Interaction of PSGL-1 and P-selectin regulates adhesion-related signaling and β-integrin activation in MM cells. (A) MM1s cells were treated with recombinant P-selectin 10μg/mL for different durations (0, 5, 10, 20, 30, and 60 minutes), lysed, and whole-cell lysates were subjected to Western blotting for pFAK, pAKT, pCoffilin, pSRC, and p-GSK3α/β. Increased adhesion-related signaling was observed after activation with recombinant P-selectin, with maximal activation at 30 minutes. (B) Recombinant P-selectin was incubated with or without GMI-1070 (500mM for 1 hour) and then applied to MM1s cells; nontreated MM cells were used as a control. Cells were then lysed and whole-cell lysates were subjected to Western blotting for pFAK, pAKT, pCoffilin, pSRC, and p-GSK3α/β. GMI-1070 reversed the induction of adhesion-related signaling in MM cells induced by recombinant P-selectin. (C) HUVECs were treated with or without GMI-1070 (500mM for 1 hour), nontreated MM1s cells were cocultured with the HUVECs for 1 hour, and MM1s cells not cocultured with HUVECs served as a control. MM cells were then separated from the HUVECs, lysed, and whole-cell lysates were subjected to Western blotting for pFAK, pAKT, pCoffilin, pSRC, and p-GSK3α/β. Coculture of MM cells with HUVECs induced adhesion-related signaling in MM cells that was reversed by GMI-1070. (D) Recombinant P-selectin was incubated with or without GMI-1070 (500mM for 1 hour) and then applied to MM1s cells; nontreated MM cells were used as a control. Cells were fixed and the expression of activated β₁-integrin was detected using FITC-labeled Ab under immunofluorescence microscopy. Increased activation of β₁-integrin was observed after activation with recombinant P-selectin, which was reversed by GMI-1070. Results are shown as an average of the percentage of cells with activated integrins, normalized to controls (P < .001; i), and in representative fluorescent images at 20× magnification and at 100× in the inset (ii).