Supplementary Figure 1: Efficient deletion of Ezh2 gene and Ezh2 protein in DCs and neutrophils.

(a) Deletion efficiency of Ezh2 gene in ex vivo isolated DCs. CD11c^high and CD11c^low cells were sorted from skin, lymph nodes and spleen of the Ezh2^f/Δ and Ezh2^f/f Cd11c-Cre mice. RNA was isolated and used for cDNA synthesis followed by specific PCR (full-length Ezh2 transcript: 543 bp, truncated Ezh2 transcript: 246bp). HPRT was used as a loading control. (b) Cell numbers of spinal cord infiltrating leukocytes (top) and percentages of DCs associated with blood vessels (bottom) at the peak of EAE disease (day 21) in Ezh2^f/f control and Ezh2^f/f Cd11c-Cre mice are shown. Error bars indicate mean ± s.d. of 3 independent experiments. *P=0.01. (c) Deletion efficiency of Ezh2 gene in neutrophils. Gr-1⁺ granulocytes including neutrophils (Nph) were isolated from bone marrow (BM) and peripheral blood (PB) of poly(I)-poly(C) treated Ezh2^f/f and Ezh2^f/f Mx1-Cre mice. RNAs from purified cells were processed as described above. (d) Generation of Ezh2-deficient BMDCs in vitro. Bone marrow cells from Ezh2^f/f and Ezh2^f/f Mx1-Cre mice pre-treated with poly(I)-poly(C) were isolated and cultured in the presence of GM-CSF and IL-4. The phenotypes of the CD11c⁺ cell populations were analyzed by FACS on day 3, 5 and 6, with or without the final 20 h LPS treatment. Filled gray area indicates the fluorescence content of the isotype control. (e) Deletion efficiency of the floxed Ezh2 locus in BMDCs. RNAs from Ezh2^f/f and Ezh2^Δ/Δ BM and BMDC were isolated and the deletion efficiency was determined as described in a. (f) Depletion of Ezh2 protein in Ezh2-deficient BMDCs. Expression of Ezh2, Suz12 and Vav1 in nucleus and cytosol was analyzed in BMDCs isolated from culture at day 6 (lane 1, 4) and day 8 with (lane 3, 6) or without LPS (lane 2, 5) stimulated maturation. Expression of lamin B and tubulin was used to control for the purity of the nuclear and cytosolic fractions, respectively.