Fig. 4. The HBXIP/Nrf2 feedback loop regulates redox homeostasis in breast cancer cells following detachment.

A MCF-7 cells were infected with pLVSIN-Nrf2 lentivirus or treated with 50 μM H₂O₂. MDA-MB-436 cells were infected with pLKO-shHBXIP lentivirus or treated with 5 μM ML385. The indicated treated cells were fractionated, and the expression of HBXIP and NQO1 was measured. B Wild-type MEFs and Nrf2^−/− MEFs infected with pLVSIN-Nrf2 lentivirus were fractionated, and the levels of Nrf2 and HBXIP were analyzed via western blotting. C Stable MCF-7 and MDA-MB-436 cells as indicated were cultured in suspension for 48 h and (a) stained with CM-H₂DCFDA and subjected to flow cytometry to determine the intracellular ROS level; (b) stained with MitoSOX Red, and the ROS levels were determined; (c) used to measure the reduced GSH levels; (d) used to measure NADPH levels; (e) used for assessment of mitochondrial membrane potential using a JC1 assay; and (f) used for ATP measurement using an ATP determination kit. The error bars indicate the ±SD values as assessed by Student’s t test. All the experiments were performed at least three times.