expression, ON-TARGETplus SMARTpool siRNA were trans- fected into PCa cells using DharmaFECTH 2 transfection reagent according to the manufacturer’s instructions. Non-targeting siRNA was used as control. For overexpression, 800 ng each expression plasmid DNA was transfected or co-transfected into PCa cells using LipofectamineTM LTX with PlusTM Reagent. Empty vector was used as blank control and to keep the total amount of plasmids DNA equal in each well. The plasmids expression irrelevant protein EGFP and Triptolide binding protein XPB were used as negative and positive control, respectively. 48 h after transfection, cells were treated with Triptolide for another 48 h. After stained with trypan blue, viable cells were counted using cellometer Auto T4 automated cell counter. Apoptosis analysis 1.56105 LNCaP or 16105 PC-3 cells per well were cultured in 6-well plate in which coverslips were plated. After attachment, cells were treated with 1 mM Triptolide or Celastrol for 24 h. Annexin V (AV)/propidium iodide (PI) co-labeling was performed using the Vybrant Apoptosis Assay Kit #3 according to the manufacturer’s instructions. The apoptotic cells were analyzed by inverted fluorescence microscopy (Leuca DM IRM) or by flow cytometry (FACSCalibur, Becton Dickinson) using FITC signal detector (FL1) and phycoerythrin emission signal detector (FL2). Data analysis was performed using Expo32 (version 1.2) software. Chemiluminescence Immumo-Assay (CLIA) LNCaP cells were cultured in 6-well plate at a density of 16105 cells per well and then treated with 1 mM Triptolide or Celastrol for 24 h or 48 h. Aliquots of medium and cell lysate from the treated cells were collected to measure the total PSA level on a chemiluminescence apparatus (Roche Cobas E601) according to protocol of CLIA. Real-time PCR LNCaP or PC-3 cells were cultured in 6-well plate and then treated with various doses of Triptolide or Celastrol for 24 h or with 0.1 mM Triptolide and Celastrol for desired time points. RNA was isolated from treated cell using Trizol Reagent following the manufacturer’s instructions. 500 ng total RNA was reverse transcribed to cDNA using High-Capacity cDNA Reverse Transcription Kits. The real-time PCR were performed on the Bio-Rad CFX 96 Real-time PCR system using SYBRH Prime- ScriptTM RT-PCR Kit II and specific primers (Figure S3). mRNA level of each gene were normalized to b-actin with DDCT method using Bio-Rad CFX Manager V1.1.308.1111 software. The relative mRNA level was calculated by dividing the normalized each gene expression of treated cells with untreated control sample. Western blot analysis LNCaP or PC-3 cells were treated with various doses of Triptolide or Celastrol for 24 h or with 0.1 mM Triptolide or Celastrol for desired times. Cell were trypsinized and washed with cold PBS. Cell pellets were lysed with lysis buffer (50 mM Tris [pH 7.5], 5 mM EDTA, 0.1% NP-40, 300 mM NaCl, with freshly added 0.5 mM phenylmethylsulfonyl fluoride and 16 Roche protease inhibitors cocktail solution) and incubated on ice for 30 minutes with gentle shake or with M-PER protein extraction solution according to the instructions (Thermo Scientific, #78501). Protein samples were separated by 12% SDS-polyacryl- amide gel electrophoresis and electrotransferred to nitrocellulose membrane. Protein blots were probed with an appropriate Triptolide Inhibits PCa Cells Proliferation primary antibody and a secondary antibody (IRDye, LI-COR) and then analyzed by quantitative immunoblot using an Odyssey Infrared Imaging System (LI-COR). a-tubulin or b-actin was used as loading control. Mouse xenograft assay 5-week-old male nude immunodeficient mice (NCRNU-M) were purchased from Animal Research Center of the Fourth Military Medical University (Xi’an, Shaanxi, China) and main- tained in a standard environment. The mice were allowed to acclimatize for at least 1 week before experiments. PC-3 cells (26106) suspended in 0.1 mL of serum-free RPMI 1640 were inoculated s.c. in the left flank of each mouse. When tumor volume 3 reached 100 mm , mice were divided randomly into control and treatment groups. Mice in treatment group were injected with Triptolide at 0.4 mg/kg daily for 15 days while control mice were injected with vehicle (DMSO). Tumor sizes were measured using calipers and their volumes were calculated using a standard formula: width26length/2, and body weight was measured every three days. The xenograft tissues were collected immediately after the animals were sacrificed and stored at 280uC for further study. Statistical analysis Results were expressed as the mean 6 SD. Student’s t test was applied to evaluate the differences between treated and control groups. For all the tests, the level of significance was set at P,0.05. Supporting Information Figure S1 Chart for the results from Figure 2C. After treatment with Triptolide or Celastrol, LNCaP cells (A) and PC-3 cells (B) were stained with AV/PI and analyzed by flow cytometry. Percentages of viable cells (AV2/PI2), early apoptotic cells (AV+/PI2) and late apoptotic/necrotic cells (AV+/PI+) are presented. (TIF) Figure S2 Triptolide enhanced cellular SUMOylation in PCa cells. (A) and (B) Triptolide enhanced SUMO-1 heterodi- mers level in PCa cells. PCa cells were treated with 1 mM Triptolide or Celastrol for 24 h, Cell pellets were lysed in lysis buffer with 20 mM NEM. Western blot were performed using a SUMO-1 monoclonal antibody. (A) Triptolide enhanced SUMO- 1 heterodimers levels in PC-3 cells. (B) Triptolide enhanced SUMO-1 heterodimers levels in LNCaP cells. (C) Triptolide decreased SUMO-1 monomer level in LNCaP cells. LNCaP cells were treated with 1 mM Triptolide or Celastrol for 24 h, Cell pellets were lysed in normal lysis buffer. Western blot were performed using a SUMO-1 monoclonal antibody. (TIF) Figure S3 Primers for plasmids construction and Real- time PCR. Specific primers used for SENP1, c-Jun, XPB and EGFP expression plasmids construction or analysis of SENP1, AR, c-Jun, and AR/c-Jun target genes mRNA levels in Triptolide- or Celastrol-treated PCa cells by Real-time PCR. (TIF) Acknowledgments We thank Dr. Guangchao Sui at Wake Forest University for critical reading of the article. We acknowledge the Key Laboratory of Agricultural Molecular Biology at Northwest A&F University for provision of the access to equipments. PLoS ONE | www.plosone.org 15 May 2012 | Volume 7 | Issue 5 | e37693