Journal: PLoS ONE

Article Title: Cysteine Dioxygenase 1 Is a Tumor Suppressor Gene Silenced by Promoter Methylation in Multiple Human Cancers

doi: 10.1371/journal.pone.0044951

Figure Lengend Snippet: , Expression of CDO1 in cell lines was examined by RT-PCR or qRT-PCR analyses. m, mock treatment. a, 5-Aza-dC treatment (5 µM for three days). β-actin was used as a loading control. Methylation status of CDO1 promoter in each cell line was examined and indicated as M for methylation, U for unmethylation, and M/U for co-existence of methylated and unmethylated alleles. CDO1 was completely methylated in all cancer cell lines since only cytosine peaks were observed in CpGs sequenced (100% methylation) while it was not methylated in HEK293 since only thymidine peaks were observed (0% methylation). CDO1 was partially methylated in MCF-12A since both methylated and unmethylated alleles were observed in 10 CpGs of the CDO1 promoters examined. When a cytosine peak were compared with a thymidine peak in each CpG of the 10 CpGs, cytosine peaks were dominant (methylated), but since these “methylated CpGs” were found in less than 50% of total CpGs (10/34), it was considered as “methylation-negative” according to the criteria described in . B . qRT-PCR was performed in cDNAs derived from patients with colon, breast, esophagus, bladder and stomach cancer (T) and patients without cancer (NN) (upper). Relative expression (Fold) was calculated by comparing the ratios of mRNA expression of CDO1 to an internal control gene, β-actin. The CDO1 expression level was determined in 10 lung cancer patients and 10 patients without cancer (lower). 2?-()*100, the expression of CDO1 relative to β-actin calculated based on the threshold cycle (C t ) as 2 −ΔCt (ΔCt = C t, CDO1 - C t,β-actin ). Experiments were done in duplicate, and values indicate means ± SD. *, P<0.05 in T- test. C , The CDO1 expression level was examined in five pairs (A ∼ E) of matched cDNA prepared from patients with colon and lung cancer. PT, primary tumor; PN, matched normal tissues.

Article Snippet: For the qRT-PCR analysis, five matched normal and tumor cDNA (A ∼ E) were purchased from Clontech Laboratories, Inc. (Mountain View, CA). cDNA panels of human normal (NN) and cancer tissue (T) derived from colon, breast, esophagus, bladder and stomach were purchased from BioChain Institute, Inc. (Hayward, CA).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Control, Methylation, Derivative Assay