Fig. 6

RAEC transfected with shERK2 and shAkt 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 one shERK2 alone. RAEC were stably transfected with shERK2 and then transiently transfected with shAkt. 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. E, cells transfected with shERK2 alone. EA, cells stably transfected with shERK2 and then transiently transfected with shAkt. (Lower Panel) Open bars are control cells not transfected. Stippled bars (ERK2 KD) RAEC stably transfected with shERK2. Black bars (ERK2 KD + shAkt) RAEC stably transfected with shERK2 and transiently transfected with shAkt. “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