Gadkin is dispensable for spreading of mature BMDCs.

<p>A. Gadkin^-/- BMDCs display no alterations in cell spreading. WT and Gadkin^-/- BMDCs were matured for 24 h with LPS. Subsequently, cells were allowed to spread on fibronectin-coated cover slips for 15 to 240 min. Cells were processed for immunofluorescence and incubated with phalloidin to stain F-actin and thus visualize cell shape. Cell size was quantified at the indicated times post-plating (N = 2 independent experiments for 15 min time point and N = 4 for 30 and 240 min time points).</p

Unaltered <i>in vivo</i> DC migration in absence of Gadkin.

<p>A,B. Skin irritant-induced DC migration to lymph nodes is not altered in the absence of Gadkin. FITC in acetone/dibutylphtalate was painted onto the shaved skin of WT and Gadkin^-/- mice. The percentage of FITC-positive DCs migrating to the draining inguinal lymph nodes was assessed by flow cytometry. A. Representative examples of gating to select FITC-positive DCs for analysis (top). Lower panels show control lymph nodes devoid of FITC-labeled DCs. B. Quantification of the percentage of FITC-positive cells in draining lymph nodes (N = 5 mice per genotype from 2 independent experiments). C,D. Labeled LPS-matured WT and Gadkin^-/- BMDCs reach the draining popliteal lymph nodes with comparable efficiency after injection into foot pads. C. Representative example of gating to select transferred TAMRA- resp. DDAO-positive BMDCs for analysis. D. Quantification of the percentage of TAMRA- and DDAO-positive WT and Gadkin^-/- BMDCs in popliteal lymph nodes (LN) (N = 3 mice per genotype from 2 independent experiments).</p

Loss of Gadkin does not affect generation of splenic DC subsets <i>in vivo</i>.

<p>WT and Gadkin^-/- spleens were processed into erythrocyte-depleted cell suspensions, and the percentage of different DC subsets relative to total splenocytes was quantified by flow cytometry using CD11c, CD8 and B220 as markers for the different DC populations (N = 3 independent experiments). Pos, positive; neg, negative; pDCs, plasmacytoid DCs.</p

Gadkin is expressed in lymphoid organs and in immune cells.

<p>A. Lysates of WT and Gadkin^-/- (labeled as KO) tissues were analyzed by immunoblotting using Gadkin- and AP-1-specific antibodies. B. Lysates of WT and Gadkin^-/- thymus, of DCs derived from WT and Gadkin^-/- bone marrow, as well as of B lymphocytes (i.e. B220-positive B cells) and T lymphocytes (i.e. CD3-positive T cells) isolated from spleen were analyzed by immunoblotting using Gadkin-specific antibodies. Detection of the constitutively expressed molecular chaperone heat shock cognate protein 70 (Hsc70) was used as loading control. B, B-lymphocytes; T, T-lymphocytes.</p

Loss of Gadkin leads to an increase of F-actin.

<p>A. AP-1 localization is not altered in the absence of Gadkin. WT and Gadkin^-/- BMDCs were matured for 24 h with LPS. Subsequently, BMDCs were allowed to spread on fibronectin-coated cover slips for 240 min prior to fixation and staining with Gadkin- and AP-1-specific antibodies. Scale bar: 10 μm. B. ARP2/3 and F-actin localization appears unaltered in the absence of Gadkin. BMDCs treated as in A. were incubated with ARP2/3-specific antibodies against the subunit p34/ARPC2 and with fluorescently labeled phalloidin to stain F-actin. Scale bar: 10 μm. C. The level of ARP2/3 is not changed in Gadkin^-/- BMDCs. Immunofluorescence-based quantification of the mean fluorescence intensity of the ARP2/3 subunit p34/ARPC2 (N = 5 independent experiments, unpaired t-test, ns = non significant). D. Gadkin^-/- BMDCs contain more F-actin. Flow cytometry-based quantification of phalloidin-stained suspension cells that had been starved for 1 h (N = 7 independent experiments, unpaired t-test, ** = p<0.01).</p

Antibodies.

<p>IF, immunofluorescence; WB, western blotting.</p><p>Antibodies.</p

Loss of Gadkin impairs BMDC chemotaxis in 2D.

<p>A,B. LPS-matured WT and Gadkin^-/- BMDCs were seeded into Transwell inserts and allowed to migrate for 3 h towards 250 ng/ml CCL19 (A) resp. 250 ng/ml CCL21 (B). Migration towards the chemokine was analyzed by flow cytometry. Values were subtracted for unspecific migration towards chemokine-free medium to determine the specific migration towards chemokine and then normalized to the average value of each experiment (N = 6 independent experiments, unpaired two-tailed t-test, * = p<0.05). C-H. LPS-matured WT and Gadkin^-/- BMDCs were seeded into a 2D chemotaxis chamber. BMDCs were allowed to migrate for 4 h towards 650 ng/ml CCL19, and their migratory behavior was evaluated based on bright-field images obtained at 2 min intervals. C,D. Migration tracks of 21 WT (C) and 23 Gadkin^-/- BMDCs (D) centered on the same starting point. Center of mass depicted as blue cross (WT: x = 10.57 μm, y = 326.38 μm, KO: x = 4.26 μm, y = 213.79 μm). Velocity (E) was quantified as well as the accumulated (F) and Euclidian distance (G) covered by the BMDCs. Directionality (H) was calculated as Euclidian distance divided by accumulated distance (N = 4 independent experiments, at least 20 cells per experiment were tracked, paired two-tailed t-test for distances, paired one-tailed t-test for velocity, * = p<0.05).</p

Macropinocytosis is unaltered in Gadkin^-/- BMDCs.

<p>Immature WT and Gadkin^-/- BMDCs were incubated with FITC-dextran for the indicated time intervals. FITC-Dextran uptake was quantified by flow cytometry (N = 2 independent experiments).</p

Gadkin is regulated in an LPS-dependent manner.

<p>A-D. LPS treatment of BMDCs increases Gadkin levels and induces its transient posttranscriptional modification. A. Immature WT BMDCs were either left untreated or incubated for 24 h with LPS. Subsequently, cells were allowed to spread on fibronectin-coated cover slips for 15 min to achieve a more uniform morphology prior to analysis. Cells were processed for immunofluorescence and labeled with Gadkin-specific antibodies. Nuclei were stained with DAPI. Insets show 2x enlarged perinuclear area. Scale bar: 10 μm. B,C. Lysates from WT BMDCs treated with LPS for the indicated times were analyzed by immunoblotting using Gadkin- and Hsc70-specific antibodies. The closely spaced upper band is indicated by red arrows. D. Quantification of immunoblot images like the one depicted in B. Gadkin levels were normalized to Hsc70 levels derived from the same blot. (N = 3 independent experiments, unpaired two-tailed t-test; * = p<0.05; a.u., arbitrary units).</p

Loss of Gadkin impairs directed BMDC migration towards CCL21 gradients in 3D collagen gels.

<p>A-D. WT and Gadkin^-/- BMDCs matured in the presence of LPS for 24 h were embedded into 1.6 mg/ml collagen gels. The upper gel surface was covered with medium containing 650 ng/ml CCL19 or 650 ng/ml CCL21, respectively. BMDC chemotaxis was monitored by bright-field real-time microscopy for 6 h. Images were obtained at 4 min intervals. Velocity of migrating cells (A) was quantified as well as the accumulated (B) and Euclidian distance (C) covered by the BMDCs. Directionality (D) was calculated as Euclidian distance divided by accumulated distance (N = 5 independent experiments, at least 20 cells were tracked per experiment, paired two-tailed t-test for comparison of WT and Gadkin^-/- BMDCs subjected to same condition, *** = p<0.001; ** = p<0.01).</p