Figure 5.

SSM activated Nrf2 signaling through promoting ERK and AKT activation in Caco-2 cells. (a) The effect of SSM on ERK, AKT, P38, PKC, and Keap1 was analyzed by western blot in Caco-2 cells treated with 0 μM SSM (0.1% DMSO) and various concentrations of SSM (10, 20, 40, and 80 μM) for 2 and 8 h. (b) Quantitative data were obtained from the densitometric quantification of immunoblots by using ImageJ software. Data were expressed as the mean ± SD (n = 5). ^∗p < 0.05 and ^∗∗p < 0.01 versus the DMSO treatment group. (c) Caco-2 cells were treated with 0.1% DMSO (control), PD98059 (5 and 10 μM), or wortmannin (5 and 10 μM) for 2 h prior to SSM treatment (40 μM) for 8 h. The protein expressions of AKT and ERK were analyzed by western blot. Quantitative data were obtained from the densitometric quantification of immunoblots using ImageJ software. Data were expressed as the mean ± SD (n = 5). ^∗p < 0.05 versus the DMSO treatment group. ^#p < 0.05 and ^###p < 0.001 versus the SSM treatment group. (d) Caco-2 cells were treated with 0.1% DMSO (control), PD98059 (5 and 10 μM), or wortmannin (5 and 10 μM) for 2 h prior to SSM treatment (40 μM) for 8 h. The protein expressions of Nrf2 and its target genes were analyzed by western blot. Quantitative data were obtained from the densitometric quantification of immunoblots using ImageJ software. Data were expressed as the mean ± SD (n = 5). ^∗p < 0.05 versus the DMSO treatment group. ^#p < 0.05, ^##p < 0.01, and ^###p < 0.001 versus the SSM treatment group. (e) Caco-2 cells were treated with 0.1% DMSO (control), PD98059 (5 and 10 μM), or wortmannin (5 and 10 μM) for 2 h prior to SSM treatment (40 μM) for 8 h. The nuclear protein expression of Nrf2 was analyzed by western blot. Quantitative data were obtained from the densitometric quantification of immunoblots using ImageJ software. Data were expressed as the mean ± SD (n = 5). ^∗∗∗p < 0.001 versus the DMSO treatment group. ^###p < 0.001 versus the SSM treatment group. (f) Caco-2 cells were treated with 0.1% DMSO (control), PD98059 (10 μM), or wortmannin (10 μM) for 2 h prior to SSM treatment (40 μM) for 8 h and then treated with 1.6 mM H₂O₂ for another 24 h. Images are displayed at 200x magnification using an OLYMPUS IX73 inverted fluorescence phase-contrast microscope. The quantification of fluorescence was done with a Flex Station 3 multifunctional microplate reader. Data were expressed as the mean ± SD (n = 5). ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001 versus the control group. (g, h) Caco-2 cells were treated with 0.1% DMSO (control), PD98059 (10 μM), or wortmannin (10 μM) for 2 h prior to SSM treatment (40 μM) for 8 h and then treated with 1.6 mM H₂O₂ for another 24 h. Cell viability was measured by MTT assay. Data were expressed as the mean ± SD (n = 5). ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001 versus the control group.