Figure 2. Depleting CDK8 promotes tumorigenesis in AN3 CA cells. AN3 CA cells were transduced with pLKO.1-shRNA targeting CDK8 or with control sequence targeting GFP. (A) The relative mRNA level of CDK8 was analyzed by qRT-PCR analysis, and (B) the CDK8 protein levels were analyzed by western blot. (C) AN3 CA cells or control cells (1.5 × 105 per well) were seeded in 6-well plate, and the number of cells per well was shown for 5 d (n = 4). (D) The effects of CDK8 reduction on cell cycle progression of the AN3 CA cells analyzed by flow cytometry analysis. (E) Colony formation experiments were conducted in 6-cm cell culture dish. 1,000 cells were seeded and allowed to grow for 2 weeks, and then the cells were stained with crystal violet (left). (F) The size and number of colonies were quantified by Fuji (ImageJ) software. (G) Tumor growth of CDK8 knockdown or control AN3 CA stable cells in 6–8-week-old female nude mice (n > 14 separate mice in each group). Cells (2 × 106) were implanted in two flanks of each mice and the volume of tumors were assessed in indicated days.