Stable heparanase silencing of RH30 and RD cell lines with shRNA. (A) HPSE mRNA expression levels in control (black bars) and silenced (hatched bars) RH30 and RD cell lines were determined by Real-time PCR. The results were normalized using GAPDH as internal control and represent the mean ± standard deviation of three samples performed in duplicate. (B) Western blot analysis was performed to demonstrate HPSE silencing between control and silenced RH30 and RD cell lines. GAPDH was included as loading control. (C) Heparanase activity in serum-free conditioned media obtained from control (black bars) and silenced (hatched bars) RH30 and RD cell lines. The enzymatic activity is expressed as nanograms of heparan sulphate (HS) removed per minute. The results represent the mean ± standard deviation of three independent experiments performed in duplicate. Platelet extract was used as positive control (PC, white bar). An asterisk (*) indicates a significant difference from control (P < 0.05).