EPC-CM effects on total sprout length of <i>ex vivo</i> aortic rings are AKT and ERK dependent.

<p>Quantitative analysis of vascular outgrowth from rat aortic rings embedded in growth factor reduced-Matrigel. Incubation with EPC-CM (CM) enhanced total sprout length from the aortic rings compared to control medium incubation (Ctr). This effect was significantly abolished by addition of [5 µM] LY294002 (CM+LY) as well as of [10 µM] PD98509 (CM+PD). Data are given as mean + s.e.m. and values are presented as percentage of control. *: p < 0.05.</p

EPC-CM effects on tubular structure formation are AKT and ERK dependent.

<p>Quantitative analysis of tubular network formation by microvascular endothelial cells on growth factor reduced Matrigel. EPC-CM incubation (CM) significantly promoted total area covered by the tube-like structures (A), total sprout length (B) and branching (C) as compared to control medium (Ctr). Supplementation of EPC-CM with the inhibitor of AKT phosphorylation (CM+LY) ([5 µM]) substantially attenuated all three parameters while the addition of the inhibitor of ERK phosphorylation (CM+PD) ([10 µM]) resulted in a significant decrease of covered area and total tube length but had no significant effect on the reduction of the number of branches. Data are given as mean + s.e.m. and values are presented as percentage of corresponding controls. *: p < 0.05.</p

Angiogenic potential of EPC-CM on <i>ex vivo</i> aortic rings.

<p>Representative microphotographs of vascular outgrowth from rat aortic rings embedded in growth factor reduced-Matrigel. Incubation with EPC-CM (CM) enhanced the formation of vascular outgrowth from the aortic rings compared to control medium incubation (Ctr). EPC-CM-enhanced capillary outgrowth was abolished by the addition of [5 µM] LY294002 (CM+LY). Similarly, incubation with EPC-CM in presence of [10 µM] PD98509 (CM+PD) significantly impaired total sprout length induced by EPC-CM. Scale bars: 200 µm and 100 µm (inserts).</p

Angiogenic potential of EPC-CM on tubular structure formation.

<p>Representative microphotographs of tubular network formation by microvascular endothelial cells on growth factor reduced Matrigel. EPC-CM incubation (CM) strikingly promoted the tube-like structure formation as compared to control medium (Ctr). Supplementation of EPC-CM with the inhibitor of AKT phosphorylation (CM+LY) ([5 µM]) substantially attenuated EPC-CM induced tube formation. Similarly, addition of the inhibitor of ERK phosphorylation (CM+PD) ([10 µM]) resulted in a decrease of tube formation, however, to a lesser degree as compared to the CM+LY group. Scale bar: 100 µm.</p

EPC-CM activates AKT and ERK pathways.

<p>Representative immunoblots <b>(A)</b> and quantitative analysis <b>(B)</b> of rBCEC4 lysates incubated with vehicle (Ctr) or EPC-CM (CM) in presence of either LY294002 (CM+LY) or PD98509 (CM+PD). Exposure of rBCEC4 to EPC-CM resulted in a marked increase of both phosphorylated AKT and phosphorylated ERK (pAKT and pERK) with regards to the total AKT and ERK levels (tAKT and tERK). Concomitant incubation with EPC-CM and the corresponding inhibitors almost completely blocked the phosphorylation of AKT and ERK. Data are given as mean + s.e.m. and values are presented as percentage of corresponding controls. *: p < 0.05.</p

Basal functions of rBCEC4 are not altered by LY294002 or PD98059.

<p>Viability (A), migration (B) and capacity to organize in tubular structures (C) by rBCEC4 was not significantly changed compared to controls (Ctr) when incubated in presence of [5 µM] LY294002 or [10 µM] PD98059. Data are given as mean + s.e.m. and values are presented as percentage of corresponding controls.</p

No co-localization of FA1/dlk1 with tyrosine hydroxylase in striatum.

<p>Representative photomicrographs of double immunofluorescence staining showing no co-localization of TH with FA1/dlk1 positive cells (arrows) in the lesioned striatum. Scale bar: 50μm.</p

Neuronal expression of FA1/dlk1 in the SNc.

<p>Double immunofluorescence staining of FA1/dlk1 and the general neuronal marker NeuN (upper row) and the astroglial marker GFAP (lower row) in the in the substantia nigra pars compacta of adult rats. Note the distinct co-localization of FA1/dlk1 with NeuN (arrows). As expected no co-localization was found for FA1/dlk1 and GFAP. Scale bar: 50 μm.</p

Co-expression of FA1/dlk1 with calbindin but not parvalbumin in the SN.

<p>Digitalized photomicrographs of FA1/dlk1-ir cells in the ventral mesencephalon of adult rats. Some calbindin (CB)-ir cells (arrowheads) showed co-localization with FA1/dlk1 (open arrowhead, upper row) in the substantia nigra pars compacta, whereas several CB-ir neurons (arrowheads, upper row) and FA1/dlk1-ir cells (arrows, upper row) did not co-localize. As expected from the distribution pattern depicted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116088#pone.0116088.g001" target="_blank">Fig. 1</a> no co-localization was detected for FA1/dlk1 with parvalbumin (PV) in the substantia nigra pars reticulata (lower row). Scale bar: 50μm.</p

FA1/dlk1-ir cells are dopaminergic projection neurons.

<p>Representative photomicrographs of double immunofluorescence stainings showing an overview of the distribution pattern of FA1/dlk1-ir cells (FA1) and the retrograde neuronal tracer Fluorogold (FG) in rat substantia nigra pars compacta (upper row). A substantial number of FA1/dlk1-ir cells were labeled for FG (arrows, middle row). Immunofluorescence images recorded at higher magnification demonstrated that a significant number of FA1/dlk1-ir cells labeled with FG co-expressed tyrosine hydroxylase (TH) (arrows, lower row). Notably, not all FA1/dlk1-ir cells expressing TH were labeled with FG (arrowheads), and as expected some TH-ir neurons did neither label for FG nor FA1/dlk1 (open arrowheads, lower row). Scale bars: 200μm (upper row), 50μm (middle and lower rows).</p