1. Molecular Therapy - Nucleic Acids
Endoglin (CD105) Silencing Mediated by shRNA Under the Control of Endothelin-1 Promoter for Targeted Gene Therapy of Melanoma 
Natasa Tesic, Urska Kamensek, Gregor Sersa, Simona Kranjc, Monika Stimac, Ursa Lampreht, Veronique Preat, Gaelle Vandermeulen, Miha Butinar, Boris Turk, Maja Cemazar 
Molecular Therapy - Nucleic Acids 
Volume 4, (January 2015)
DOI: /mtna
Copyright © 2015 American Society of Gene & Cell Therapy Terms and Conditions

2. Figure 1
Gene electrotransfer of anti-CD105 plasmids had antiproliferative and antiangiogenic effects on murine SVEC4-10 endothelial cells. (a) CD105 mRNA level after gene electrotransfer was determined by qRT-PCR. (b) Migration of cells after the treatments was determined by wound healing assay. (c) Invasion of cells was determined by determining the absorbance of cells that travelled through Matrigel covered cell inserts. (d) Proliferation of cells was followed for 96 hours by Alamar Blue assay. Proliferation of cells in each experimental group was normalized to day 0. (e) Tube formation assay by plating of cells on Matrigel covered wells and the formation of capillary like structures was assessed by staining with calcein AM 1 hour after cell seeding. (f) Cell adhesion was performed 48 hours after treatment by plating the cells on Matrigel covered wells, which were washed after 3 hours and the absorbance of the attached cells was determined by Alamar Blue assay. Migration, invasion, and adhesion of cells from each experimental group were normalized to the untreated control group. Each bar represents the mean ± SEM for three independent experiments. *P < 0.05 compared to all other groups. N.S., not statistically significant. (e) Scale bar = 500 µm.
Molecular Therapy - Nucleic Acids 2015 4, DOI: ( /mtna )
Copyright © 2015 American Society of Gene & Cell Therapy Terms and Conditions

3. Figure 2
Gene electrotransfer of anti-CD105 plasmids reduced proliferation of murine B16F10-luc melanoma cells. (a) Representative images of cell proliferation taken at day 4 with a DP72 CCD camera (Olympus) connected to a BX-51 microscope (Olympus). (b) Relative cell proliferation. Proliferation of cells in each experimental group was normalized to day 0. Each symbol represents the mean ± SEM for three independent experiments. *P < 0.05 compared to all other groups. Scale bar = 200 µm.
Molecular Therapy - Nucleic Acids 2015 4, DOI: ( /mtna )
Copyright © 2015 American Society of Gene & Cell Therapy Terms and Conditions

4. Figure 3
Two consecutive treatments with anti-CD105 plasmids had an antitumor effect in murine B16F10-luc melanoma. (a) CD105 mRNA level 48 hours after the second therapy. (b) The antitumor effect of anti-CD105 plasmids. CD105 mRNA level was normalized to the CD105 mRNA level of the untreated control group. The arrows represent the day of the treatment. Each bar represents the mean ± SEM for a group of three to six mice. Each symbol represents the mean ± SEM for a group of six mice; *P < 0.05 compared to the EP. N.S., not statistically significant.
Molecular Therapy - Nucleic Acids 2015 4, DOI: ( /mtna )
Copyright © 2015 American Society of Gene & Cell Therapy Terms and Conditions

5. Figure 4
Treatment of murine B16F10-luc melanoma with anti-CD105 plasmids prevented metastasis development. (a) Representative images from each group captured with the bioluminescence noninvasive whole body imaging system IVIS at day 21. (b) Percentages of metastases free mice after local tumor treatment with anti-CD105 plasmids at the end of follow-up. (c) Evaluation of the percentage of metastasis free mice by days. Each bar represents the mean ± SEM for a group of 12 mice. *P < 0.05 compared to Ctrl, EP, pET-antiCD105, and pControl. N.S., not statistically significant.
Molecular Therapy - Nucleic Acids 2015 4, DOI: ( /mtna )
Copyright © 2015 American Society of Gene & Cell Therapy Terms and Conditions

6. Figure 5
Two consecutive treatments with anti-CD105 plasmids increased tumor necrosis and reduced proliferation of tumor cells. (a) Representative images of HE staining of tumor sections. Scale bar = 500 µm. (b,c) Representative images of tumor sections stained with Ki-67 proliferation marker. (b) Scale bar = 500 µm. (c) Scale bar = 50 µm.
Molecular Therapy - Nucleic Acids 2015 4, DOI: ( /mtna )
Copyright © 2015 American Society of Gene & Cell Therapy Terms and Conditions

7. Figure 6
Two consecutive treatments with anti-CD105 plasmids reduced the number of blood vessels in murine B16F10-luc melanoma that expressed high levels of CD105. (a) B16F10-luc tumor was immunohistochemically stained for CD105 demonstrating high expression of CD105. Scale bar = 50 µm. (b,c) The number of CD31-positive blood vessels was reduced after two consecutive treatments with anti-CD105 plasmids in comparison with control. *P < 0.05 compared to control. N.S., not statistically significant. Scale bar = 50 µm.
Molecular Therapy - Nucleic Acids 2015 4, DOI: ( /mtna )
Copyright © 2015 American Society of Gene & Cell Therapy Terms and Conditions