Figure 3. Oncogenic Kras-induced ROS drive ADM through nuclear factor-κB.

A: Primary mouse pancreatic acinar cells were isolated, lentivirally-infected with control or Kras^G12V harboring virus and seeded on collagen in presence of MitoQ (500 nM), as indicated. After 48 hours, cells were isolated, nuclear extracts prepared and an EMSA performed to measure NF-κB binding activity. B: Primary mouse pancreatic acinar cells were isolated, co-infected with lentivirus harboring control or Kras^G12V virus and adenovirus harboring a NF-κB-luciferase reporter gene. Cells were seeded on collagen in presence of MitoQ (500 nM), as indicated. After 48 hours, cells were isolated and a luciferase assay performed to measure NF-κB activity. C: Primary mouse pancreatic acinar cells were isolated from LSL-Kras^G12D mice, infected with adeno-null or adeno-cre, and then seeded in 3D collagen explant culture in presence of BMS345541 (labeled: BMS) at indicated doses. At day 5, ducts formed (ADM events; number of ducts per field) were counted. D: Primary mouse pancreatic acinar cells were isolated from wildtype mice, infected with adenovirus harboring control (null), NF-κB1 or NF-κB2, and then seeded in 3D collagen explant culture. At day 5, brightfield pictures were taken (4x magnification; show is a representative picture; bar = 100 μm) and newly-formed duct-like structures formed were counted. In B–D, * indicates statistical significance (p<0.05) as compared to control; ** as compared to Kras^G12V.