Triptolide Inhibits PCa Cells Proliferation Figure 8. Hypothetic mechanisms of anti-PCa effects of Triptolide. Triptolide suppresses SENP1, AR and c-Jun expression, induces restoration of SUMOylation/deSUMOyaltion balance, inhibitions of the AR and c-Jun mediated transcription, which may all contribute to the anti-PCa effect of Triptolide. Other genes or proteins may involve in mechanism of Triptolide effect (see Discussion for detail). doi:10.1371/journal.pone.0037693.g008 siRNAs and DNA plasmids ON-TARGETplus SMARTpool siRNAs for human SENP1 (L- 006357-00), human c-Jun (L-003268-00), human AR (L-003400- 00) and Non-targeting control siRNA were purchased from Dharmacon RNAi Technologies. Flag-EGFP, Flag-SENP1, Flag- c-Jun and Flag-XPB were made by standard cloning method. SENP1, c-Jun and XPB coding DNA fragments without ATG were amplified by PCR using the cDNA reverse transcription from LNCaP total mRNA as template. EGFP coding fragment was cloned from pEGFPN1. Cloned fragments were subcloned into BamHI and EcoRI digested pcDNA3 vector with a Flag tag. The sequences of constructs were confirmed by DNA sequencing. The AR expression plasmid Psl2-HA-AR was a gift from Dr. Guangchao Sui (Wake Forest University). Cell culture LNCaP and PC-3 cells, purchased from Institute of Basic Medical Sciences Chinese Academy of Medical Sciences, were maintained in RPMI1640 (GIBCO) supplemented with 10% fetal bovine serum, 100 units/ml of penicillin and streptomycin, and incubated at 37uC with 5% CO2. various doses of Triptolide or Celastrol, using DMSO as negative control and no treatment as blank control. Cells were gently trypsinized and staining with trypan blue dye. The viable cells were counted using cell counting chamber every 24 h for 7 days. For viability assay, 56103 LNCaP or 36103 PC-3 cells per well were plated in 96-well plate and cultured until attachment, then treated with various doses of Triptolide or Celastrol for 48 h, using DMSO as negative control. After adding 100 ml medium containing 10 ml MTT solution (5 mg/ml stock in PBS) per well, the plates were incubated at 37uC with 5% CO2 for 4 h. Aspirating the medium, the crystals formed in each well were dissolved in 100 mL DMSO and mix on Shaker for 1 min. The absorbance of each well was measured on a Multilabel counter (PerkinElmer Victor3TM 1420) at 595 nm. Each treatment was performed in triplicate and experiments were repeated over 4 times. Data were expressed as percentage of growth inhibition as follows: relative cell viability = (A595 (treated)-A595 (blank))/(A595 (control)-A595 (blank)). 50% inhibitory concentration (IC50) was calculated from viability assay data with GraphPad Prism 5.04 (GraphPad Software, Inc) using a sigmoidal dose-reponse nonlin- ear regression analysis. Cell proliferation assay and viability assay LNCaP and PC-3 cells were plated in 24-well plate at a density of 16104 per well and cultured until attachment, then treated with Transient transfection and treatment LNCaP or PC-3 cells were plated in 6- or 12-well plate and cultured until attachment. To knockdown SENP1, c-Jun or AR PLoS ONE | www.plosone.org 14 May 2012 | Volume 7 | Issue 5 | e37693