Fig 1.
The effects of kahweol on H2O2-induced oxidant stress in hepatocytes.
AML12 cells were treated with kahweol (10 or 20 μM) for 19 h and then incubated with H2O2 (1mM) for a further 5 h. (A) DCF-DA staining of cells treated with 1 mM H2O2 and with or without 10 μM kahweol. Data in the bar graph are means ± SEM of three independent measurements. * p < 0.05 compared with the control, # p < 0.05 compared with the H2O2 treatment. (B) The viability of AML12 cells treated with or without H2O2 and kahweol, as determined by a CCK-8 assay. (C) FACScan flow cytometry analyses of AML12 cells treated with or without H2O2 and kahweol. Apoptosis was determined based on the sub-G1 population. (D) Western blot analyses of cleaved caspase 3 and HO-1 levels in AML12 cells treated with or without kahweol (10 or 20 μM) and 1 mM H2O2. * p < 0.05 compared with the control, # p < 0.05 compared with the H2O2 treatment. Con, control; Kah, kahweol.
Fig 2.
The effects of kahweol (Kah) on Nrf2 and HO-1 expression.
AML12 cells were treated with 20 μM kahweol and primary hepatocytes were treated with 40 μM kahweol. (A) Representative real-time RT-PCR analyses of Nrf2 mRNA expression in AML12 cells (left) and primary hepatocytes (right). * p < 0.05, * p < 0.05 compared with the control for each time. (B) Western blot analyses showing the effect of kahweol on Nrf2 expression in whole cell extracts of AML12 cells (left) and primary hepatocytes (right). Data in the bar graph are means ± SEM of three independent measurements. * p < 0.05 compared with the control for each time. (C) Western blot analysis of cytoplasmic and nuclear extracts of AML12 cells (left) and primary hepatocytes (right), showing the effects of kahweol on Nrf2 expression. Kahweol was treated for 24 hours. Data in the bar graph are means ± SEM of three independent measurements. * p < 0.05 compared with the control. (D) Western blot analyses showing the effect of kahweol on HO-1 expression in AML12 cells (left) and primary hepatocytes (right). Data in the bar graph are means ± SEM of three independent measurements. * p < 0.05 compared with the control for each time.
Fig 3.
The effect of kahweol (Kah) on Keap1 expression.
AML12 cells were treated with 20 μM kahweol and primary hepatocytes were treated with 40 μM kahweol. (A, B) Real-time RT-PCR (A) and western blot (B) analyses of Keap1 mRNA and protein levels, respectively, in AML12 cells (left) and primary hepatocytes (right). Data in the bar graphs are means ± SEM of three independent measurements. * p < 0.05 compared with the control for each time.
Fig 4.
The effect of kahweol (Kah) on p62 expression.
AML12 cells were treated with 20 μM kahweol and primary hepatocytes were treated with 40 μM kahweol. (A,B) Real-time RT-PCR (A) and western blot (B) analyses of p62 mRNA and protein levels, respectively, in AML12 cells (left) and primary hepatocytes (right). Data in the bar graph are means ± SEM of three independent measurements; * p < 0.05 compared with the control for each time. (C) Primary hepatocytes were transfected with a p62-specific siRNA (siRNA-p62; 100 nM) or a control siRNA (siCon; 100 nM), treated with 40 μM kahweol for 24 h, and then analyzed by western blotting. Data in the bar graph are means ± SEM of three independent measurements. * p < 0.05 compared with the control siRNA. # p < 0.05 compared with the siRNA-p62.
Fig 5.
The effect of kahweol (Kah) on Keap1 protein degradation.
(A) AML12 cells (left) were treated with 20 μM kahweol and primary hepatocytes (right) were treated with 40 μM kahweol in the absence or presence of E64d (10 μM), MG132 (0.5 μM), or chloroquine (CQ; 10 μM). Data in the bar graph are means ± SEM of three independent measurements. * p < 0.05 compared with the control. # p < 0.05. (B) Western blot analyses of Keap1 and HO-1 protein levels, in primary hepatocytes from ATG7f/f Alb-Cre+ (autophagy-deficient) mice. Data in the bar graph are means ± SEM of three independent measurements. * p < 0.05 compared with the ATG7f/f, # p < 0.05 compared with the ATG7f/f-Cre+.