DMP1 blocks the cell cycle in G₁, modulates the expression of cell cycle–related proteins, and induces p27^Kip1 through CD44 ligation. (A) Cell-cycle analysis of serum-starved HUVECs treated with DMP1 (50 nmol/L) and mimosine (200μM). Mimosine and non-serum–released cells were used to assess for G₁ arrest. Error bars represent the means ± SD of 3 replicates of a representative experiment (n = 3). **P ≤ .001 and ***P ≤ .0005 vs control serum-released cells; n.s., not significant. (B) S-phase cell-cycle analysis of serum-starved HUVECs incubated with blocking antibodies to CD44 and α_Vβ₃, with IgG used as a control, followed by treatment with DMP1 (50 nmol/L), as described in “Methods.” Error bars represent the means ± SD of 3 replicates of a representative experiment (n = 2). **P ≤ .001 vs control or DMP1 condition; n.s., not significant. (C) Western blot analysis with an antibody to p27^Kip1 using total lysates from HUVECs treated with increasing concentrations of DMP1. (D) Western blot analysis with antibodies to p21^Cip1 using total lysates from DMP1-treated cells. (E) Western blot analysis with antibodies to pRb using total lysates from DMP1-treated cells. (F) Western blot analysis with an antibody to p27^Kip1 using total lysates from DMP1-treated cells. Before DMP1 treatment (50 nmol/L) for 24 hours, cells were incubated for 1 hour in the presence of 10 μg/mL of anti-CD44 and anti-α_Vβ₃ blocking antibodies or IgG as a control. (G) Western blot analysis with antibodies to p27^Kip1 and P-p27^Kip1(Ser¹⁰) using total lysates from DMP1-treated HUVECs. All Western blotting results were evaluated by densitometric scanning, corrected with respect to β-actin expression, and expressed relative to the value obtained with the corresponding control (arbitrarily set as 1). These relative protein level values are shown in italics below the lanes. Western blots were performed at least 2 times with similar results. Equal protein loading was assessed by anti–β-actin immunoblotting.