1% Triton 100 in PBS for 2 min on ice. The TUNEL assay was carried out following the manufacturers instruction. Immunofluorescence Cells were grown, treated with 50 nM Mcl 1 siRNA, and fi ed as previously described, and stained using rabbit polyclonal anti LC3 antibody for LC3 staining. The LC3 dots were quantified www.selleckchem.com/products/CP-690550.html using the Image J software command analyze particles, which counts and measures objects in thresholded images as we previously described. Determination of cell viability Cell viability was determined by the WST 8 kit from Dojindo Labs. siRNA was transfected 18 h after cell seeding in a 96 well plate and viability assessed 24, 48 and 72 h after transfection. Briefly, 10ul of the tetrazo lium substrate was added to each well and plates were incubated at 37 C for 1 h after which the absorbance at 450 nm measured.

All e periments were done in tripli cate and repeated at least three times. Quantitative real time PCR RNA isolation was performed using the mirVana RNA isolation kit. cDNA synthesis was carried out using 1 ug of total RNA using the miScript II RT Kit or High Capacity cDNA Reverse Transcription Kits. Real time PCR was performed using the miScript SYBR green PCR kit ac cording to the manufacturers instructions. Mcl 1 primers primers were purchased from Qiagen. 18S and U6 were used as internal controls for quantifying Mcl 1 and miR 204 levels respectively. Relative levels of Mcl 1 or miR 204 were assessed using the Ct method. Dual Luciferase reporter assay and 3UTR binding site mutagenesis MIA PaCa 2 and S2 VP10 cells were seeded in 24 well plates immediately prior to transfection.

The Mcl 1 derived miR 204 binding site or a binding site deletion in the 3UTR was inserted into the psiCheck2 e pressing firefly luciferase plasmid and transfected into MIA PaCa 2 or S2 VP10 cells Drug_discovery using Attractene following manufacturers instruc tions. The miR 204 mimic was co transfected where indicated. Forty eight hours post transfection, cells were assayed for both firefly and renilla luciferase using the dual luciferase glow assay. Human tumor enograft model Three de identified human tumors were implanted sub cutaneously into SCID animals. Once tumor size reached 500 mm3, tumors were dissected and cut into 10 mm3 pieces, which were then subcutaneously implanted into both flanks of additional SCID mice. One animal was treated with saline and the other with the water soluble prodrug of triptolide, Minnelide for 7 days. Animals were sacrificed 7 days after start of the treatment and RNA e tracted from tumors was evaluated for Mcl 1 and miR 204 e pression. All e periments were performed in accordance with institutional guidelines and approved by the animal care and use committee at the University of Minnesota.