![]() ![]() Transfection efficiency was determined by pCMV- β-gal transfection and staining of cells for β-galactosidase activity. The plasmid pcDNA3-Pax7 was transfected into 293T cells using Qiagen Effectene reagent (Qiagen Inc., Valencia, CA, USA). 293T cells transfected with Pax7 plasmid served as a positive control. The following primers were used: G3PDH (human) primers: sense 5′-TGAAGGTCGAGTCAACGGATTTGGT-3′ and antisense 5′-CATGTGGGCATGAGGTCCACCAC-3′, with an expected PCR product of 983 bp and PAX7 primers: forward 5′-GCTCCGGGGCAGAACTACC-3′ and reverse 5′-GCACGCGGCTAATCGAACTC-3′, with expected PCR product of 436 bp. A 2- μl portion of cDNA was used for the PCR experiment. As a positive control, each RNA sample was tested for the expression of the housekeeping gene G3PDH. As a negative control against genomic DNA contamination, each RNA sample was tested by PCR following an RT step in the absence of RT. ![]() Reverse transcriptase-PCR (RT-PCR) was performed using SuperScript III First-Strand Synthesis System for RT-PCR following the manufacturer’s protocols (Invitrogen), using random hexamer primers to generate cDNA strands. RNA was isolated from freshly growing cells using Trizol (Invitrogen). Reverse transcriptase-PCR for Pax7 Expression in Melanoma Cell Lines The membranes were stripped using Restore Western Blot Stripping Buffer (Pierce Biotechnology, Rockford, IL, USA), checked for complete removal of antibodies, and reanalyzed for β-tubulin expression (1:400 mouse monoclonal antibody E7 University of Iowa Hybridoma Bank) as a loading control. For western blot analysis detection of PAX6 and β-tubulin, cell lysate (40 μg/well) was loaded on 4–12% gradient gels, transferred to PVDF membranes, and probed with PAX3 mouse monoclonal antibody (1:100 dilution University of Iowa Hybridoma Bank, Iowa City, IA, USA) and western blotted (Western Breeze Chemiluminescent kit Invitrogen, Carlsbad, CA, USA). The mouse fibroblast 3T3 and human pancreatic cancer cell line CFPAC1 are available from ATCC (Manassas, VA, USA). We studied the B16 murine melanoma cell line and eight human melanoma cell lines: 537, 624, 888, A375, HIM2, SK23, SKMEL5, and SKMEL28. Our data suggest that PAX3-expressing melanomas may be less environmentally dependent and more genetically linked.Īll cell lines were obtained through the University of Chicago Cancer Research Center. Further analysis of our melanoma set revealed that PAX3 expression is strongly correlated with younger patients with low or no evidence of sun damage. We also find that PAX3 is commonly expressed in primary melanoma samples (2 1/58) but significantly less frequently in benign pigmented lesions (9/75). ![]() ![]() Here, we find PAX3 expression in 8/8 melanoma cell lines. This mechanism may also contribute to the uncontrolled cell growth and loss of terminal differentiation in melanomas. Based on this function, PAX3 promotes a melanocytic phenotype but blocks terminal differentiation. The transcription factor PAX3 has a well-established role in the development of melanocytes during embryogenesis, and has recently been characterized as a molecular switch in the mature melanocyte. Although the histogenesis of the tumor is not well understood, it is thought to originate from a rare melanocyte stem cell that resides in the skin. Siddiqi, Camille Rizkallah, Trudi Hanley, Neon Cobran, Gary A.Melanoma is responsible for an estimated 62 000 new American cancer diagnoses and is projected to cause nearly 8000 deaths in 2008 alone. Germain, Peter Mensah, Arthur Holden, Michael Sinelnikoff, John Dunn-Hill, Dennis St John, Neil Napier, Dylan Smith, Maurizio Terrazzano, Robert Paradis, Kwasi Songui, Alexandra Beaton, Frédéric Smith, Loucas Minchillo, Nicholas Minchillo, Tom Rack, David Francis, James Bradford, Andrew Shaver, Robin Wilcock, Kent McQuaid, Marcel Jeannin, Jere Gillis, Jeremy Thibodeau, Tyrone Benskin, Robert Maillet, Patrick Sabongui, Leon Laderach, Dave Lapommeray, Vervi Mauricio, Charles Papasoff, Isabelle Champeau, Veronique-Natale Szalankiewicz, Maéva Nadon, David Thibodeau, David Schaap, Jean Michel Paré, Stewart Myiow, Andreanne Ross, Sara Giacalone, Ariadne Bourbonnière, Isabelle Fournel, Sandrine Merette-Attiow, Elisabeth Etienne, Danielle Hubbard, Ruan Vibegaard, Genevieve Guilbault, Bonnie Mak, Amélie Sorel, Caroline Aspirot, Gina Gagnon, Tania Trudell, Stéphanie Aubry, Mercedes Leggett, Stephania Gambaroff, Chanelle Lamothe, Sabrina-Jasmine Guilbault, Manny Cortez Tuazon, Cindy, Atif Y. All actors – Gerard Butler, Lena Headey, Dominic West, David Wenham, Vincent Regan, Michael Fassbender, Tom Wisdom, Andrew Pleavin, Andrew Tiernan, Rodrigo Santoro, Giovani Cimmino, Stephen McHattie, Greg Kramer, Alex Ivanovici, Kelly Craig, Eli Snyder, Tyler Neitzel, Tim Connolly, Marie-Julie Rivest, Sebastian St. ![]()
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