Betulinic Acid Induces ROS-Dependent Apoptosis and S-Phase Arrest by Inhibiting the NF-κB Pathway in Human Multiple Myeloma

Figure 5

BA induced inhibition of the NF-κB pathway, resulting in accumulation of ROS in U266 cells. (a, b) The expression levels of NF-κB p65, p-NF-κB p65, p-IKKα/β, IκBα, and p-IκBα in U266 cells were evaluated by Western blotting after exposure to 40 μM BA for the indicated times or after exposure to the indicated concentrations for 12 h. (c) U266 cells incubated with 40 μM BA or 0.1% DMSO for 12 h were analyzed for NF-κB p65 expression and distribution by immunofluorescence; . (d) Representative flow cytometry plots using DCFH-DA after treatment with 40 μM BA for 12 h in the presence or absence of 10 ng/ml TNF-α and 5 μM BAY 11-7082. (e) Quantitative analysis of ROS levels with or without TNF-α and BAY 11-7082 in BA-treated U266 cells. vs. the control group; ^#vs. the BA-treated group; and ^##vs. the BA-treated group. (f) Quantitative analysis of survival rates by a CCK-8 assay via comparison between groups in the presence or absence of TNF-α and BAY 11-7082 and exposure to BA. vs. the control group; ^##vs. the BA-treated group.

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