Figure 2. Expression of IFN-a and TNF-a in A498 xenografts after rIL-22 treatment. Western blot assay was performed to detect the expression of IFN-a and TNF-a in the A498 cell xenografts. Neither the expression of IFN-a nor TNF-a were increased significantly in A498 cell xenografts treated with rIL-22 (P.0.05 compared with control). N = 2. doi:10.1371/journal.pone.0020382.g002 of the cell cycle (Fig. 3A). At a dose of 400 ng/ml rhIL-22, about 74% of the cells were arrested in the G2/M phase (Fig. 3B). These results show that the extent of cell cycle arrest is dependent on the dose of rhIL-22. Taken together the MTT results and the cell cycle arrest data, these results show that IL-22 inhibits A498 tumor cell proliferation both in vitro and in vivo IL-22 receptor detection by western blot assay IL-22R expression has been found in many cancer cells [22– 25]. Concerning the growth inhibition of A498 cells both in vivo and in vitro after IL-22 exposure, we reasoned that IL-22R might be expressed and might play a functional role in A498 cells. Therefore, we studied the expression of the IL-22R and functional consequences of IL-22 exposure in A498 cells. To examine the expression of IL-22R in A498 cells, we performed a western blot assay using mouse IL-22 Ra1 antibody, which displayed a measurable IL-22R expression in A498 cells. We also selected HepG2 cells and human B cells served as positive control and negative control cells of IL-22R respectively, which demonstrates a selective expression of IL-22R in different cells (Fig. 4). IL-22 regulates the STAT1 and ERK1/2 signaling pathways We investigated the downstream intracellular signal mediators of IL-22 action in A498 cells. Western blotting assays showed that STAT1 was phosphorylated from the 5th minute after exposure of the cells to rhIL-22 and that the phosphorylation of STAT1 (p-STAT1) reached its peak level 30 minutes after rhIL-22 exposure. In contrast to STAT1, ERK1/2 was dephosphorylated in response to rhIL-22; this effect was maximal 30 min after rhIL- 22 exposure (Fig. 5A). Both the phosphorylation of STAT1 and the dephosphorylation of ERK1/2 were amplified with increasing doses of rhIL-22 (Fig. 5B). In this procedure, the concentration of STAT1 in A498 cells was stable regardless of extension the rhIL- 22 exposure time. The results demonstrate that rhIL-22 activates STAT1 and inhibits ERK1/2 pathways in A498 cells in a time- and dose-dependent manner and that the biological functions of the receptors for these pathways show time-dependent STAT1 activation and ERK1/2 deactivation evoked by rhIL-22. The output of STAT1 protein in these cells treated with IL-22 is not Interleukin22 Suppress Renal Cell Carcinoma Growth Figure 3. A498 cell cycle arrest in the G2/M phase after rhIL-22 exposure. A. After 50 ng/ml rhIL-22 exposure for 24 h, significantly more A498 cells accumulated in the G2/M phase of the cell cycle. B. With increasing doses of rhIL-22, more cells accumulated in the G2/M phase of the cell cycle. N = 3. doi:10.1371/journal.pone.0020382.g003 significantly increased indicating that STAT1 protein is not extra synthesized in gene level during this procedure. To confirm that p-STAT1 has an anti-RCC effect on A498 cells, the expression of STAT1 was knocked down by STAT1 specific siRNA transfection. It revealed that p-STAT1 was maintained in a lower level in transfected cells after IL-22 Figure 4. Expression of IL-22R on the surface of cells by western blot assay. Western blot assay was performed to detect the expression of IL-22R on the surface of cells using mouse IL-22 Ra1 antibody. The results showed a measurable expression of IL-22R on the surface of A498 cells compared with that of HepG2 cells and human B cells, which served as positive control and negative control respectively. N = 3. doi:10.1371/journal.pone.0020382.g004 PLoS ONE | www.plosone.org 3 May 2011 | Volume 6 | Issue 5 | e20382