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. 2021 Sep 24;11:728047. doi: 10.3389/fonc.2021.728047

Figure 5.

Figure 5

Pharmaceutical NRF2 activation promotes NF-кB activation in vitro and in vivo. (A, B) NRF2 activator ALA promotes NF-кB activation in BC cells. (A) 4T1 cells were treated with ALA (0, 40, and 60 μM), and p-65 and p-p65 expressions were detected by Western blotting. (B) NF-кB transcriptional activation was analyzed by luciferase reporter gene assay. (C, D) ALA enhances NF-кB-associated proteins expression in vitro. 4T1 cells were treated with ALA (0, 40, and 60 μM) for 4–6 h. (C) NF-кB regulatory proteins and (D) NF-кB-responsive targets were detected by Western blotting. β-Actin was a loading control. (E–G) ALA induces NF-кB activation and its downstream targets expression in vivo. 4T1-bearing NOD-SCID mice were treated with ALA (80 mg/kg) via intraperitoneal (i.p.) administration. IHC staining was used to detect (E) p-65 and p-p65, (F) IL-6 and TNF-α, and (G) ICAM-1 and VCAM-1 in lung and liver metastatic tissues, respectively. Data are presented as mean ± SD of three independent experiments. Scale bar: 50 μm. Representative images are shown. *p < 0.05, ** p < 0.01, and ***p < 0.001 between the indicated groups determined by unpaired Student’s t-test or the one-way analysis of variance (ANOVA).