Supplementary Figure 3: Arp2/3 decreases the migration of iDCs but enhances their antigen uptake capacity.

(a) Immunoblot blot analysis of Arpc4 expression in BMDCs nucleofected with Arpc4-specific siRNA. (b,c) LifeAct-GFP density maps obtained from iDC or LPS-DC silenced (b) or inhibited (c) for Arp2/3. LPS-DCs were treated with the Arp2/3 inhibitor CK666 (25 μM) or silenced for Arpc4. Scale bar: 2.5 μm. 1 representative experiment out of 2 is shown. (d) 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. (e) Mean instantaneous speed of DCs migrating in micro-channels and treated or not with CK666 as in c (n = 244, 192, 231 and 230 for iDC, iDC-CK666, LPS-DC and LPS-DC CK666 respectively). 1 representative experiment out of 3 is shown. (f) Immunoblot analysis of Arpc2 and tubulin expression in tamoxifen-treated DCs. TomatoFP⁺ (Arpc2WT) and conditional GFP⁺ (Arpc2KO) iDCs were sorted from 10-days- DC cultures in which tamoxifen was added during the last 8 days. (g) Analysis of the migration of Arpc2WT and KO iDCs under an agarose gel. Cells were imaged during 200 min and their cell speed was quantified after tracking. The analysis was performed on cells that had migrated >100 μm (n = 80 and 163 for Arpc2 WT and Arpc2 KO respectively). 1 representative experiment out of 2 is shown. (h) Analysis by flow cytometry of surface expression of the DC activation marker CD86 performed as in d. (i) Quantification of the size (left) and number (right) of vesicles present at the front of Arpc2WT and KO DCs migrating in micro-channels filled with fluorescent ovalbumin (n = 36 and 37 for Arpc2WT and Arpc2 KO respectively). Graphics show mean and error bars correspond to S.E.M. (j) Macropinocytic capacity of migrating DCs treated with the Arp2/3 inhibitor CK666 (n = 62 and 41 for iDC and iDC CK666 respectively). 1 representative experiment out of 3 is shown. (k) Macropinocytic capacity of iDCs and LPS-DCs (n = 39 and 33 for iDC and LPS-DC respectively). 1 representative experiment out of 2 is shown. (l) Density maps obtained from LifeAct-GFP DCs fixed while migrating in micro-channels and stained for Arp2 and GFP. Scale bar: 2.5 μm. In the box plots of panels e,g,j and k 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.