Role of p21 gene in colony formation and cellular growth in vitro and tumorigenicity in vivo . To see if p21 expression reverses the phenotype for ACVR1B gene homozygous deletion, we created a p21-overexpressed Sui68 cell line (homozygous deletion of ACVR1B gene and wild-type SMAD4 gene). (A) Western blot analyses. The overexpression of p21 was confirmed using western blot analyses in the Sui68/p21 cell line. β-actin was used as an internal control. (B) Colony formation of Sui68- transfectant cell lines. The colony formation in the Sui68/p21 cell line was suppressed, compared with that in the control cell line (EGFP, 28.0 ± 9.17 vs. p21, 0.89 ± 0.19, P = 0.035*). Columns, mean of independent triplicate experiments; Bars, SD; *P < 0.05. (C) Cellular growth of Sui68- transfectant cell lines. The cellular growth was evaluated using an MTT assay. Cellular growth in the Sui68/p21 cell line was suppressed, compared with Sui68/EGFP (0 h, P = 065; 24 h, P = 0.074; 48 h, P = 0.053; 72 h, P = 0.030*). Lines, mean of independent triplicate experiments; Bars, SD; *P < 0.05. (D) Tumorigenesis in vivo. To evaluate tumorigenicity in vivo, a suspension of 5 × 106 Sui68-transfectant cells (in 50 μL PBS) were subcutaneously inoculated into both flanks of nude mice (n = 7). Sui68/EGFP exhibited a significantly elevated level of tumorigenesis (Sui68/EGFP 14/14 vs. Sui68/p21 8/14, P = 0.016). (E) Tumor growth in vivo. To evaluate the tumor growth, a suspension of 5× 106 Sui68-transfectant cells (in 50 μL PBS) with 50 μL of Matrigel were subcutaneously inoculated into the right flanks of nude mice (n = 5). Sui68/EGFP exhibited a larger tumor volume than Sui68/p21 on day 15 (Sui68/EGFP, 507.0 ± 83.5 mm3 vs. Sui68/p21, 276.5 ± 95.0 mm3; P = 0.0036*). Lines, mean of five tumors; Bars, SD; *P < 0.05.