Perlecan and the SHH-GLI1 pathway. A. Decreased Perlecan and SHH signaling in Perlecan RNAi treated LNCaP cells. Expression of Perlecan, and the SHH signaling molecules PTCH1 and GLI1 as determined by Real Time PCR. Black columns represent control samples, Grey columns represent Perlecan RNAi treated cells. All expression normalized to β-actin levels. Real Time PCR studies were run with an n = 9. Error bars indicate standard deviation. B. Gli-1 transfection restores BrdU Proliferation in Perlecan RNAi treated cells. Percent BrdU incorporation normalized to levels of BrdU incorporation in control (scrambled RNAi treated) cells. BrdU analysis was done with n = 6. Error bars indicate standard deviation. C. Immunoprecipitation with anti-Perlecan antibody pulls down SHH. Co-immunoprecipitation of SHH and Perlecan from equal amounts of medium conditioned by 80% confluent cells. Size marker is indicated. Due to modifications, mature SHH runs as an approximately 22 kD band. Note the increased amount of bound SHH in the C4-2 and C4-2B cell lines. D. Relative expression of the SHH pathway components in LNCaP series cells. Black columns represent SHH mRNA, grey columns represent PTCH mRNA, with expression presented as ratios with respect to expression in LNCaP cells. While SHH is lower, PTCH is higher in the androgen insensitive metastatic cell lines C4-2 and C4-2B compared to LNCaP. All mRNAs by QRT-PCR were normalized to Beta-actin.