BDNF Contributes to Cisplatin Resistance Figure 1. CDDP-resistant HNSCC showed up-regulation of TrkB and BDNF expression. A and B, IC50 values for parental cells and its CDDP-resistant cells, OSC-19, A, and HN-5, B. Cell viability was measured by MTT assay 3-day after incubation with CDDP. Each point shows mean 6 SDE. IC50 values were calculated with the prism software form the GraphPad Software. C and D, Lysates from parental cells and its CDDP-resistance cells were analyzed by western blot assay. After lysates were separated with 12% (BDNF) and 8% (TrkB) SDS-PAGE gels membrane was exposed to proper antibodies. E. The expression levels of TrkB and BDNF were determined with OSC-19 parental (OSC-19P) and its CDDP-resistance cells (OSC- 19CR). Total RNA was extracted from both OSC-19P and OSC-19CR cells and RT-PCR was performed as described at materials and methods. Cloned products were separated with 1% Agarose gel. F. After 24 hours cell culture, medium was collected and the concentration of BDNF was determined with ELISA kit. doi:10.1371/journal.pone.0030246.g001 fresh culture media grown for 48 hours before collecting the medium. This medium was centrifuged at 1000 g for 10 minutes and used for determining BDNF concentration. Immunohistochemistry Tissues for immunochemistry were obtained from mice as previously reported [16]. Immunohistochemistry was conducted as previously described [17], using anti-XiAP (1:100, catalogue # IMG-5770; IMGENEX, San Diego CA, USA), anti-Ki67 (1:50, Dako, Carpinteria, CA, Catalogue # M7240), or anti-Bim antibody (1:100, catalogue # 2933; Cell Signaling, Danvers, MA, USA). Quantification of immuohistochemistry in mouse tumor xenografts was done by counting positive cells in high power field (2006) using Image-Pro MC 6.1 (Media Cybernetics, Inc., PLoS ONE | www.plosone.org 3 January 2012 | Volume 7 | Issue 1 | e30246