7 cells plated in 6 cm dishes were pre incubated in serum free DM

7 cells plated in 6 cm dishes were pre incubated in serum free DMEM for 2 h before stimulated with PS F2. At various time after stimulation, whole cell lysates were prepared by treating cells with 200 ul of SDS PAGE sample buffer. To prepare cytoplasmic and nuclear extracts, cells were STI 571 harvested and resuspended in 150 ul of hypotonic buffer and incubated on ice for 15 min. The samples were then mixed with 10 ul of 10% NP 40 and centrifuged at 16,000 g for 30 sec. The supernatant representing the cytosolic fraction was collected, and the pellet containing the nuclei was resuspended in 50 ul of nuclear extract buffer and incubated at 4 C for 15 min with vigorous shaking. After centrifugation at 16,000 g for 5 min, the supernatant representing the nuclear fraction was collected and stored at ?20 C.

Western blot analysis Cell lysates in SDS PAGE sample buffer were heated at 95 C for 5 min, separated by 12. 5% SDS PAGE, and transferred to a nitrocellulose membrane. The mem brane was blocked with 5% bovine serum albumin in Tris buffered saline containing 0. 05% Tween 20 and incubated with primary antibodies specific for JNK, p38, ERK, phospho JNK, phospho p38, phospho ERK, NF ��B, I ��B, B actin, or histone deacetylase 1 at 4 C for overnight. The mem brane was then incubated with horseradish peroxidase conjugated secondary antibodies and visualized with an enhanced chemiluminescence kit and a chemiluminescence imaging system. Densitometric analysis of band in tensities was performed using the ImageJ software. Statistical analysis Statistical analysis was performed using an unpaired, two tailed Students t test and a P 0.

05 was considered significant. Data are reported as mean and SEM. Background Oral cancers are malignancies arising from either tongue, lip, gingivae, palate, salivary glands, buccal mu cosa or floor of the mouth, and accounts for an esti mated 2. 08% of total cancer cases worldwide in 2011. About 90% of oral cancers are squamous carcinomas, with main treatment options that in clude surgery followed by radiotherapy and adjuvant chemotherapy. Even though oral cancers are rela tively preventable, diagnosed patients often face a low five year survival rate of 58%, which has remained un changed over the past three decades despite recent treat ment advances.

Presently, platinum based drugs such as cisplatin, remains one of the most commonly used chemotherapeutic agents available for the treat ment of advanced oral cancers. While CDDP treat ment often results in initial responses and disease stabilization, its long term success is hindered by the de velopment of drug Dacomitinib resistance and dose limiting toxicities through the occurrence of DNA cross linking in sur rounding non cancerous cells. Thus, there is an on going need for modified CDDP combination regimes that can ideally reduce overall dose toxicity through chemo sensitization of oral cancer cells.

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