qRT-PCR primers used for these experiments.

<p>qRT-PCR primers used for these experiments.</p

qRT-PCR results on flash frozen chondrosarcoma tumors from the UW Sarcoma Tissue Bank relative to positive Control Cell Lines Mel375 (for NY-ESO-1 and PRAME) and JJ (For LAGE-1s).

<p>qRT-PCR results on flash frozen chondrosarcoma tumors from the UW Sarcoma Tissue Bank relative to positive Control Cell Lines Mel375 (for NY-ESO-1 and PRAME) and JJ (For LAGE-1s).</p

qRT-PCR expression in the chondrosarcoma cell line FS, JJ, 105KC and SW1353 with and without treatment using 5-Aza-dC for 48 hours.

<p>Treated cells rested 48 hours prior to RNA extraction. Primers are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032165#pone-0032165-t001" target="_blank">Table 1</a>. Data is presented relative to a reference sample: either Mel375 for NY-ESO-1 and PRAME or JJ for LAGE-1s normalized to GAPDH. Asterisk indicates statistical significance of p<0.05. Note that all CT antigens were significantly increased following 5-Aza-dC in all chondrosarcoma lines with the exception of LAGE-1s expression in the 105KC and JJ. These cell lines both strongly expresses LAGE-1s without treatment and were not changed significantly by 5-Aza-dC. The JJ cell line is not displayed in 1C as it is used for a reference value for LAGE-1s. Error bars describe variation between multiple values within a single experiment. Experiments were repeated at least three times to ensure reproducibility.</p

Chromium Release using two A*0201 expressing chondrosarcoma cell lines: FS and JJ.

<p>Asterisks indicate statistical significance of p<0.05. Each cell line had significantly increased lysis using antigen specific effectors following treatment with 5-Aza-dC with the exception of JJ in combination with NY-ESO-1/LAGE-1s effectors at high E∶T ratios. JJ is highly sensitive to NY-ESO-1/LAGE-1 specific effectors without 5-Aza-dC treatment and the fact that increased lysis was not seen at high E∶T ratios probably represents a maximum lysis that can be detected for the JJ cell line with these effectors in the given conditions. To control against non-specific cell lysis, targets were tested with a MART-1 specific CTL clone used at an E∶T ratio of 25∶1. Minimal killing was seen testing the control effectors against either treated or untreated targets. No cell line had increased lysis following 5-Aza-dC treatment using the control MART-1 effectors (p>0.1). To be sure the MART-1 specific cells were effective, we also targeted the MART-1 expressing cell line Mel526 during each experiment resulting in over 40% lysis (not shown). Error bars describe variation between multiple values within a single experiment. Experiments were repeated at least three times to ensure reproducibility.</p