/Luc plasmid. Dual-luciferase reporter assays were performed on the lysed cells co-transfected with rad-51 or survivin promoter/Luc (firefly luciferase) and phRL-SV (hRenilla luciferase) 48 h after co-transfection. Reporter gene activation was determined as a relative ratio of firefly luciferase to hRenilla luciferase activity. (D) Analysis of the reporter activity of survivin and rad-51 promoter in An-miR-451 (or Anti-miR-NC) and pcDNA/c-Myc (or pcDNA/control)-transfected SPC-A1 cells used above-mentioned methods. (E1-2) Upregulation of miR-451 decreased the amount of c-Myc binding to the promoters of survivin and rad-51.ChIP assay was performed with antibody directly against c-Myc in SPC-A1/DTX cells transfected with pcDNA/control or pcDNA/miR-451, repectively. ChIP-derived DNA was amplified after immunoprecipitation by qRT-PCR with primers designed to amplify the sequences containing the putative c-Myc-binding sites. Data were adjusted with qRT-PCR products that were amplified with input DNA before immunoprecipitation and determined relative to miR-NC group. (F1-2) silencing of c-Myc decreased the amount of c-Myc binding to the promoters of survivin and rad-51. Chip assays were performed used above-mentioned methods. (G) Analysis of the reporter activity of survivin and rad-51 promoter in pcDNA/miR-NC or pcDNA/miR-451-transfected SPC-A1/DTX cells co-transfected with pcDNA/control or pcDNA/c-Myc used above-mentioned methods. (H) Western blotting detection of rad-51 and survivin protein expression in pcDNA/miR-NC or pcDNA/miR-451-transfected SPC-A1/DTX cells co-transfected with pcDNA/c-Myc (or pcDNA/control), or Anti-miR-451 (or Anti-miR-NC)-transfected SPC-A1 cells co-transfected with sh-control or sh-c-Myc#3. GAPDH was used as an internal control. Results represent the average of three independent experiments (mean±SD). *P< 0.05, **P< 0.01 and N.S >0.05.