isobologram is a graphical representation of the interaction between two drugs and is formed by plotting the individual drug doses required to achieve a single agent effect on their respective x and y axes, a line connecting the two points is drawn and the concentrations of the two drugs used in combination to achieve the same effect are plotted on the isobologram. Combination data points that fall on the line represent an additive interaction, whereas points above or below represent antagonism or synergy, respectively. Lentivirus constructs The lentiviruses encoding siRNA against the three GADD45 family members have been previously described [34]. The LVsiRNA GFP construct (control) was kindly donated by Dr. Oded Singer (Salk Institute for Biological Studies, San Diego, CA). The lentivirus encoding siRNA against mda-7/IL-24 gene was cloned using Advantage 2 PCR kit (Clontech, Mountain View, CA), and the virus was generated by using a previously described methodology [34]. The following siRNA oligonucleotide for mda- 7/IL-24 was used: 59CTGTCTAGACAAAAACTTTGTTCT- CATCGTGTCATCTCTTGAATGACACGATGAGAACAAA- GGGGGATCTGTGGTCTCATACA-39. Animal experiment Severe combined immunodeficient (SCID)-beige mice were purchased from Taconic (Germantown, NY) and housed in a pathogen-free environment. The animals were randomly divided into 3 groups (n = 7 per group). 8-week-old female SCID-beige mice were fed with one of the experimental diets supplemented with 200 ppm of Sulindac Sulfide, 100 ppm of Diclofenac or control solvents for 2 weeks. Immediately before implantation, SKOV-3 cells were trypsinized and resuspended in MEM with 10% fetal bovine serum. Cell viability was determined by trypan blue exclusion and a single cell suspension with .90% viability was used for implantation. SKOV-3 cells (26106 cells in 50 ml) were carefully injected subcutaneously as described previously [23,34] and animals continued on experimental diets. Tumor size (volume) was measured every 5 days, starting at day 35 after implantation. The experiment was finished when the average tumor weight in the control animals reached 2–5% of the body weight. The diets were prepared by Research Diets, Inc. (New Brunswick, NJ). Body weight and food intake were measured weekly. All procedures with animals were reviewed and approved by the Institutional Animal Care and Use Committee at the Beth Israel Deaconess Medical Center according to NIH guidelines. Supporting Information Figure S1 Multiple NSAIDs induce apoptosis and mda- 7/IL-24 gene expression in ovarian cancer cells. CAOV-3 and SW626 ovarian cancer cell lines after treatment with 5 mM Aspirin, 200 mM Ibuprofen, 1 mM Acetaminophen, 200 mM Naproxen, 200 mM NS-398, 200 mM Diclofenac, 50 mM Finaste- ride, 200 mM Flufenamic acid, 40 mM Meloxicam, 50 mM Ebselen, 20 nM Flurbiprofen, 50 mM Sulindac Sulfide and 50 mM Sulindac Sulfone or DMSO as control. (A) Apoptosis assay of ovarian cancer cells after NSAID treatment. Data means 6 s.d. of triplicate independent experiments for each treatment. (B) Real time PCR analysis of mda-7/IL-24 expression in SKOV-3 cells after 24 hours treatment with different NSAIDs. Each sample was normalized to hGAPDH. (C) NSAIDs mediated induction of mda-7/IL-24. Western-blot analysis using antibody against mda-7/ IL-24 (kindly provided by Dr. Paul B. Fisher, Virginia Common- NSAIDs and NF-kB Inhibitors in Ovarian Cancer wealth University, School of Medicine) shows that treatment with 50 mM Sulindac Sulfide induces mda-7/IL-24 expression in a cell line with silenced GFP control gene, while cells with silenced mda- 7/IL-24 and treated with 50 mM Sulindac Sulfide shows abrogation of its induced expression mediated by NSAIDs. (TIF) Figure S2 Synergistic effects of NSAID used in combi- nations. (A) Apoptosis assay of CAOV-3 and SW626 cells after treatment with 10 mM Sulindac Sulfide, 40 mM Diclofenac, 25 mM Ebselen or 40 mM Naproxen and a combination thereof or DMSO. Apoptosis was measured 24 hours post-treatment. Data means 6 s.d. of triplicate independent experiments for each treatment. (TIF) Figure S3 NSAIDS treatment of ovarian cancer cells induces GADD45 family gene expression. Real time PCR analysis of GADD45a (A) and GADD45c (B) expression after NSAIDS treatment with 5 mM Aspirin, 200 mM Ibuprofen, 1 mM Acetaminophen, 200 mM Naproxen, 200 mM NS-398, 200 mM Diclofenac, 50 mM Finasteride, 200 mM Flufenamic acid, 40 mM Meloxican, 50 mM Ebselen, 20 nM Flurbiprofen, 50 mM Sulindac Sulfide and 50 mM Sulindac Sulfone or DMSO as control. Total RNA was collected from SKOV-3, CAOV-3 and SW626 cells 24 hours after treatment. Normalization of each sample was carried out by measuring the amount of hGAPDH cDNA. (C) Real time PCR analysis of GADD45 a and c expression after treatment with 200 mM Diclofenac and infection with lentivirus encoding siRNA against mda-7/IL-24 or GFP genes. Total RNA was collected from SKOV-3 cell lines after 24 hours after treatment. (TIF) Figure S4 NSAIDs and NF-kB inhibitor activities in ovarian cancer cells. (A) NSAIDs and NF-kB inhibitors induce PARP activation. Western Blot analysis using anti PARP antibody (Santa Cruz) of cell lysates from CAOV-3 cells treated with 50 mM Sulindac Sulfide, 100 mM Diclofenac, 200 mM IKK inhibitor II Wedelolactone (7-Methoxy-5,11,12-trihydroxy-coumestan) or DMSO. (B) Real-time PCR analysis of CAOV-3 cells treated with 200 mM IKK inhibitor II Wedelolactone (7-Methoxy- 5,11,12-trihydroxy-coumestan) or DMSO shows induced expres- sion of GADD45 a and c genes, (C) while western Blot analysis using anti-phospho JNK antibody of cell lysates from CAOV-3 cells treated with 200 mM IKK inhibitor II Wedelolactone (7- Methoxy-5,11,12-trihydroxy-coumestan) or DMSO shows activa- tion of JNK. (TIF) Methods S1 (DOCX) Acknowledgments Description of Real Time methodology. We are grateful to Dr. Inder M. Verma and Dr. Oded Singer for help with the generation of lentiviruses. Author Contributions Conceived and designed the experiments: LFZ RET AC JZ DS PBF TAL. Performed the experiments: LFZ RET RGC AC JC MB FMS YW XG LL JZ. Analyzed the data: LFZ RET RGC AC JC MB FMS YW XG LL JZ DS PBF TAL. Contributed reagents/materials/analysis tools: RGC DS PBF. Wrote the paper: LFZ RET DS PBF TAL. PLoS ONE | www.plosone.org 9 September 2011 | Volume 6 | Issue 9 | e24285