Fig. 6: The Nrf2-HMOX1 axis is involved in defence against ferroptosis and cisplatin resistance.

A Total RNA was extracted from A549/DDP cells following Nrf2 knockdown, and RT-PCR was performed to quantify the transcript levels of Nrf2 and its downstream target genes, including HMOX1. B, C Western blot analysis of Nrf2, HMOX1, SLC7A11, ACSL4, and GPX4 following Nrf2 knockdown in A549/DDP cells. D, E Western blot analysis of Nrf2, HMOX1, ACSL4, SLC7A11, and GPX4 in A549 (A) and A549/DDP (D) cells treated with various concentrations of cisplatin for 48 h. F, G A549/DDP cells transfected with negative control (NC) or Nrf2-shRNA were treated with CoPP, an inducer of HMOX1, for 48 h. Western blot analysis was conducted to assess HMOX1 expression levels in both cell lines. H Intracellular ROS levels were assessed via DCFH-DA (10 μM) following cisplatin treatment alone or cotreatment with the HMOX1 inducer CoPP and analyzed via flow cytometry. I Changes in the intracellular lipid peroxidation levels were analyzed with an MDA detection kit combined with a microplate reader. J The intracellular GSH level was measured via a GSH and GSSG assay kit. K Cell viability was assessed via a Cell Counting Kit following treatment with cisplatin alone or in combination with the HMOX1 inducer CoPP (10 μM). All WB experiments were repeated three times with similar results. The grayscale values of the protein bands were quantified relative to the β-actin loading control via ImageJ software. The error bars represent the standard deviations from three replicates. Statistical analysis was performed via unpaired t-tests, with *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 indicating significance relative to the control or differently treated groups.