Figure 5

Target validation in vitro and in vivo. (A) Validation of mRNA levels of a subset of PDGF-regulated genes (HMOX1, PDGFRB, CYR61, CXCL12, GDF15 and DIAPH3) in pBSMC. (B, C) PDGF-induced changes in expression of the CXCL12, CYR61 and HMOX1 genes were evaluated in the context of MYC (B) and JNK/JUN (C) inhibition, using real time RT-PCR analysis. (D) Protein level changes in PDGFRβ, CYR61 and GDF15 in response to PDGF treatment for different times were verified by immunoblot analysis. Data are representative of three independent trials. (E) Sensitivity of PDGF-induced changes in PDGFRβ and CYR61 and to inhibition of JNK and MYC was assessed by immunoblot analysis. The long exposure is included to appreciate differences in sensitivity of PDGFRβ to JNK and MYC inhibition. (F) Immunoblot analysis of whole bladder tissue (WB, n = 2, pooled) or bladder smooth muscle (BSM, n = 5, pooled) from mice subjected to sham surgery (Sh) or outlet obstruction (Obs) were blotted with the indicated antibodies; serum-depleted pBSMC treated without (-) or with 1 nM PDGF-BB for 2 h (+) were included as negative and positive controls respectively. (G) Bladder smooth muscle tissues from sham-operated (Sham, n = 3) or obstructed mice (Obs, n = 3) were subjected to real-time RT-PCR analysis for the indicated transcripts.