Si jun or scrambled sirna

Unless otherwise indicated, 1×10⁵ cells were seeded into 12-well plates and transfected with 30nM miR-125b mimic or negative control mimic, 15nM miR-125b inhibitor or negative control inhibitor, and 15nM si-Jun or scrambled siRNA (Life Technologies) in antibiotic-free RPMI media. 24hrs post-transfection cells were re-seeded into 6-well plates for expansion. In some experiments cells were pre-treated with Tanshinone IIA. 72hrs later cells were irradiated at 0-6Gy. Cells were immediately seeded into 6-well plates at ∼100–900 cells per well, depending upon γ-irradiation condition from the lowest number of cells (100-200) at 0Gy, (275) at 2Gy, (400) at 4Gy, to the highest (900) at 6 Gy in order to produce colonies with clearly defined borders and minimal overlapping. After 10 days of undisturbed growth, colonies were fixed, stained with crystal violet, and counted on a dissection scope (Zeiss). Plating efficiency and surviving fraction were calculated from technical quadruplicates of each γ-irradiation dose and miRNA condition. Experiments were performed a minimum of three times. Statistical significance was evaluated by two-way ANOVA (two-tailed) of the data for each transfection or drug treatment across all doses.