Pig pseudo attP sites are in favor of robust transgene expression. (A) Extracellular EGFP expression. Medium containing extracellular EGFP was harvested for EGFP fluorescence and IGF-I detection. IGF-I was internal control for input normalization. IGF-I concentration was measured by a porcine ELISA kit. Sp, the supernatant of PK15 cell line as negative control. (B) Intracellular EGFP level by western blot. Transgenic cells were harvested and quantified by a BCA protein quantification kit. Anti-EGFP antibody was used to detect the intracellular EGFP expression, and anti-β-actin antibody was used as internal control. MW of EGFP and pig β-actin is 27 kD and 43 kD, respectively. They are shown by arrows. PK15 cell lysate was used as negative control. The intensity of the bands was quantified by VisionCapture and converted into Prism to create the bar graph. (C) Cell proliferation assay. 103 cells were seeded in a 96-well plate in triplicate. 10 μl Alamarblue indicator (Gibco) was added into the medium (with a final concentration of 10% v/v). The plate was incubated for additional 5 hours. The optical densities were measured at 570 nm and 630 nm in a micro-plate reader. Reduction percentage was calculated according to the formula provided in the instruction. Such a procedure was repeated in continuous 15 days. The reduction of four time points (day1, day5, day10 and day15) was indicated. Fresh medium was used as blank control. PK15 is the wild-type cell line. R1, R2 and R3 are randomly integrated transgenic cell lines.