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. 2020 Dec 22;2020:6670464. doi: 10.1155/2020/6670464

Figure 2.

Figure 2

Induction of nrf2 expression and nuclear translocation by TNF-α through inducing ROS. The mRNA expression (a) and nuclear protein level (b, c) of nrf2 in RA-FLS were detected by real-time PCR and Western blotting after TNF-α (25 ng/mL) stimulation for 0, 3, 6, and 24 h. Histone-3 was used as the housekeeping gene in Western blotting. N = 3. (d) Nrf2 nuclear translocation (as shown by white arrows) after TNF-α (25 ng/mL) stimulation for 0, 3, 6, and 24 h was examined by immunofluorescence staining. Representative pictures (magnification ×200) from different groups and partially enlarged pictures (magnification ×800) are shown. Scale bar represented 50 μm. (e) Intracellular ROS levels in RA-FLS stimulated with TNF-α (25 ng/mL) for 0, 0.5, 1, 2, and 3 h and tert-butyl hydroperoxide (TBHP, 50 μM) (positive control) for 3 h were detected using DCFDA probe. Scale bar represented 100 μm. (f) The nuclear protein level of nrf2 in RA-FLS after TNF-α (25 ng/mL) stimulation for 3 h with or without N-acetylcysteine (NAC, 5 μM) pretreatment for 1 h was detected by Western blotting. Histone-3 was used as the housekeeping gene. Data were shown as the mean ± SD; ∗p < 0.05 and ∗∗p < 0.01.