Figure 5

Nrf2 activation leads to decreased proliferation of TNF-α pre-treated synoviocytes. HSE cells were co-transfected with pGL3-ARE/phRL-TK and either pre-treated (3 h) with 10 ng/mL TNF-α and then stimulated for a further 6 h with 6.25 μM sulforaphane (SFN) or only stimulated for 6 h with 6.25 μM SFN without TNF-α pre-treatment. Afterwards, dual luciferase reporter gene (DLR) assay was carried out (A-C). A real-time cell analysis (RTCA) experiment was performed with HSE cells stably transduced with either a shRNA targeting Keap1 mRNA or a shNonTarget control construct. Twenty-four h after seeding, the cells were treated with 10 ng/mL TNF-α and were observed for further 24 h by the RTCA device (D). The Cell Index was determined at three time-points (TP) (E) and the slope of the curve was calculated for three characteristic phases of proliferation (F). All experiments were performed with n = 8. Graphs represent mean + SD. Statistical significances are indicated as $P < 0.05, $$P < 0.01, $$$P < 0.001 vs. control; ###P < 0.001 vs. all other groups; *P < 0.05, **P < 0.01, ***P < 0.001 as indicated. (DLR: one-way ANOVA with Bonferroni multiple comparison post hoc test; RTCA analysis: two-way ANOVA with Bonferroni post hoc tests, GraphPad Prism 5 software; GraphPad Software, La Jolla, CA, USA.).