Fibulin-1 was silenced in bladder cancer by promoter hypermethylation. A) fibulin-1 expression in five bladder cell lines with or without 5-aza-dC treatment was determined by real-time RT-PCR. The results are the average of three independent experiments normalized to GAPDH levels. B) fibulin-1 methylation status in 5 bladder cell lines and two matched pairs of normal (N)/tumor (T) bladder tissues were analyzed by MSP. (M) amplification using primers specific for methylated DNA. (U) amplification using primers specific for unmethylated DNA. IVD (in vitro methylated DNA) and ddH2O were positive and negative controls, respectively. C) qPCR was used to analyze fibulin-1 expression in two matched pairs of bladder tissues mentioned in B). D) Pyrosequencing of fibulin-1 promoter region two matched pairs of bladder tissues mentioned in B), the average methylation rate of 5 CpG was listed. E) The correlation analysis of fibulin-1 expression and methylation in 139 NMIBC patient samples.