OPCML-associated RTK modulation affects downstream signaling. OPCML-associated RTK modulation affects downstream signaling. A, Western blots of total and phospho HER2 and EGFR protein from SKOBS-V1.2 (vector control), BKS-2.1 cells, and SKOBS-3.5 were subjected to a 60-minute EGF (50 ng/mL) time course demonstrating profound abrogation of tHER2, HER2, and EGFR phosphorylation (Y-1248 and Y-1173, respectively) in BKS2.1 and SKOBS-3.5. OPCML transfection was also associated with abrogation of EGF phospho-activation of ERK 1 and 2 (T-202/T-204) and Akt (S-473), more profoundly in BKS-2.1 line. Representative Western blots were run in parallel with equivalent loadings of the same lysate preparation for each of the 3 cell lines and are defined by their β-tubulin bands. BKS2.1 tERK/pERK (left) is from the same blot as tERK/pERK for SKOBS-V1.2/SKOBS3.5 on the right, and therefore the same SKOBSV1.2 empty vector–transfected control data are shown alongside for ease of interpretation (original blot shown in Supplementary Fig. S9). BKS2.1 and SKOBS V1.2 tAkt/pAkt are from the same blot: removal of irrelevant additional sample lanes, for ease of interpretation, is indicated by black boxes. SKOBS V1.2 and SKOBS 3.5 tAkt/pAkt (bottom right) are a separate representative blot. B, the same cells subjected to a 60-minute FGF1 (10 ng/mL) time course again demonstrate loss of total and phospho (Y-766) FGFR1. C, analysis of shRNA clones by quantitative reverse-transcription PCR for OPCML showing 2 clones exhibiting reduced OPCML mRNA levels relative to the PLKO-1 and PLKO-2 (empty–vector clones) and shSCRAMbled controls. We focused on 2 clones; *sh464–23, which exhibited 60% knockdown, and **sh339–24, which exhibited 95% knockdown of OPCML. D, Western blot analysis demonstrated that total HER2 and EphA2 are strongly upregulated in the shRNA lines with 95% OPCML knockdown. E, Western blots showing the upregulation of HER2 and EphA2 upon 95% knockdown of OPCML selectively on serum and on exposure to ligand stimulation of cells. F, cell proliferation assay shows that OSE-C2-sh339–24 line with 95% knockdown of OPCML exhibits greater rates of proliferation at 48, 72, and 96 hours compared with PLKO-2 control (*P = 0.002, **P = 0.009, and ***P = 0.003, respectively) compared with shRNA scrambled and empty–vector PLKO controls in 10% FBS. No such growth rate difference was demonstrated in 0.5% FBS, underscoring the RTK ligand dependency of OPCML effect (data not shown). Arthur B. McKie et al. Cancer Discovery 2012;2:156-171 ©2012 by American Association for Cancer Research