Supplementary Figure 8: Ezh2 regulates integrin-dependent, but not integrin-independent, adhesion and migration of leukocytes.

(a) Cervical lymph node cells were prepared from Ezh2^f/f and Ezh2^f/f Cd11c-Cre mice 24h after FITC painting onto the dorsum of ears. CD11c⁺FITC⁺ DCs were analyzed by FACS. Representative plots (left) and percentages of FITC-positive DCs from 7 pairs of mice (right) are shown. Red line indicates the average percentages of FITC⁺ cells in control and Ezh2-deficient mice. (b) Control and Ezh2-deficient mature bone marrow derived DCs and BM neutrophils isolated from poly(I)-poly(C) treated Ezh2^f/f and Ezh2^f/f Mx1-Cre mice were plated on poly-L-lysine coated slides for 1 h and F-actin was visualized by phalloidin. F-actin (green), talin1 (red), DAPI (blue) Data shown are representative from 1 of the 2 independent experiments. Scale bars, 10 µm. (c) Epidermal Langerhans cells (LCs) from control Ezh2^f/f and Ezh2^f/f Cd11c-Cre mice were identified by MHCII staining in steady-state and upon oxazolone stimulations. Representative images of more than 3 independent experiments are shown. Left to right, ***p=6.6x10^-5, 8.5x10^-5. Scale bars, 100 µm. (d) Migratory LCs (CD11c⁺MHCII^hiEpCAM⁺CD103^-) in skin draining lymph nodes from control Ezh2^f/f and Ezh2^f/f; Cd11c-Cre mice were determined by FACS and cell numbers were calculated. *p=0.01, **p=0.002 (e) LCs as in c isolated from epidermis were plated on laminin coated slides for 2 h and identified by MHCII staining. The adhesion structures were determined by anti-talin1 antibody. Cell area was quantified from images. Thirty cells per mouse were scored. ***p=1.3x10^-5. Scale bars, 5 µm. (f) Various adhesion structures in control and Ezh2-deficient LCs were quantified according to the criteria specified in Supplementary Fig. 4e. Forty cells per mouse were scored. Error bars in the figures c-f represent mean ± s.e.m. of 3 independent experiments with 3 mice per group (two-tailed student’s t-test with equal variance). Left to right, ***p=1.8x10^-7, 7.0x10^-6, 1.5x10^-5. (g) Migration of control (filled bars) and Ezh2-deficient mature BMDCs (open bars) as defined in b was determined on surfaces coated with various concentrations of fibronectin. Time-lapse images were taken every 5 min for 2 h. Error bars in the figure represent mean ± s.e.m. of around 169-300 cells pooled from 2-3 independent experiments. ***p=2.5x10^-18. (h) Migration of control (filled bars) and Ezh2-deficient DCs (open bars) was determined as described in g except some cells were treated with ROCK inhibitor Y27632 (Y27, 30 µM) or myosin II inhibitor blebbistatin (ble, 10 µM) while imaging. ***p=3.6x10^-13. (i) Control (filled bars) and Ezh2-deficient mature BMDCs (open bars) were allowed to adherent to slides coated with indicated concentrations of fibronectin for 2 h. Cell areas were calculated using ImageJ. Error bars in h and i represent mean ± s.e.m. of around 200 cells polled from 2 independent experiments. ***p=3.6x10^-19. (j) Representative images from i are shown. Control (Ezh2^f/f) and Ezh2-deficient (Ezh2^Δ/Δ) mature BMDCs were cultured on surfaces coated with different concentrations of fibronectin (FN) in the presence or absence of Y27632. Focal adhesions were visualized by anti-paxillin staining (red), F-actin by Alexa Fluor 488® phalloidin and nucleus by DAPI (blue). Scale bars, 10 µm.