Truncated rPP terminating with Ala⁴⁸² (PP-(Ala terminal)) induced cell adhesion and migration.

<p>(A) The fluorescence intensity (FI) of Alexa-Fluor 488 conjugated rPP-ΔSSD and rPP-(Ala terminal). One hundred microliters of serially-diluted Alexa-Fluor 488 conjugated rPP-ΔSSD (Alexa-rPP-ΔSSD) and rPP-(Ala terminal) (Alexa-rPP-(Ala terminal)) were added onto black 96-well plates and FI was measured on a fluorescence microplate reader. Their FI values were equivalent. (B) Flow cytometry analysis of Alexa-rPP-ΔSSD and Alexa-rPP-(Ala terminal) binding to MG63 cells. MG63 cells were incubated with Alexa-rPP-ΔSSD and Alexa-rPP-(Ala terminal) (250 nM). rPP-(Ala terminal) binding to MG63 cells was apparently higher than that of rPP-ΔSSD and control cells, which were incubated with medium only. (C) MG63 and MC3T3-E1 cells were seeded onto rPP, rPP-(Ala terminal), rPP-RGE-(Ala terminal), and rC-DMP-1 (100 nM) with MnCl₂ (1 mM) in serum-free medium. The number of attached cells was evaluated as described above. Each value represents the mean of triplicate determinations; bars mean ±SD. (D) Organization of actin stress fibers on rPP, rPP-(Ala terminal), rPP-RGE-(Ala terminal), and rC-DMP-1. MG63 cells were seeded onto glass plates precoated with rPP (box 1), rPP-(Ala terminal) (2), rPP-RGE-(Ala terminal) (3), rC-DMP-1 (4), or none (5) (100 nM) in serum-free medium. Actin fibers were visualized by FITC-phalloidin 1 hr after seeding. Bar = 10 µm. (E) rPP-(Ala terminal) was able to induce haptotaxis cell migration. Haptotaxis cell migration was measured in MG63 cells toward rPP, rPP-(Ala terminal), rPP-ΔSSD, and rC-DMP-1 in serum-free medium. After the 12-hr incubation, cells were fixed and stained. Migration was quantified by counting the number of cells that migrated through filters. Each value represents the mean of 10 randomly selected fields at 200× magnification; bars mean ±SD. Statistical analysis was performed by a one-way ANOVA, followed by Dunnett's test. ***p<0.001 significantly lower than PP-(A terminal)-coated wells.