Cell culture and adenovirus infection Primary human umbilical vein endothelial cells (HUVECs) were collected and cultured as previously described [11]. The CT26 mouse colon carcinoma and B16-F10 mouse melanoma cell lines were PF-6463922 chemical structure purchased from the American Type Culture Collection

(ATCC, Rockville Maryland, USA) and cultured in RM1640 medium (Gibico BRL, Grand Island, New York, USA) supplemented with 10% FBS and 100 μg/ml amikacin. 2.5 × 105 CT26 or B16-F10 cells were plated into 6-well plates and grew to 70%~80% confluence. The cells were washed three MK-4827 mouse times gently by serum-free medium and infected with Ad-PEDF or Ad-null (both at MOI50, 2.5 × 107 pfu per 5 × 105 cells) in 0.5 ml serum-free medium, CB-5083 research buy with normal saline as the non-infection control. After incubation for 90 minutes at 37°C, 1.5 mL complete medium with 10% FBS was

added to wells. Supernatants were collected after further culture for 72 hours and stored at -80°C for further analysis. Western blotting analysis Western blot analysis was performed as described previously [12]. Briefly, the supernatant was concentrated by super filter (5 kDa, Minipore) and mixed with an equal volume of sodium dodecyl sulfate (SDS) sample buffer. The proteins were separated by SDS-polyacrylamide gel electrophoresis (PAGE) and electronically transferred onto a polyvinylidene difluoride membrane (PVDF, Bio-Rad, Richmond, CA, USA). The blots were probed with a mouse anti-human PEDF monoclonal antibody (3:1000, mAb; R&D Systems, Boston, Massachusetts, USA) plus a peroxidase-conjugated secondary antibody, goat anti-mouse IgG (1:10,000, Thalidomide ZSGB-BIO, Beijing, China). The protein bands were visualized using an enhanced chemiluminescence (ECL) detection system (Pierce, Rockford, Illinois, USA). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) colorimetric assay The

MTT Assay was used to determine the effect of PEDF derived from Ad-PEDF infected cells on human umbilical vein endothelial cells (HUVECs). Three types of supernatants from B16-F10 cells infected with Ad-PEDF, Ad-Null or NS, respectively, were prepared as described above. Each type of supernatant was further diluted into a series of 1/2 dilutions in six tubes (from 1:2 to 1:64). Each supernatant dilution was added into triplicate wells (50 μl/well) of HUVECs which were seeded on 96-well plates on the previous day (2 × 103 cells in 50 μl complete medium per well). The cells were incubated at 37°C in 5% CO2 for 72 hours. Then, each well received 10 μl MTT solution (5 mg/mL). After a 4-hour incubation, the media was removed and 150 μl dimethyl sulfoxide was added. After 20 min of incubation, the OD value was determined by a microplate reader (3550-UV, BIO-RAD, USA)[13]. The following formula was used to calculate the inhibition rate of HUVEC proliferation: [1 - (experimental group OD value - negative control OD value)/(positive control OD value - negative control OD value)] × 100%.