Xenograft study in Sui66- and Sui73-transfectant cell lines. (A) Tumorigenesis in vivo. To evaluate tumorigenicity in vivo, a suspension of 5 × 106 Sui66-transfectant cells (in 50 μL PBS) were subcutaneously inoculated into both flanks of nude mice (n = 7). Tumor formation was assessed every 2 or 3 days. Sui66/sh-ACVR1B exhibited a significantly elevated level of tumorigenesis in vivo (Scr-1 1/14 vs. ACVR1B-1 8/14, P = 0.013* and Scr-2 2/14 vs. ACVR1B-2 10/14, P = 0.0063*). (B) Tumor growth in vivo. To assess the tumor growth in vivo, a suspension of 5 × 106 Sui73-transfectant cell lines (in 50 μL PBS) with 50 μL of Matrigel were subcutaneously inoculated into the right flank of nude mice (n = 5). The tumor volume was assessed every 2 or 3 days. Sui73/shACVR1B exhibited a significantly elevated tumor volume (Sui73/Scr-1, 167.7 ± 59.1 mm3 vs. Sui73/ACVR1B-1, 275.0 ± 56.3 mm3; P = 0.018* on day 36 and Sui73Scr-2, 105.7 ± 27.2 mm3 vs. Sui73/ACVR1B-2, 217.3 ± 81.8 mm3; P = 0.020* on day 29, respectively). Lines, mean of 5 tumors; Error bars, SD; *P < 0.05. (C) Western blot analyses of the tumors and immunostaining. The expressions of p21 were clearly elevated in cancer cells in shScr-inoculated tumors, compared with the expression levels in shACVR1B cells. The expressions of Ki67 were also clearly elevated in the cancer cells in the shACVR1B-inculated tumors. β-actin was used as an internal control.