1α,25-Dihydroxycholecalciferol (Vitamin D3) Induces NO-Dependent Endothelial Cell Proliferation and Migration in a Three-Dimensional Matrix Cell Physiol Biochem 2013;31:815-822 - DOI:10.1159/000350099 Fig. 1. NO production. Quantification of NO production measured by means of Griess method and expressed as percentage of control values. NO production was evaluated after 3 minutes stimulation in the presence of different concentrations of Vit. D (1-100 nM). Black bars = cells without ZK159222 addition, gray bars = cells + 10 nM ZK159222 (P<0.05). © 2013 S. Karger AG, Basel - CC BY-NC 3.0

1α,25-Dihydroxycholecalciferol (Vitamin D3) Induces NO-Dependent Endothelial Cell Proliferation and Migration in a Three-Dimensional Matrix Cell Physiol Biochem 2013;31:815-822 - DOI:10.1159/000350099 Fig. 2. PAE cells proliferation. A) Determination of PAE proliferation in the presence of different concentrations of Vit. D (1-100 nM) after 24 hours of incubation. Results are expressed as n. cells/mm2 ± S.D. Black bars = cells without L-NAME addition, gray bars = cells + 10 mM L-NAME. * p<0.05; ** p<0.001. B) Optical microscopy images of control and Vit. D (10 nM) treated cells both in absence or presence of 10 mM L-NAME after 24 hours of incubation, stained with toluidine blue. Magnification = 10X. Scale bar = 150 µm. © 2013 S. Karger AG, Basel - CC BY-NC 3.0

1α,25-Dihydroxycholecalciferol (Vitamin D3) Induces NO-Dependent Endothelial Cell Proliferation and Migration in a Three-Dimensional Matrix Cell Physiol Biochem 2013;31:815-822 - DOI:10.1159/000350099 Fig. 3. PAE cells migration into a three-dimensional matrix. A) Determination of PAE migration after 7 days of incubation in the presence of different concentrations of Vit. D (1-100 nM). Results are expressed as n. cells/HPF ± S.D. * p<0.05. B) Determination of control and Vit. D (100 nM) treated cells migration both in the presence or absence of 10 mM L-NAME after 7 days of incubation. *p<0.0001 compared to control sample; # p<0.05 compared to Vit. D 100 nM alone. © 2013 S. Karger AG, Basel - CC BY-NC 3.0

1α,25-Dihydroxycholecalciferol (Vitamin D3) Induces NO-Dependent Endothelial Cell Proliferation and Migration in a Three-Dimensional Matrix Cell Physiol Biochem 2013;31:815-822 - DOI:10.1159/000350099 Fig. 4. Optical microscopy images of control and Vit. D (100 nM) treated cells both in the presence or absence of 10 mM L-NAME after 7 days of incubation, stained with Hoechst 33342. Magnification = 10X. Scale bar = 60 µm. © 2013 S. Karger AG, Basel - CC BY-NC 3.0

1α,25-Dihydroxycholecalciferol (Vitamin D3) Induces NO-Dependent Endothelial Cell Proliferation and Migration in a Three-Dimensional Matrix Cell Physiol Biochem 2013;31:815-822 - DOI:10.1159/000350099 Fig. 5. MMP-2 production. A) Representative zymography of cell growth medium form PAE cells migrated into the three-dimensional matrix for 7 days in the presence of different Vit. D concentrations (1-100 nM). B) Densitometric quantification of MMP-2 expression. C) Representative zymography of cell growth medium from control and Vit. D (100 nM) PAE cells migrated into the three-dimensional matrix for 7 days, both in the presence or absence of 10 mM L-NAME. D) Densitometric quantification of MMP-2 expression. © 2013 S. Karger AG, Basel - CC BY-NC 3.0