Kwon et al. BMC Cancer 2010, 10:392 http://www.biomedcentral.com/1471-2407/10/392 18 hours culture in complete media, cells were stimulated with PMA (phorbol 12-myristate 13-acetate) and iono- mycin (P+I) for 4 hours in the presence or absence sev- eral dose of cinnamon extract from 0.1 mg/ml to 0.5 mg/ ml. Luciferase activity measured by dual luciferase assay system (Promega) is expressed relative to expression of the cotransfected Renilla luciferase promoter (phRL-null; Promega) to control for transfection efficiency. RNA isolation, cDNA synthesis, quantitative RT-PCR and standard RT-PCR Total RNA was prepared using TRI Reagent (Molecular Research Center) according to the manufacturer’s proto- col. For reverse transcription, cDNA was generated using 1 μg of total RNA, oligo (dT) primer (Promega) and Improm-II Reverse Transcriptase (Promega) in a total volume of 20 μl. One μl of cDNA was amplified using the following RT-PCR primer sets: L32 (5′-GAGGACCAA- GAAGTTCATCAG-3′ and 5′-GCACAGTAAGATTT GTTGCAC-3′), BcL-xL (5′-GACAAGGAGATGCAGG- TATTGG-3′ and 5′-TCCCGTAGAGATCCACAAAAGT- 3′), Bcl-2 (5′-ATGCCTTTGTGGAACTATATGGC-3′); Bak (5′-GTGACCTGCTTTTTGGCTGAT-3′ and 5′-GG TCTCTACGCAAATTCAGGG-3′); Bax (5′-TGAAGAC AGGGGCCTTTTTG-3′ and 5′-AATTCGCCGGAGAC ACTCG-3′); Bim (5′-CCCGGAGATACGGATTGCAC-3′ and 5′-GCCTCGCGGTAATCATTTGC-3′); Bad (5′-AA GTCCGATCCCGGAATCC-3′ and 5′-GCTCA CTCGGC TCAAACTCT-3′) and 5′-GGTATGCACCCAGAGTGAT GC-3′), and Survivin (5′-CTACCGAGAACGAGCCT- GATT-3′ and 5′- AGCCTTCCAATTCCTTAAAGCAG-3′). Preparation of nuclear extracts Cell lines or cells isolated from tumor tissues were washed twice with ice cold PBS and incubated in 1ml of lysis buffer (10 mM Tris/HCl, 3 mM CaCl2, 2 mM MgCl2) containing a protease inhibitor cocktail (Roche) for 10 mins on ice. Then the cells were vortexed gently and incubated in 1ml of NP-40 buffer (10 mM Tris/HCl, 3 mM CaCl2, 2 mM MgCl2, 1% NP-40) for 5 mins at 4°C, and the suspension was centrifuged at 3000 rpm for 10 mins at 4°C. Nuclei was washed with 1ml of Buffer A (20 mM Hepes-KOH, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT, 0.5 mM PMSF), and 100 μl of Buffer C (20 mM Hepes-KOH, 25% Glycerol, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 5 mM DTT, 0.5 mM PMSF, 1% Triton X-100) was added to the pellet and vortexed vig- orously at 4°C for 10 mins. Nuclear debris was removed by centrifugation at 13000 g for 5 mins. Protein concen- trations were determined by the Bradford Assay (Bio- Rad). The nuclear extract was confirmed by immunoblot- ting with anti-Lamin B and anti-Tubulin beta. For single cell suspension of tumor tissues, tumor tissues from each group was homogeized with homogenizer (Fluko). Page 3 of 10 Immunoblotting Proteins were resolved by 10% (for NF B and AP1) or 15% (for caspase-3, Bcl2, Bcl-xL, Bad, Bax, Bak, Bim and Sruvivin) SDS-PAGE gels, transferred onto a PVDF membrane (Bio-RAD) and subjected to Western blot analysis using anti-NF B (Abcam), anti-pc-JUN (Santa- Cruz), anti-caspases-3 (Abcam), anti-Bcl-2 (Abcam), anti-Bcl-xL (Cell signaling), anti-Survivin (Cell signal- ing), anti-Bad (Abcam), anti-Bax (Abcam), anti-Bak (Abcam), anti-Bim (Abcam) and peroxidase-conjugated secondary antibodies (DAKO). Proteins were visualized with a chemiluminescence kit (Amersham Bioscience). The levels of Tubulin (anti-tubulin; Santa Cruz), beta- actin (anti-beta-actin; Abcam) and Lamin B (anti-lamin B; SantaCruz) detected by relevant antibodies were monitored as a loading control. Melanoma induction and anti-tumor assay Mouse melanoma B16F10 (1 × 106 cells/0.1ml) cells were injected subcutaneously (s.c) into the flanks of C57BL/6 mice (6 weeks old male). One week after the injection, mice were divided into two groups (10 mice/ each group) and orally treated with either 10 mg/dose (400 μg/g mouse weight) of cinnamon extract in 100 μl of PBS or same volume of PBS alone as a sham control for 30 days. During the treatment period, the tumor size was measured with vernier calipers every 2 days, and tumor volumes were calculated using the standard for- mula: width2 × length × 0.52. Mice were sacrificed for further analysis after 30 days of treatment. DNA fragmentation assay Genomic DNA isolation was performed with gDNA puri- fication kit (Solgent, Korea). Briefly, mouse tumor tissues from the differentially treated group were collected, pooled, and 5 mg of tumor tissues from each group were transferred. They were dissolved in 300ml of cell lysis buf- fer with 25 mg of proteinase K for 4 hours at 55°C, and then mixed with 100ml of protein precipitation solution. Then solution was centrifugated at 14000 rpm for 3 mins. After centrifugation, DNAs was precipitated, washed with isopropanol and 70% ethanol. DNA pellets were dissolved in 100 μl of DNA hydration solution. Finally, fragmented DNAs (10 ml) were visualized in 2% agarose gels. Nuclear staining Cells seeded on the glass in 12 well plate were incubated with cinnamon extract for 72 hours, washed with PBS and fixed with 4% paraformaldehyde for 15 mins at RT. Fixed cells were incubated in PBS (pH 7.4) containing 200mg of DNase-free RNase (Sigma) for 30 mins at 37°C and stained with 2 mg/ml of Hoechst for 10 mins at 37°C. Nuclear morphology of the cells was observed under fluorescence microscope.