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. Author manuscript; available in PMC: 2009 Sep 15.

Published in final edited form as: Nat Genet. 2007 Jan 9;39(2):237–242. doi: 10.1038/ng1972

a. Real-time qRT/PCR analysis of genes frequently hypermethylated in adult cancers following treatment EC cells with all-trans retinoic acid (ATRA, 2uM) for 0 (untreated), 1, 3, 6, 9, and 12 days. PCR reactions were performed in triplicate, and average threshold cycle for altered gene expression with ATRA treatment was normalized to GAPDH. Fold change (log scale) for each gene over untreated Tera-2 cells was calculated using the formula fold change = -log[c^t(treatment)-c^t(untreated)]. Representative results of two or more independent experiments are shown. Genes are divided into two groups by threshold cycle: low to medium expression (top panel - note high cycle threshold number for the PCR, average; 30.8)) and genes with high basal expression (note low threshold cycle number, average; 21.4).

b. Immunohistochemistry of teratocarcinoma tumor grown in NOD/SKID mice. Immunostaining was performed by the Johns Hopkins Immunopathology Department according to established protocols on paraffin embedded section using antibodies to CD34 (40x):endothelial cells, chromogranin (40x); neuroendocrine, cytokeratin (10x): epithelial, alpha fetoprotein (AFP; 20x): yolk sac development, Glial fibrillary acidic protein (GFAP; 20x): glial cells, and Myogenin (40x): muscle.

c. qRT/PCR was performed for RNA from 5×10⁶ Tera-2 cells grown as xenographs in nod-skid mice until tumors reached approximately 1.5 centimeters in diameter. Fold expression change was calculated as described above, and results from Tera2 cells treated with ATRA (2uM) for 12 days are shown for comparison.

a. Real-time qRT/PCR analysis of genes frequently hypermethylated in adult cancers following treatment EC cells with all-trans retinoic acid (ATRA, 2uM) for 0 (untreated), 1, 3, 6, 9, and 12 days. PCR reactions were performed in triplicate, and average threshold cycle for altered gene expression with ATRA treatment was normalized to GAPDH. Fold change (log scale) for each gene over untreated Tera-2 cells was calculated using the formula fold change = -log[c^t(treatment)-c^t(untreated)]. Representative results of two or more independent experiments are shown. Genes are divided into two groups by threshold cycle: low to medium expression (top panel - note high cycle threshold number for the PCR, average; 30.8)) and genes with high basal expression (note low threshold cycle number, average; 21.4).

b. Immunohistochemistry of teratocarcinoma tumor grown in NOD/SKID mice. Immunostaining was performed by the Johns Hopkins Immunopathology Department according to established protocols on paraffin embedded section using antibodies to CD34 (40x):endothelial cells, chromogranin (40x); neuroendocrine, cytokeratin (10x): epithelial, alpha fetoprotein (AFP; 20x): yolk sac development, Glial fibrillary acidic protein (GFAP; 20x): glial cells, and Myogenin (40x): muscle.

c. qRT/PCR was performed for RNA from 5×10⁶ Tera-2 cells grown as xenographs in nod-skid mice until tumors reached approximately 1.5 centimeters in diameter. Fold expression change was calculated as described above, and results from Tera2 cells treated with ATRA (2uM) for 12 days are shown for comparison.