Figure 6. Induction of protrusion... | Rockefeller University Press

Figure 6.

$Figure 6. Induction of protrusion formation and enhancement of directional migration by TNFα-EEV fractions is dependent on GPCR signaling and CX3CL1. (a) Quantitation of transmigrated human mature MMDCs from the upper cell culture insert into the lower chamber well of a transwell assay. PTX-treated or untreated MMDCs were loaded together with EEV-free supernatants or TNFα-EEV fractions into the upper cell culture insert (n = 3; unpaired two-tailed t test). (b) Immunofluorescence of CX3CL1 (green) and ALIX (red; left) or CD9 (red; right) in primary human LECs. Cell nuclei are stained with DAPI (blue; n = 5). Yellow regions of interest were used for Manders colocalization analyses. White region of interest is a zoom. Arrows indicate colocalization of CX3CL1 with either ALIX or CD9. (c) Immunofluorescence of the lymphatic vessel marker podoplanin (green) and CX3CL1 (red) in human renal transplant rejections (fluorescence profiles across the respective vessels are plotted beneath the merged images; x axis: cross-sectional distance; y axis: CX3CL1- or podoplanin-specific mean immunofluorescence intensities; n = 6). Bars, 5 µm. (d) Anti-CX3CL1 immunoblot of ss-EEV fractions and TNFα-EEV fractions probed with an antibody to the C terminus of CX3CL1 (n = 5). Molecular masses are given in kilodaltons. (e) Flow cytometry contour plot of unstained beads, isotype control IgG-coated beads stained with fluorescent SYTO RNASelect-labeled TNFα-EEV fractions and anti-CX3CL1 IgG-coated beads stained with fluorescent SYTO RNASelect-labeled TNFα-EEV fractions. X axis indicates forward scatter. Y axis indicates mean fluorescence intensity (MFI) of SYTO RNASelect–labeled EEVs (n = 6). (f) Quantitation of SYTO RNASelect MFI of beads described in e (n = 6; unpaired two-tailed t test). (g) Quantitation of transmigrated MMDCs from the upper cell culture insert into the lower chamber well of a transwell assay. MMDCs were loaded together with EEV-free supernatants or TNFα-EEV fractions and control IgGs or anti-CX3CL1 IgGs into the upper cell culture insert (n = 3; unpaired two-tailed t test). (h) Quantitation of transmigrated MMDCs from the upper cell culture insert into the lower chamber well of a transwell assay. MMDCs were loaded together with EEV-free supernatants or TNFα-EEV fractions derived from 5-mispair control morpholino oligonucleotide–treated LECs or CX3CL1-specific morpholino oligonucleotide–treated LECs into the upper cell culture insert (n = 2; unpaired two-tailed t test). (i) Migration analyses of MMDCs in a 3D collagen matrix migration assay. Cells were exposed to gradients of EEV-free supernatants plus CCL19 (n = 1,070), TNFα-EEV fractions derived from 5-mispair control morpholino oligonucleotide–treated LECs plus CCL19 (n = 654) or TNFα-EEV fractions derived from CX3CL1-specific morpholino oligonucleotide–treated LECs plus CCL19 (n = 958). Red columns indicate chemotactic displacement. Blue columns indicate chemotactic index. Data are obtained from at least 218 cell tracks per experiment and three independent experiments that were pooled for analysis (unpaired two-tailed t test with Welch’s correction). (j) Migration analyses of MMDCs in a confined microenvironment migration assay in the presence of EEV-free supernatants (n = 1,464), TNFα-EEV fractions derived from 5-mispair control morpholino oligonucleotide–treated LECs (n = 9,380), or TNFα-EEV fractions derived from CX3CL1-specific morpholino oligonucleotide–treated LECs (n = 5,588). Red columns indicate circularity of cell shape. Blue columns indicate migratory angle change. Green columns indicate speed of migration. Data are obtained from at least 488 time points and from three independent experiments that were pooled for analysis (unpaired two-tailed t test with Welch’s correction). Values represent means ± SEM. ns, P > 0.05; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.$

Induction of protrusion formation and enhancement of directional migration by TNFα-EEV fractions is dependent on GPCR signaling and CX3CL1. (a) Quantitation of transmigrated human mature MMDCs from the upper cell culture insert into the lower chamber well of a transwell assay. PTX-treated or untreated MMDCs were loaded together with EEV-free supernatants or TNFα-EEV fractions into the upper cell culture insert (n = 3; unpaired two-tailed t test). (b) Immunofluorescence of CX3CL1 (green) and ALIX (red; left) or CD9 (red; right) in primary human LECs. Cell nuclei are stained with DAPI (blue; n = 5). Yellow regions of interest were used for Manders colocalization analyses. White region of interest is a zoom. Arrows indicate colocalization of CX3CL1 with either ALIX or CD9. (c) Immunofluorescence of the lymphatic vessel marker podoplanin (green) and CX3CL1 (red) in human renal transplant rejections (fluorescence profiles across the respective vessels are plotted beneath the merged images; x axis: cross-sectional distance; y axis: CX3CL1- or podoplanin-specific mean immunofluorescence intensities; n = 6). Bars, 5 µm. (d) Anti-CX3CL1 immunoblot of ss-EEV fractions and TNFα-EEV fractions probed with an antibody to the C terminus of CX3CL1 (n = 5). Molecular masses are given in kilodaltons. (e) Flow cytometry contour plot of unstained beads, isotype control IgG-coated beads stained with fluorescent SYTO RNASelect-labeled TNFα-EEV fractions and anti-CX3CL1 IgG-coated beads stained with fluorescent SYTO RNASelect-labeled TNFα-EEV fractions. X axis indicates forward scatter. Y axis indicates mean fluorescence intensity (MFI) of SYTO RNASelect–labeled EEVs (n = 6). (f) Quantitation of SYTO RNASelect MFI of beads described in e (n = 6; unpaired two-tailed t test). (g) Quantitation of transmigrated MMDCs from the upper cell culture insert into the lower chamber well of a transwell assay. MMDCs were loaded together with EEV-free supernatants or TNFα-EEV fractions and control IgGs or anti-CX3CL1 IgGs into the upper cell culture insert (n = 3; unpaired two-tailed t test). (h) Quantitation of transmigrated MMDCs from the upper cell culture insert into the lower chamber well of a transwell assay. MMDCs were loaded together with EEV-free supernatants or TNFα-EEV fractions derived from 5-mispair control morpholino oligonucleotide–treated LECs or CX3CL1-specific morpholino oligonucleotide–treated LECs into the upper cell culture insert (n = 2; unpaired two-tailed t test). (i) Migration analyses of MMDCs in a 3D collagen matrix migration assay. Cells were exposed to gradients of EEV-free supernatants plus CCL19 (n = 1,070), TNFα-EEV fractions derived from 5-mispair control morpholino oligonucleotide–treated LECs plus CCL19 (n = 654) or TNFα-EEV fractions derived from CX3CL1-specific morpholino oligonucleotide–treated LECs plus CCL19 (n = 958). Red columns indicate chemotactic displacement. Blue columns indicate chemotactic index. Data are obtained from at least 218 cell tracks per experiment and three independent experiments that were pooled for analysis (unpaired two-tailed t test with Welch’s correction). (j) Migration analyses of MMDCs in a confined microenvironment migration assay in the presence of EEV-free supernatants (n = 1,464), TNFα-EEV fractions derived from 5-mispair control morpholino oligonucleotide–treated LECs (n = 9,380), or TNFα-EEV fractions derived from CX3CL1-specific morpholino oligonucleotide–treated LECs (n = 5,588). Red columns indicate circularity of cell shape. Blue columns indicate migratory angle change. Green columns indicate speed of migration. Data are obtained from at least 488 time points and from three independent experiments that were pooled for analysis (unpaired two-tailed t test with Welch’s correction). Values represent means ± SEM. ns, P > 0.05; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.