Figure 2. Primary human LECs release... | Rockefeller University Press

Figure 2.

$Figure 2. Primary human LECs release EEVs. (a) Transmitted EM (TEM) of human skin LECs in situ (left: antipodoplanin-conjugated gold particles) and in vitro (middle and right). MVBs (middle, unstained) and exocytosis of CD63+ vesicles (right, anti-CD63–conjugated gold particles) into the basolateral stroma (n ≥ 5). (b) Experimental setup to collect basolateral LEC culture supernatant from the lower chamber for the enrichment of basolateral EEVs (yellow dots). (c) TEM of vesicles of basolateral EEV fractions. Right: Anti-CD63–conjugated gold particles (n ≥ 21). Bars, 100 nm. (d) Diameters of vesicles in basolateral EEV fractions collected from ss (n = 21) and TNFα-stimulated LECs (n = 26). Data are obtained from at least seven TEM images per experiment and from three independent experiments that were pooled for analysis (unpaired two-tailed t test with Welch’s correction). (e) BCA-derived protein concentrations of EEV fractions collected over 24 h from ss and TNFα-stimulated LECs in the absence or presence of 10 µM GW4869 (n = 3; unpaired two-tailed t test). (f) Anti-CD9 immunoblot of EEV fractions from equal volumes of basolateral ss and TNFα-stimulated LEC culture supernatants (n = 3). (g) Mean absolute vesicle numbers of EEV fractions isolated from 1 ml basolateral LEC culture supernatant of ss and TNFα-stimulated LECs (n = 3; unpaired two-tailed t test). Left graph, vesicle numbers plotted against diameter size intervals; right graph, vesicle numbers of all sizes. (h) Coomassie blue–stained electrophoresis gel (top) and quantitation of relative protein abundance (as measured by densitometric analyses; bottom) of density gradient centrifugation subfractions of EEV fractions isolated from basolateral culture supernatants of ss and TNFα-stimulated LECs (n = 3). (i) Anti-CD63–stained immunoblot (top) and quantitation of relative protein abundance (as measured by densitometric analyses; bottom) of density gradient centrifugation subfractions of EEV fractions isolated from basolateral culture supernatants of ss and TNFα-stimulated LECs (n = 3). Values represent means ± SEM. ns, P > 0.05; *, P ≤ 0.05; **, P ≤ 0.01.$

Primary human LECs release EEVs. (a) Transmitted EM (TEM) of human skin LECs in situ (left: antipodoplanin-conjugated gold particles) and in vitro (middle and right). MVBs (middle, unstained) and exocytosis of CD63⁺ vesicles (right, anti-CD63–conjugated gold particles) into the basolateral stroma (n ≥ 5). (b) Experimental setup to collect basolateral LEC culture supernatant from the lower chamber for the enrichment of basolateral EEVs (yellow dots). (c) TEM of vesicles of basolateral EEV fractions. Right: Anti-CD63–conjugated gold particles (n ≥ 21). Bars, 100 nm. (d) Diameters of vesicles in basolateral EEV fractions collected from ss (n = 21) and TNFα-stimulated LECs (n = 26). Data are obtained from at least seven TEM images per experiment and from three independent experiments that were pooled for analysis (unpaired two-tailed t test with Welch’s correction). (e) BCA-derived protein concentrations of EEV fractions collected over 24 h from ss and TNFα-stimulated LECs in the absence or presence of 10 µM GW4869 (n = 3; unpaired two-tailed t test). (f) Anti-CD9 immunoblot of EEV fractions from equal volumes of basolateral ss and TNFα-stimulated LEC culture supernatants (n = 3). (g) Mean absolute vesicle numbers of EEV fractions isolated from 1 ml basolateral LEC culture supernatant of ss and TNFα-stimulated LECs (n = 3; unpaired two-tailed t test). Left graph, vesicle numbers plotted against diameter size intervals; right graph, vesicle numbers of all sizes. (h) Coomassie blue–stained electrophoresis gel (top) and quantitation of relative protein abundance (as measured by densitometric analyses; bottom) of density gradient centrifugation subfractions of EEV fractions isolated from basolateral culture supernatants of ss and TNFα-stimulated LECs (n = 3). (i) Anti-CD63–stained immunoblot (top) and quantitation of relative protein abundance (as measured by densitometric analyses; bottom) of density gradient centrifugation subfractions of EEV fractions isolated from basolateral culture supernatants of ss and TNFα-stimulated LECs (n = 3). Values represent means ± SEM. ns, P > 0.05; *, P ≤ 0.05; **, P ≤ 0.01.