Supplementary Figure 3: Defective migration of Ezh2-deficient leukocytes is not caused by impaired responsiveness to chemoattractant.
(a) Migration of neutrophils on ICAM-1-coated slides in the absence (black bar) or presence (white bar) of fMLP. Control and Ezh2-deficient neutrophils isolated from the bone marrow of poly(I)-poly(C) treated Ezh2f/f and Ezh2f/f Mx-Cre mice were purified using murine neutrophil isolation kit (Miltenyi). Purified neutrophils were seeded to ICAM-1-coated chamber slides and stimulated with fMLP (10-5 M) for 10 minutes in 37ºC. Non-adherent cells were removed, adherent cells were used in parallel for time-lapse imaging at 30-sec interval for 30 min. Phase contrast images were captured using Zeiss inverted microscope at 10x magnification with temperature and CO2 controls. Only cells that were not clustering and could be tracked for the whole time duration were taken into account. Cell tracking was done using Image J software, with Manual Tracking, and Chemotaxis Plugin. About 100 cells from 3 random fields were tracked in each experiment. Statistical analysis of distance was compiled using a “Chemotaxis Plug-in tool” from ImageJ. Error bars represent mean + s.d., ***P=1.1x10-19. (b) Control (solid line filled square) and Ezh2-deficient (dashed line open circle) bone marrow neutrophils were rested at room temperature for 1 hour before pre-warming them at 37ºC for 10 min and stimulated in solution with fMLP for indicated time points. Actin contents were measured by phalloidin staining followed by FACs analysis. (c) Activation of integrin downstream signaling molecules in control and Ezh2-deficient neutrophils. Neutrophils were stimulated as described in b. Equal amount of lysate was resolved with SDS-PAGE, and probed with indicated antibodies. (d) The surface expression of CCR7 on control (blue line) and Ezh2-deficient CD11c+MHCIIhi mature DCs (red line) derived from the bone marrow of poly(I)-poly(C) treated Ezh2f/f and Ezh2f/f Mx-Cre mice were analyzed by flow cytometry. Isotype control is indicated by filled area.
