p38 MAPK in Epithelial-Mesenchymal Transition Figure 12. Expression of E-cadherin, CK, a-SMA, vimentin in HK-2 cells by Western blot and RT-PCR analysis. (A–B)a-SMA(A) and E- cadherin(B) were detected by Western blot of the HK-2 cells incubated in 5.5 mM glucose (lane 1), 30 mM glucose(lane 2), 30 mM glucose+AP-1 inhibitor(lane 3).(C–D)mRNA of CK(C) and vimentin(D) was measured by RT-PCR of the HK-2 cells incubated in 5.5 mM glucose (lane 1), 30 mM glucose(lane 2), 30 mM glucose+AP-1 inhibitor(lane 3). Values represent the mean 6 SD. aP,0.05 vs. 5.5 mM glucose, bP,0.05 vs. 30 mM glucose. doi:10.1371/journal.pone.0022806.g012 p38 MAPK and AP-1; alterations in cell morphology; loss of the epithelial markers, E-cadherin and CK; de novo expression of vimentin and a-SMA; and an increase in fibronectin levels. The siRNA-mediated down-regulation of p38 expression prevented AP-1 activation and the high glucose-induced EMT. In addition, the suppression of the AP-1 transcriptional complex blocked the high glucose-induced EMT. The inhibition of the p38/AP-1 signaling pathway limited the development of the EMT in vitro and suppressed the expression of Snail, which is a key EMT- regulatory gene. Taken together, this study suggests the p38 MAPK pathway may play an important role in high glucose- induced EMT through the activation of AP-1 in tubular epithelial cells. Materials and Methods Antibodies and chemical inhibitors Anti-phospho-p38 antibodies and anti-p38 antibodies were purchased from Cell Signaling Technology (USA). Anti-E- cadherin, anti-a-SMA, anti-vimentin, anti-CK and anti-TGF-b1 antibodies were purchased from Santa Cruz Biotechnology, Inc. (USA), and [6]-Gingerol was purchased from Wako Ltd. (Japan). PLoS ONE | www.plosone.org 9 July 2011 | Volume 6 | Issue 7 | e22806