Figure 2
Decreased expression of endogenous PTN following transfection of CAM cells with pCDNA3.1 carrying AS-PTN results in decreased angiogenesis. A. Equal amounts of protein extracts of chicken embryo CAM 24 h after application of pCDNA3.1 alone (PC) or carrying AS-PTN (AS) were analyzed by SDS-PAGE, followed by Western blot analysis for PTN and actin. B. Haematoxylin & eosin staining of CAM paraffin sections 24 h after application of 1 μg of pCDNA3.1 alone (PC) or carrying AS-PTN (AS). Untreated CAM paraffin sections at the same developmental stage were used as control. C. Different amounts of pCDNA3.1 alone or carrying AS-PTN were applied on the CAM and 48 h later the number of vessels was estimated using image analysis software. Results are expressed as mean ± S.E.M. of the % change of the total vessel length in treated with pCDNA3.1 carrying AS-PTN (AS) compared with the tissue treated with pCDNA3.1 vector alone (PC) at the same quantity (control). Asterisks denote a statistically significant difference (unpaired t-test) from the control. ***P < 0.001. The pictures are representative, showing the vessel network of the chicken embryo CAM after treatment with 1 μg pCDNA3.1 alone (PC) or carrying AS-PTN (AS). D. Representative pictures of Western blot analyses from three independent experiments for phosphorylated ERK1/2 (pERK1/2) and total ERK1/2 (tERK1/2) in protein extracts of chicken embryo CAM 24 h after application of pCDNA3.1 alone (PC) or carrying AS-PTN (AS). Phospho- and total protein amounts were quantified by densitometric analysis of the corresponding band in each lane, and the ratio pERK1/2/tERK1/2 was calculated in each lane. Results are mean ± S.E.M. of the % change of ERK1/2 phosphorylation in treated with pCDNA3.1 carrying AS-PTN (AS) compared with the tissue treated with pCDNA3.1 vector alone (PC) at the same quantity (control). Asterisks denote a statistically significant difference (unpaired t-test) from the control. *P < 0.05. E. Representative pictures of Western blot analyses from three independent experiments for PROX-1 and actin in protein extracts of chicken embryo CAM 24 h after application of pCDNA3.1 alone (PC) or carrying AS-PTN (AS).