Supplementary Figure 4: Rapid spreading and activation of Ezh2-deficient innate leukocytes.
(a) Control (Ezh2f/f) and Ezh2-deficient (Ezh2Δ/Δ) mature DCs derived from poly(I)-poly(C) treated Ezh2f/f and Ezh2f/f Mx1-Cre mice were serum-starved and plated onto RGDS-coated plates for the indicated periods of time. Adherent cells were fixed and stained using crystal violet and DAPI. Data shown are representative of 3 independent experiments with triplicates. The corresponding statistical analysis is shown in Fig. 3d. (b) The surface expression of integrins α5β1, α4ββ1, αXβ2, and αMβ2 in control (blue line) and Ezh2-deficient DCs (red line) were analyzed by flow cytometry. Isotype control is indicated by filled area. Gated mature DCs (CD11c+MHCIIhi) are shown in these histograms. (c) Control and Ezh2-deficient mature BMDCs as in a were plated onto RGDS coated plate for indicated time points. Equal amount of lysate was resolved by the standard SDS-PAGE, and probed with indicated antibodies. “L” indicates cells adherent to poly-L-lysine coated dishes for 45 min. (d) Control (Ezh2f/f) and Ezh2-deficient (Ezh2Δ/Δ) neutrophils isolated from the BM of poly(I)-poly(C) treated Ezh2f/f and Ezh2f/f Mx1-Cre mice were rested as described in Supplementary Fig. 3b and plated onto ICAM-1 coated dishes for indicated time points. Activation of indicated molecules was determined by specific antibodies. (e) Control and Ezh2-deficient mature DC as in a adherent to VCAM-1-coated slide were stained for talin1 (red) and statistical analysis of various adhesion structures (bottom) in control (filled bars) and Ezh2-deficient DCs (open bars) were performed. Nascent adhesions (NAs), focal complexes (FCs), focal adhesions (FAs) and fibrillar adhesions (FBs) were measured by setting the size limits of the measured particles from 0.05-200 µm2. The classifications are as follows: NA <0.25 µm2, FC <0.5 µm2, FA <1.5 µm2, FB > 5 µm2. Error bars represent Mean ± s.e.m. of cells pooled from 3 independent experiments. (Two tailed student’s t test, *p=0.041 (left), 0.008, ***p=0.0002 (left), 5x10-8). (f) The expression of Paxillin, p-Paxillin (Y118) or overall phosphorylated protein (Py99) in control and Ezh2-deficeint DCs on fibronectin coated slides was determined by immunofluorescence staining and the images were acquired either with confocal or TIRF microscopy. (g) Immunofluorescence staining of indicated proteins in control and Ezh2-deficeint neutrophils on ICAM-1 coated slides. Paxillin (red), p-Paxillin (Y118) (green), DAPI (blue). Data shown in this figure are either representative from 1 of 3 (a, b, e (top), f) or 2 (g, d with 3 mice per group) independent experiments or summary (e, bottom) of data acquired from 3 mice per group. Scale bars in e, f, g represent 10 µm.
