Fig. 8.

VW-MSC-specific paracrine action for mediating radio-protection especially of endothelial cells. a A membrane-based sandwich immunoassay (Proteome Profiler Human XL Cytokine Array Kit, RnD Systems) was used for the parallel determination of the relative levels of selected human cytokines and chemokines in cell culture supernatants (conditioned medium, CM) of fibroblasts, VW-MSCs, Ctrl-transduced fibroblasts (CtrlTdFib) and iVW-MSCs. Equal protein amounts were run on each array. Profiles of detected signals were quantified by densitometry and related to the reference signal (set as 1). Results from one representative experiment out of two independent biological replicates (supernatants) measured in duplicates each were presented as heatmap. b Endothelial cell migration was investigated after irradiation and subsequent introduction of a thin wound in confluent monolayers by scratching with a pipette tip. Wound closure was determined for the different supernatant treatments by measuring the migration distance after 8 h. Data are shown as mean ± SEM of three independent experiments measured in duplicates each. ***P ≤ 0.001, **P ≤ 0.01 *P ≤ 0.05 by two-way ANOVA followed by post hoc Sidak’s test. c Cell cycle phases (G0/G1 and G2/M) of irradiated endothelial cells and subsequent conditioned medium (CM) treatment was further analyzed after 24 h. Data are shown as mean ± SEM of three independent experiments measured in duplicates each. **P ≤ 0.01 *P ≤ 0.05, by two-way ANOVA followed by post hoc Tukey’s test. (D) Cultured endothelial cells were exposed to irradiation with 15 Gy, subsequently cultured in normal growth media (NGM) or NGM supplemented with CM of fibroblasts, VW-MSCs, Ctrl-transduced fibroblasts (CtrlTdFib) and iVW-MSCs. Expression levels of the indicated proteins were analyzed in whole protein lysates using Western blot analysis at 96 h after radiation and subsequent growth factor treatment. Representative blots from four different experiments are shown (n = 3). e Cultured normal lung tissue fragments embedded in growth factor reduced matrigel were exposed to irradiation with 15 Gy, subsequently cultured in normal growth media (NGM) alone or NGM supplemented with conditioned media (CM; ratio 1/1), which were derived from cultured fibroblasts, VW-MSCs, Ctrl-transduced fibroblasts and iVW-MSCs. Expression levels of the indicated proteins were analyzed in whole protein lysates using Western blot analysis at 5 days after radiation and subsequent CM treatment. Representative blots from four different experiments are shown (n = 4)