BrdU incorporation assay The BrdU incorporation assay was carried out using Cell Proliferation ELISA kits from Roche (Indianapolis, IN). Briefly, 2000 HCT116 cells or 3000 SW620 cells were cultured in 96-well microplates in a final volume of 100 ul culture medium per well, drugs added and cells incubated for 48 hours. BrdU labeling solution was added to a final concentration of 10 uM, and cells incubated for an additional 4 hours at 37uC. The culture medium was removed and each well washed twice with 250 ul wash medium containing 10% serum. Cells were fixed and treated with nucleases working solution for 30 min at 37uC. Incorporated BrdU was detected using anti-BrdU-POD, Fab fragments and peroxidase substrate ABTS. Sample absorbance was measured at 405 nm against background control, using a Victor 3 multiwell plate reader. Inhibition rate was calculated for each well as (A405 control cells - A405 treated cells)/A405 control cells6100% (A405: OD value at 405 nm) Senescence detection assay SA-b-gal enzymatic activity was detected using a Senescence Cell Staining kit from Sigma-Aldrich (St. Louis, MO). Briefly, 10,000 cells were plated to 12-well plate, incubated at 37uC overnight and treated with control vehicle or drugs. Forty eight hours after treatment, cells were washed with PBS (pH 6.0), fixed overnight, and stained with 5-bromo-4- chloro-3-indolyl-h-D- galactopyranoside (X-Gal) overnight at 37uC. Ten fields were randomly selected and cells counted. Senescent rate was calculated for each field as positive cells/all cells6100%. Tunel assay Tunel assay was carried out using in situ cell death detection kit from Roche. Briefly, 10,000 cells were plated to 12-well plate, incubated at 37uC overnight and treated with control vehicle or drugs. Forty eight or 96 hours after treatment, control and drug- treated cells were trypsinized and resuspended in 100 ul fixation solution for 30 minutes Cells were permeabilized, washed and incubated in TUNEL assay mixture at 37uC for 1 hour. Cells were washed, incubated in 10 ul 16 PBS+0.5 mg/ml propidium iodide (PI) for 10 min and subject to microscopic analysis. Ten fields were randomly selected and cells counted. Apoptotic rate was calculated for each field as positive cells/all cells6100%. Western blot Protein extracts were resolved by Nupage 4–12% Bis-Tris Gel (Invitrogen), samples electro-blotted onto PVDF membrane (Invitrogen), and membranes blocked and incubated with primary antibody. Donkey anti-rabbit or anti-mouse (GE Healthcare, NJ) antibodies were conjugated to horseradish peroxide to reveal immunocomplexes by enhanced chemiluminescence (Pierce, IL). The detected bands were quantified using Image J v1.43 as instructed in the software manual. ChIP assay Chromatin immunoprecipitaton was performed with ChIP-IT kits (Active Motif, Carlsbad, CA). Briefly, cells were fixed and chromatin sonicated to 0.3–2 kb with most at 0.8 kb. A negative IgG provided in the kit was used as a negative control antibody. Chromatin samples were immunoprecipitated using depicted antibodies, RNA removed by addition of Rnase A and cross-links removed by incubating at 65uC for 4 hours. Proteins were removed by treating with proteinase K, and DNA purified and subjected to realtime PCR detection. PTTG1 and Drug-Induced Senescence Luciferase assay p21 promoter activity assay was carried out using Dual Luciferase Reporter System (Promega) according to the manufac- turer’s protocol. Briefly, ten thousand cells were seeded in 24-well plates and incubated at 37uC overnight. Each well was co- transfected with 200 ng luciferase plasmids (pGL3-Basic as control, p21 promoter with/out p53 binding sites) and 5 ng pRL-TK plasmids (Promega) encoding Renilla (internal control to assess transfection efficiency). After 24 hours, whole-cell lysates were collected and measured using an Orion Microplate Luminometer (Berthold Detection System). Transfections were performed in triplicate and repeated three times to assure reproducibility. All luciferase reads were normalized to corre- sponding Renilla value. Relative luciferase activity was calculated using the following formula: Relative luciferase levels = Luc- promoter/Luc-pGL3 basic. Subcutaneously xenografted tumor Animal protocols were approved by the Cedars Sinai Institu- tional Animal Care and Use Committee (protocol # 2603). 5- to 6-week-old female athymic mice were separated into two groups, one for HCT116 WT and another for HCT116 PTTG12/2 cells. Cells (0.1 ml Matrigel containing 16106 cells) were injected subcutaneously into the lumbar area at left side to generate tumors. Tumors were allowed to grow when the first tumor reaching ,100 mm3, at which time mice were further randomized to 2 treatment groups, with 10 evaluable tumors in each group: (a) control and (b) drug treated. Doxorubicin was administered iv through the tail vein (10 mg/kg) at day 1 after grouping. Mice were monitored daily for signs of toxicity and weighed twice weekly. Tumor sizes were measured twice weekly by caliper measurements using the following formula: tumor volume = (length6width2) /2 as previously reported [30,31]. Relative tumor growth was calculated by relative tumor size of treated mice divided by relative tumor size of control mice (T/C). At the end of the study, 3 weeks after cell injection, animals were euthanized under CO2 inhalation followed by cervical dislocation to ensure death (consistent with the Panel on Euthanasia of the American Veterinary Medical Association). Tumor tissue was collected, weighed and cut into three pieces for analysis. One was used for mRNA extraction, one was frozen immediately in liquid nitrogen for protein analysis and the third one fixed with formaldehyde, embedded in paraffin and sectioned. Immunofluorescence Tissue sections were deparaffinized in xylene, hydrated in graded ethanol, and heated in Target Retrieval Solution (DakoCytomation) to retrieve antigen at 95uC for 40 minutes. Slides were permeabilized with 1% triton-X-100 in PBS for 30 minutes, and incubated in blocking buffer (10% goat serum, 1% BSA, 0.1% triton-X-100 in PBS) for 1 hour. After washing with PBS, slides were hybridized at 4uC overnight with antibodies against indicated proteins. Primary antibody was omitted for negative controls. Alexa Fluor anti-mouse antibody with green fluorescence or anti-rabbit antibody with red fluorescence (Molecular Probes, Carlsbad, CA) were used as secondary antibodies and incubated at room temperature avoiding light for 1 hour. Slides were mounted with ProLong Gold Antifade Reagent with DAPI (Invitrogen), and nuclei dyed by DAPI with blue fluorescence. Senescence activity in tissue samples was assessed by measuring p21 expression and b-gal activity. Ten fields were randomly selected and cells counted. Senescent or apoptotic rate was calculated for each field as positive cells/all cells6100%. PLoS ONE | www.plosone.org 2 August 2011 | Volume 6 | Issue 8 | e23754