Ding to a conclusion that Notch1 gene is a p53 GDC-0152 site target with a role in human tumor suppression through negative regulation of Rho effectors [19]. Furthermore, Notch mutation study of head and neck squamous cell carcinomas also suggests that Notch1 may function as a tumor suppressor gene rather than an oncogene in this tumor [20]. Although the function of Notch3 is highly indicated to squamous cell differentiation[21,22], studies of Notch1function in response to hypoxia in squamous cell carcinoma cell lines and large series of clinicopathological correlation of Notch1 in human squamous cell carcinomas are still missing. In this study we intended to firstly assess the Notch family expression in three squamous esophageal cancer cell lines and a virus transformed squamous esophageal epithelial cell line, so that the most differentially expressed Notch protein(s) in the cancer and virus transformed cell lines could be identified for further functional and clinicopathological studies, in order to better understand their clinical correlation in a series of 156 patients with ten-year followup.Cell Cultures (DSMZ, Germany) and maintained in 5 CO2 in RPMI 1640 medium supplemented with 10 fetal bovine serum and 100 U/ml penicillin G and 100 mg/ml streptomycin at 37uC with saturated moisture.Quantitative RT-PCRCells in 80 confluent in culture were collected for quantitative RT-PCR analyses of Notch family members. Total RNA was prepared using the RNeasy Micro Kit (QIAGEN, Cat#: 74004) and converted into double-stranded cDNA with 0.5 mg RNA into a 10 ml total volume using the RT2 First Strand Kit (QIAGEN, Cat. No. 330401). Quantitative real time PCR was carried out by ABI 7900 HT machine using the RT2 Profiler PCR Array Human Notch Signaling Pathway kit (QIAGEN, Cat. No. PAHS-059ZA). The thermal cycling conditions were 95uC for 10 minutes, followed by 40 cycles of 95uC for 15 seconds, and 60uC for 1 minute. To verify the NOTCH1 sequence, conventional RT-PCR with two primer pairs which were used in earlier studies and sequencing of the PCR product were performed since information for primer sequences from the QIAGEN kit was not available. The forward and reward Notch1 primers for the first primer pair (a) are 59-GGGTCCACCAGTTTGAATGG-39 and 59-GTTTGCTGGCTGCAGGTTCT-39, respectively, giving product of 306 bp [23]. The forward and reward Notch1 primers for the second primer pair (b) are 59-CTACCTGTCA GACGTGGCCT-39 18325633 and 59-CGCAGA GGGTTGTATTGGTT-39, respectively, giving a product of 357bp [24]. Sequencing of both forward and reward PCR products was performed with BigDye Terminator v1.1 Cycle Sequencing Kit and and GBT440 chemical information analyzed with ABI PRISM 3130 Genetic Analyzer (Applied Biosystems) after the PCR products were purified with BigDye XTerminator Purification Kit (GE Healthcare Life Science, Uppsala, Sweden).Western blot analysisThe Western blotting procedure was published before [18]. Membranes were blocked with 5 non-fat dry milk in TBST for 60 minutes and incubated with the primary antibodies at optimal dilution in TBST/2.5 milk overnight at 4 uC, i.e. goat antiGAPDH (0.2 mg/ml, R D, UK), mouse anti-Oct3/4 (1 mg/ml R D, UK), mouse anti-Sox2 (1 mg/ml R D, UK), HIF-1a (1 mg/ ml R D, UK), HIF-2a (1 mg/ml R D, UK), rabbit anti-Notch1 (1 mg/ml, Cell Signalling, UK) and mouse monoclonal antiHes-1 (1 mg/ml, Abcam, UK). The membranes were then incubated with corresponding secondary HRP-conjugated antibodies before the immuno-complexes were visualized by enhanced che.Ding to a conclusion that Notch1 gene is a p53 target with a role in human tumor suppression through negative regulation of Rho effectors [19]. Furthermore, Notch mutation study of head and neck squamous cell carcinomas also suggests that Notch1 may function as a tumor suppressor gene rather than an oncogene in this tumor [20]. Although the function of Notch3 is highly indicated to squamous cell differentiation[21,22], studies of Notch1function in response to hypoxia in squamous cell carcinoma cell lines and large series of clinicopathological correlation of Notch1 in human squamous cell carcinomas are still missing. In this study we intended to firstly assess the Notch family expression in three squamous esophageal cancer cell lines and a virus transformed squamous esophageal epithelial cell line, so that the most differentially expressed Notch protein(s) in the cancer and virus transformed cell lines could be identified for further functional and clinicopathological studies, in order to better understand their clinical correlation in a series of 156 patients with ten-year followup.Cell Cultures (DSMZ, Germany) and maintained in 5 CO2 in RPMI 1640 medium supplemented with 10 fetal bovine serum and 100 U/ml penicillin G and 100 mg/ml streptomycin at 37uC with saturated moisture.Quantitative RT-PCRCells in 80 confluent in culture were collected for quantitative RT-PCR analyses of Notch family members. Total RNA was prepared using the RNeasy Micro Kit (QIAGEN, Cat#: 74004) and converted into double-stranded cDNA with 0.5 mg RNA into a 10 ml total volume using the RT2 First Strand Kit (QIAGEN, Cat. No. 330401). Quantitative real time PCR was carried out by ABI 7900 HT machine using the RT2 Profiler PCR Array Human Notch Signaling Pathway kit (QIAGEN, Cat. No. PAHS-059ZA). The thermal cycling conditions were 95uC for 10 minutes, followed by 40 cycles of 95uC for 15 seconds, and 60uC for 1 minute. To verify the NOTCH1 sequence, conventional RT-PCR with two primer pairs which were used in earlier studies and sequencing of the PCR product were performed since information for primer sequences from the QIAGEN kit was not available. The forward and reward Notch1 primers for the first primer pair (a) are 59-GGGTCCACCAGTTTGAATGG-39 and 59-GTTTGCTGGCTGCAGGTTCT-39, respectively, giving product of 306 bp [23]. The forward and reward Notch1 primers for the second primer pair (b) are 59-CTACCTGTCA GACGTGGCCT-39 18325633 and 59-CGCAGA GGGTTGTATTGGTT-39, respectively, giving a product of 357bp [24]. Sequencing of both forward and reward PCR products was performed with BigDye Terminator v1.1 Cycle Sequencing Kit and and analyzed with ABI PRISM 3130 Genetic Analyzer (Applied Biosystems) after the PCR products were purified with BigDye XTerminator Purification Kit (GE Healthcare Life Science, Uppsala, Sweden).Western blot analysisThe Western blotting procedure was published before [18]. Membranes were blocked with 5 non-fat dry milk in TBST for 60 minutes and incubated with the primary antibodies at optimal dilution in TBST/2.5 milk overnight at 4 uC, i.e. goat antiGAPDH (0.2 mg/ml, R D, UK), mouse anti-Oct3/4 (1 mg/ml R D, UK), mouse anti-Sox2 (1 mg/ml R D, UK), HIF-1a (1 mg/ ml R D, UK), HIF-2a (1 mg/ml R D, UK), rabbit anti-Notch1 (1 mg/ml, Cell Signalling, UK) and mouse monoclonal antiHes-1 (1 mg/ml, Abcam, UK). The membranes were then incubated with corresponding secondary HRP-conjugated antibodies before the immuno-complexes were visualized by enhanced che.
