Figure 4. Platelet-Derived TGFβ1 and Platelet-Bound Factors Cooperate to Promote Metastasis.
(A) Concentration of TGFβ measured by ELISA in the conditioned medium of MC38GFP or Ep5 cells incubated with buffer, platelets, releasate from activated platelets (releasate), or the pellet fraction from activated platelets (pellet) for 40h (n=4–6).
(B) Detection of phospho-Smad2 and total Smad2 protein levels by immunoblotting in Ep5 cells treated as in (A). β-tubulin was used as loading control.
(C) Relative luciferase activity (RLU) in Ep5 cells stably expressing a luciferase reporter under the control of the SBE promoter and treated as in (A) for 20h (n=2).
(D) Numbers of metastatic foci at the surface of lungs (2 largest lobes) 14 days after tail-vein injection of MC38GFP or Ep5-ZsGreen cells pretreated with buffer, platelets, releasate from activated platelets (releasate), or the pellet fraction from activated platelets (pellet) for 40h (n=5–17).
(E) Numbers of tumor cells at the surface of lungs 48h after tail-vein injection of MC38GFP or Ep5-ZsGreen cells pretreated as in (D). Each bar represents the mean ± SEM number of cells/view field (3X) (n=5–13). *p<0.05, **p<0.01 vs buffer were determined by Student’s t-test.
(F) Phase-contrast micrographs of MC38GFP and Ep5 cells treated as in (A) for 24h. Scale bar=50μm.
(G) Relative fold change in mRNA expression in MC38GFP or Ep5 cells treated as in (A) for 40h. Values are normalized to Gapdh expression (n=3).
(H) Zymography for MMP-9 in the conditioned medium from Ep5 cells treated as in (A) for 40h.
For panels A, C, D and G, bars represent the mean ± SEM, and ns (p>0.05), *p<0.05, **p<0.01, ***p<0.001 vs buffer were determined by one-way ANOVA followed by Tuckey’s post test.