Figure 3.

Mechanism of CD44v6 induced YB-1 expression. (A) Time course results are shown of CD44v6 and YB-1 protein expressions after transfecting the v6 rescue plasmid (described in the Methods section) into v6 Mu1 SW948-FR CICs. Expression of proteins at 0 h was not shown because of the absence of CD44v6 in the protein lysate. (B) Effects of knocking out CD44v6 in in v6 Mu1 CICs of sensitive and resistant SW948 cells on PGE2 production (analyzed by ELISA as described in Methods) in the presence and absence of FOLFOX treatment are shown. (C) 17-P-T-PGE2 (synthetic PGE2) induces YB-1 in CICs that were treated with PGE2 at 5 μM for the indicated times. YB-1 expression levels were determined by immunoblotting with anti-YB-1 antibody; β-tubulin as loading control. (D) Effects are shown of CD44v6 Mu1 knockout on PGE2 and FOLFOX induced YB-1 expression. CICs isolated from SW948-S and SW948-FR that were previously transfected with either v6 Mu1 or vector for 48 h and then treated with or without synthetic PGE2 or FOLFOX. YB-1 expression levels were determined by immunoblotting with anti-YB-1 antibody; β-tubulin as loading control. (E) Effects are shown of PGE2/EP1 receptor, and of mTOR signaling on YB-1 expression. CICs were either transfected with in v6 Mu1 or vector for 48 h and then treated with or without EP1 inhibitor (5 µM AH6809) or mTOR inhibitor (10 nM PP242) for 2 h. They were then cultured in serum-free medium for 16 h and treated with synthetic PGE2 at 5 μM, or 1 × FOLFOX for 24 h. The cell lysates were processed for YB-1 and β-tubulin (as loading control). PGE2 secretion data in Figure 3B represent means ± SD; n = 4–6; * p < 0.05 compared to either vehicle control, vector + FOLFOX treatment group, or vector control group.