Fig. 7

Curcumin decreased Nrf2 by activating GSK3β but independent of Nrf2/Keap1 in AsT cells. A BEAS-2B or AsT cells were untreated or were pretreated with 10 μM MG132 for 2 h after which cells were washed and treated with 50 μM CHX. At different incubation times, cells were lysed and protein levels were evaluated by western blot using Nrf2 specific antibody. Expression of β-actin was evaluated as a loading control. B BEAS-2B or AsT cells were treated with 10 μM MG132 for 2 h before cell lysates were collected. Anti-Nrf2 immunoprecipitates were analyzed by immunoblot with anti-Nrf2 and anti-Keap1 antibodies for detection of Nrf2-conjugated Keap1, whole protein lysates were collected and Nrf2, Keap1 and β-actin expression were examined by immunoblotting. C AsT cells were transfected with HA-Keap1. 24 h after transfection cells were then treated with 25 μM curcumin for 24 h before cell lysates were collected. HA, Keap1, Nrf2 and HO-1 expression were examined by immunoblotting. D AsT cells were transfected with control and GSK3β siRNA. After 24 h transfection, cell lysates were collected. GSK3β, Nrf2 and HO-1 expression were examined by immunoblotting. E AsT cells were transfected with control and GSK3β siRNA. 24 h after transfection cells were then treated with 25 μM curcumin for 24 h. Cell viabilities were detected by staining with MTT assay. AsT cells were co-treated with 25 μM curcumin and 5 μM SB216763 for 24 h. Cell lysates were collected and p-GSK3β s9, GSK3β, Nrf2, PARP and cleaved PARP expression were examined by immunoblotting (F). Cell viabilities and cell apoptosis were detected by staining with MTT assay and PI/Annex V assay, individually (G). The data are expressed as the mean ± SD of three independent experiments (n = 9). *p < 0.05, **p < 0.01 vs control