Supplementary Figure 4: Actin distribution at the back of DCs relies on the Formin mDia1 and is required for fast and persistent migration.

(a) LifeAct-GFP density maps of iDCs migrating in the presence or absence of the formin inhibitor Smifh2 (25 μM). 1 representative experiment out of 3 is shown. Scale bar: 2.5 μm. (b) Immunofluorescence analysis of mDia1 expression in LPS-DCs migrating in micro-channels analyzed by spinning disk microscope (100×). Scale bar: 5 μm. (c) Density maps obtained from LifeAct-GFP DC fixed while migrating in micro-channels and stained for mDia1 and GFP (2 independent experiments). Scale bar: 2.5 μm. (d) Quantitative PCR analysis of mDia1 expression in DCs nucleofected with mDia1-specific siRNA. Graphic shows mean and error bars correspond to S.E.M. (e) Immunoblot analysis of mDia1 and tubulin expression in immature mDia1WT and KO DCs. 1 representative experiment out of 3 is shown. (f) Mean instantaneous speed of LPS-DCs migrating in micro-channels and treated with different doses of Smifh2 (n = 125, 154, 134 and 141 for iDC, LPS-DC, LPS-DC 12 μM and LPS-DC 25 μM respectively). 1 representative experiment out of 3 is shown. (g) 10% of the highest instantaneous speed values of DCs migrating in micro-channels (n = 210, 238, 233 and 179 for iWT, LPS-WT, imDia1KO and LPS-mDia1KO respectively). 1 representative experiment out of 5 is shown. (h) Analysis by flow cytometry of surface expression of the DC activation marker CD86. iDCs were incubated 30 min with LPS (100 ng/ml), washed and cultured overnight. 1 experiment out of 4 is shown. (i) Mean instantaneous speed of mDia1WT and KO migrating in micro-channels and treated with CK666 (25 μM) (n = 119, 107, 52 and 111 for iWT, iWT CK, imDia1KO and imDia1KO CK respectively). 1 representative experiment out of 2 is shown. In the box plots of panels f, g and i the bars include 90% of the points, the center corresponds to the median and the box contains 75% of the data. The Mann-Whitney test was applied for all statistical analyses.