Fig 2: PI3K inhibitor partly reverses the proliferation and migration of Nrf3 overexpressing triple-negative breast cancer cells. (A) P110α, AKT, P-AKT, mTOR, and p-mTOR expression following treatment with the PI3K inhibitor. (B-D) CCK-8 assays and animal experiments showed that the proliferative ability of MDA-MB-231/Nrf3 cells was partly reduced when treated with the PI3K inhibitor. The migratory capacity was detected using (E and F) Transwell migration assays and (G and H) wound healing assays. The results suggest PI3K inhibitor partly reverses the effect of Nrf3 on migration in MDA-MB-231 cells. *P<0.05, **P<0.01 and ***P<0.001. Nrf3, nuclear factor erythroid 2-related factor 3; OD, optical density.

Fig 3: Nrf3 is upregulated in triple-negative breast cancer, and its expression is negatively associated with survival prognosis. (A) Differential expression of Nrf3 mRNA levels in breast cancer and the paracancerous tissues. Normal tissues, n=112; tumor tissues, n=1,066. (B) Nrf3 mRNA expression levels in normal breast tissue and different pathological types of breast cancer tissues. Normal tissues, n=112; tumor tissues, n=467; luminal, n=471; Her2+, n=29; TNBC, n=117. (C) Differential expression of Nrf3 mRNA levels in breast cancer and the paracancerous tissues. (D) Kaplan-Meier survival analysis between low and high Nrf3 expression breast cancer patients. Nrf3-low, n=54; Nrf3-high, n=72. P=0.025. (E) Representative images of immunohistochemical staining and the (F) immunohistochemical score in 105 breast cancer patients with different pathological types. TNBC, n=35; Her2+,0 n=35; luminal, n=35. *P<0.05, **P<0.01 and ****P<0.0001. Nrf3, nuclear factor erythroid 2-related factor 3; TNBC, triple-negative breast cancer; TCGA, The Cancer Genome Atlas; GTEx, Genotype-Tissue Expression; BRCA, breast cancer.

Fig 4: Nrf3 regulates PI3K/AKT/mTOR signaling and EMT-related protein expression. (A). Gene set variation Analysis was performed based on RNA sequencing data. (B) The genes related to proliferation and migration were clustered and displayed as a heat map. (C) quantitative PCR was used to verify the relative expression levels of EMT-related genes normalized to actin. (D) Western blot analysis showed the relative expression of the proteins in the PI3k/Akt/mTOR pathway. (E) Relative expression of EMT-related proteins. *P<0.05, **P<0.01 and ***P<0.001.

Fig 5: Overexpression of Nrf3 contributes to the proliferation and migration of TNBC, while knockdown of Nrf3 has the opposite effect. (A-D) The proliferative capacity of Nrf3 overexpression and knockdown was determined using a CCK-8 assay. (E and F) Transwell migration experiments and (G and H) wound healing assays showed that Nrf3 overexpression increased the migration of breast cancer cells, whereas Nrf3 knockdown reduced migration. *P<0.05 and ***P<0.001. Nrf3, nuclear factor erythroid 2-related factor 3; TNBC, triple-negative breast cancer; OD, optical density; sh, short hairpin.