p38 MAPK in Epithelial-Mesenchymal Transition Figure 13. The expression of Snail mRNA in HK-2 cells by RT-PCR. (A)The levels of Snail mRNA were detected by RT-PCR of the HK-2 cells cultured in 5.5 mM glucose (lane 1), 30 mM glucose (lane 2), 30 mM glucose+Cont siRNA (lane 3), 30 mM glucose+p38 siRNA for 24 h (lane 4), 30 mM glucose DMEM+p38 siRNA for 48 h(lane 5).(B)The levels of the Snail mRNA were detected by RT-PCR of the HK-2 cells incubated in 5.5 mM glucose (lane 1), 30 mM glucose (lane 2), and 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.g013 Cell culture and experimental conditions Experiments were performed with HK-2 cells (American Type Culture Collection number CRL-2190), which are human proximal tubular epithelial cells immortalized by transduction with human papillomavirus type 16 E6/E7 genes. Cells were cultured in Dulbecco’s modified Eagle’s medium (low glucose) (Gibco, UK) that was supplemented with 10% fetal calf serum (Sigma, USA). The cells were seeded at 1.56106 cells/10 cm diameter dishes for 3–4 days at 37uC in a humidified atmosphere containing 5% CO2. Transfection of siRNA To silence the p38 MAPK gene expression, siRNA oligonucle- otides with the following sequences were designed: p38 siRNA, 59-GAAGCTCTCCAGACCATTT-39 and negative Cont-siRNA, 59 -GACTTCATAAGGCGCATGC-39. The HK-2 cells were cultured for 24 h before the transfection. After a 24-h or 48-h incubation with p38 siRNA or a 24-h incubation with Cont- siRNA, the cells were cultured with high glucose (30 mM glucose) for an additional 48 h. Cells that were not transfected and were exposed to normal glucose (5.5 mM glucose) or high glucose (30 mM glucose) for 48 h were considered to be the controls. Transfection was processed in 24-well plates with 40 nM siRNAs using Lipofectamine2000 (Invitrogen). p38 MAPK knockdown was confirmed by Western blot analysis and reverse transcription- PCR (RT-PCR). SDS-PAGE and Western blot analysis Cells were washed twice with PBS and lysed with 2X SDS- PAGE sample buffer. Next, 50 mg of total protein was separated by 10% SDS-PAGE and transferred to nitrocellulose membranes; the membranes were then blocked with 1% polyvinylalcohol in PBS containing 0.2% Tween 20 for 10 min and incubated at 4uC overnight with primary antibodies (diluted to various concentra- tions in blocking buffer (5% or 1% skim milk in PBS-Tween (0.2% Tween 20)) against the following target proteins: p38, phospho- p38 (1:1000), E-cadherin (1:200), a-SMA (1:200), CK (1:200), Vimentin (1:600), TGF-b1 (1:500) and b-actin (1:2000). The membranes were then washed three times with PBS-Tween for 10 min and incubated with species-specific peroxidase-conjugated secondary antibodies (Santa Cruz, CA) diluted in blocking buffer (5% skim milk in PBS-Tween) for 2 h at room temperature. Specific bands were detected using the ECL system (Amersham) and the Bio-Rad electrophoresis image analyzer (Bio-Rad, Hemel Hampstead, UK). RNA extraction and reverse transcription Total RNA was extracted from the HK-2 cells using the Tripure reagent (Roche, Germany) according to the manufactur- er’s instructions. RNA samples were qualified by the measurement of the optic absorbance at 260 and 280 nm with a resultant A260/ A280 ratio that ranged from 1.8 to 2.0, which indicated a high purity of the extracted RNA. The concentration of total RNA was calculated according to A260. Aliquots of total RNA (1.0 mg each) from each sample were reverse transcribed into cDNA according to the instructions of the First Strand cDNA Synthesis Kit (Takara, China). Reverse Transcriptase-PCR Semiquantitative reverse transcriptase-PCR (RT-PCR) was used to determine the steady-state mRNA levels of Snail, CK and vimentin. Briefly, after reverse transcription of renal total RNA, cDNA was used as a template for the PCR reactions using gene-specific primer pairs. Generally, 30 to 35 cycles for amplification in the linear range were used. Quantification of PCR products was performed using densitometry, and the relative PLoS ONE | www.plosone.org 10 July 2011 | Volume 6 | Issue 7 | e22806