Expression of PTGF-β in conditioned medium and requirement of intact TGFβ signaling pathway for growth inhibition. (A) Expression of PTGF-β in the conditioned medium collected from PTGF-β-expressing DLD-Cl7 cells, but not from Dn1-3 vector control. Conditioned medium was collected and subjected (85 μg) to Western blot analysis using Flag-tag antibody. (B) PTGF-β-induced growth inhibition of TGF-β-sensitive but not TGF-β-resistant cells. The target cells used are TGF-β-sensitive Mv1Lu or Du145 cells as well as TGF-β-resistant cells, including RKO (TβRII mutation), R1B/L17 (TβRI mutation), and MDA-MB468 (Smad4-null). Conditioned medium was collected from PTGF-β expressing clone, DLD-Cl7 or the vector control, and Dn1-3 cells and was concentrated and added into the target cells at various protein concentrations, followed by BrdUrd incorporation assay. All results were presented as percent of growth rate ± standard error of the mean from three independent experiments, each run in quadruplet. The growth rate was calculated with the control group (without any addition of conditioned medium) setting as 100%.