RC-1 cells were collected with trypsin (Sigma-Aldrich), passed through a cell strainer (Becton Dickinson, Franklin Lakes, NJ), and put into a 48-well collagen type 1–coated glass bottom chamber (Asahi Technoglass, Chiba, Japan) filled with 400 μL MEM with 10% FBS, 4 × 104cells/well. A plasmid solution 0.5 μL was added to the medium. Immediately thereafter, US was exposed to the medium using a 6-mm probe generated by an US machine (Sonitron 2000; Richmar, Inola, OK). During exposure, the medium-containing cells were gently stirred by a magnetic stirrer (300 rpm). To induce MBs, perflutren protein type A microsphere (Optison; Amersham Health, Princeton, NJ)—a well-established, second-generation US medical diagnostic product with robust capability—was used. It is an albumin-shelled US contrast agent composed of approximately 5 to 8 × 108 MBs per milliliter measuring between 2 and 4.5 μm in diameter and filled with octaperfluoropropane. The indicated percentage was added to the plasmid solution (0.5, 0.25, or 0.1 μL), gently mixed, and left for 60 seconds. Immediately thereafter, the mixed solution was added to the dish and exposed to US as described.