Fig. 5

RAEC transfected with shAkt and shERK2 and then stimulated with insulin or TNFα alone or in combination exhibit increased VCAM-1 protein expression greater than stimulated, mock transfected cells, but not greater than cells transfected with shAkt alone. RAEC were stably transfected with shAKT and then transiently transfected with shERK2. Subsequently, cells were stimulated with insulin (10 nM, 1 h) or TNFα (10 ng/mL, 6 h) or in combination. (Upper Panel) C, control RAEC, no shRNA. A, cells transfected with shAkt alone. AE, cells stably transfected with shAkt and then transiently transfected with shERK2. (Lower Panel) Open bars are control cells not transfected. Stippled bars (Akt KD) RAEC stably transfected with shAkt. Black bars (Akt KD + shERK2) RAEC stably transfected with shAkt and transiently transfected with shERK2. “NST”, cells not stimulated with insulin or TNFα. Total VCAM-1 is expressed as fold increase above negative controls and represents the mean ± SEM of five independent experiments. Alpha-tubulin was used to normalize VCAM-1 profiles. #, P < 0.05 verses non-stimulated controls. *, P < 0.05 versus stimulated controls of similar treatment group without shRNA