Journal: Cell Death Discovery

Article Title: The Nrf2-HMOX1 pathway as a therapeutic target for reversing cisplatin resistance in non-small cell lung cancer via inhibiting ferroptosis

doi: 10.1038/s41420-025-02564-z

Figure Lengend Snippet: A , B In vitro viability assay of parental A549 cells and cisplatin-resistant A549/DDP cells. A549 and A549/DDP cells in the logarithmic growth phase were seeded in 96-well plates and exposed to varying concentrations of cisplatin (0.0096 μM to 30 μM) for 48 h, after which cell viability was assessed. The data points represent averages of two replicates, with the curve showing the mean ± SEM. C In vitro clonogenic assay. A549 or A549/DDP cells were treated with DMSO or various concentrations of cisplatin for 48 h. After treatment, the cells were cultured for an additional 11–14 days without drugs, fixed with paraformaldehyde, and stained with crystal violet. Colonies were imaged via an inverted microscope. D The colonies were counted via ImageJ software. Colonies with more than 50 cells were scored via ImageJ. The experiments were conducted in triplicate ( n = 3). E , F A549 and A549/DDP cells were treated with cisplatin for 48 h and then stained with propidium iodide (PI). Immunofluorescence and flow cytometry were performed to compare the degree of cell death following cisplatin treatment. The error bars represent the standard deviations of three replicates. Analysis was performed via unpaired t- tests, with **** p < 0.0001 relative to the control or differently treated groups.

Article Snippet: The NSCLC cisplatin-resistant cell line A549/DDP (CL-0519) and its parental cell line A549 (CL-0016) were purchased from Procell Life Science & Technology Co., Ltd.

Techniques: In Vitro, Viability Assay, Clonogenic Assay, Cell Culture, Staining, Inverted Microscopy, Software, Immunofluorescence, Flow Cytometry, Control

Journal: Cell Death Discovery

Article Title: The Nrf2-HMOX1 pathway as a therapeutic target for reversing cisplatin resistance in non-small cell lung cancer via inhibiting ferroptosis

doi: 10.1038/s41420-025-02564-z

Figure Lengend Snippet: Transmission electron microscopy images of lung cancer cells, including A549/DDP ( A , C , E ) and A549 cells ( B , D , F ). The cells were treated with DMSO ( A , B ), the ferroptosis inducer erastin (10 μM), or cisplatin (20 μM) ( E , F ) for 48 h. The white arrowheads indicate alterations in mitochondrial ultrastructure, characterized by smaller mitochondria, less tubular morphology, and darker-stained membranes with distinct disruption of inner membrane folding. At least 10 cells were examined per treatment condition. Scale bar = 1 μm.

Article Snippet: The NSCLC cisplatin-resistant cell line A549/DDP (CL-0519) and its parental cell line A549 (CL-0016) were purchased from Procell Life Science & Technology Co., Ltd.

Techniques: Transmission Assay, Electron Microscopy, Staining, Disruption, Membrane

Journal: Cell Death Discovery

Article Title: The Nrf2-HMOX1 pathway as a therapeutic target for reversing cisplatin resistance in non-small cell lung cancer via inhibiting ferroptosis

doi: 10.1038/s41420-025-02564-z

Figure Lengend Snippet: A – D A549 and A549/DDP cells in the logarithmic growth phase were seeded in 6-well plates and treated with cisplatin for 48 h. Subsequently, 10 μM DCFH-DA ( A , B ) or 2 μM C11-BODIPY581/591 ( C , D ) fluorescent probes were added to the medium in each well for 30 min to ensure coverage of the cell layer by the probe. Then, green fluorescence was observed via an immunofluorescence microscope ( A – C ), or the cells were collected to analyze the levels and differences in intracellular ROS and lipid peroxidation via flow cytometry ( B – D ). The error bars represent the standard deviation from three replicates. Analysis was performed via an unpaired t -test, with **** p < 0.0001 relative to the control or differently treated groups. E A549 and A549/DDP cells were seeded in 6-well plates, followed by stimulation with cisplatin (20 μM) alone or in combination with the ferroptosis inducer erastin (10 μM) or the iron chelator DFO (50 μM) for 48 h. Immunofluorescence staining with propidium iodide (PI) was performed to compare the differences in cell death levels after different treatments. F Flow cytometry was used to analyze the degree of cell death after different cisplatin treatments. The error bars represent the standard deviation from three replicates. Analysis was performed via an unpaired t -test, wi t h **** p < 0.0001 relative to the control or differently treated groups.

Article Snippet: The NSCLC cisplatin-resistant cell line A549/DDP (CL-0519) and its parental cell line A549 (CL-0016) were purchased from Procell Life Science & Technology Co., Ltd.

Techniques: Fluorescence, Immunofluorescence, Microscopy, Flow Cytometry, Standard Deviation, Control, Staining

Journal: Cell Death Discovery

Article Title: The Nrf2-HMOX1 pathway as a therapeutic target for reversing cisplatin resistance in non-small cell lung cancer via inhibiting ferroptosis

doi: 10.1038/s41420-025-02564-z

Figure Lengend Snippet: A , B Western blotting was performed to determine the expression levels of Nrf2 and NQO1 in both NC- and Nrf2-shRNA-infected A549/DDP cells. The grayscale values of the protein bands, including those of Nrf2 and NQO1, were quantified relative to the β-actin loading control via ImageJ software. C , D A549/DDP cells were treated with 20 μM cisplatin for 48 h following Nrf2 knockdown. The number of PI-positive cells exposed to cisplatin was determined via fluorescence microscopy and flow cytometry. E – G A549/DDP cells were treated with cisplatin (20 μM) alone or with the ferroptosis inducer erastin (10 μM) or the iron chelator DFO (50 μM) for 48 h. E PI-positive cells exposed to cisplatin were stained and counted via flow cytometry. F The intracellular ROS level was assessed via an intracellular total ROS activity assay kit (orange fluorescence) following various treatments and analyzed via flow cytometry. G Changes in the intracellular lipid peroxidation levels were determined via an MDA detection kit and a microplate reader. The error bars represent the standard deviation from three replicates. Statistical analysis was performed via unpaired t -tests: ** p < 0.01, *** p < 0.001, **** p < 0.0001 relative to the control or differently treated groups.

Article Snippet: The NSCLC cisplatin-resistant cell line A549/DDP (CL-0519) and its parental cell line A549 (CL-0016) were purchased from Procell Life Science & Technology Co., Ltd.

Techniques: Western Blot, Expressing, shRNA, Infection, Control, Software, Knockdown, Fluorescence, Microscopy, Flow Cytometry, Staining, Activity Assay, Standard Deviation

Journal: Cell Death Discovery

Article Title: The Nrf2-HMOX1 pathway as a therapeutic target for reversing cisplatin resistance in non-small cell lung cancer via inhibiting ferroptosis

doi: 10.1038/s41420-025-02564-z

Figure Lengend Snippet: A A conserved Nrf2 activity signature in lung cancer cells identified through a comprehensive multi-dataset analysis ( GSE118841 : Nrf2 knockdown; GSE118842 : Nrf2 activation). B Single-sample Gene Set Enrichment Analysis (ssGSEA) demonstrating dynamic NRG scores across different treatment groups (DMSO, RSL3, and RSL3 combined with Fer-1) based on the dataset ( GSE247883 ). C – F RNA sequencing analysis of A549/DDP cells subsequent to Nrf2 knockdown. C Bubble plot illustrating KEGG enrichment analysis of pathways significantly altered in the control and Nrf2-knockdown groups. D GSEA based on the KEGG database revealed significant differences in a set of genes related to ferroptosis pathways between the control and Nrf2-knockdown groups. E Volcano plot depicting genes that were differentially expressed between the control and Nrf2-knockdown groups. Genes with a false discovery rate (FDR) parameter below 0.05 and an absolute fold change ≥2 were deemed differentially expressed. F Bar chart created with the ggplot2 (v.3.5.1) package highlighting the top 10 downregulated genes relatated to ferroptosis following Nrf2 knockdown.

Article Snippet: The NSCLC cisplatin-resistant cell line A549/DDP (CL-0519) and its parental cell line A549 (CL-0016) were purchased from Procell Life Science & Technology Co., Ltd.

Techniques: Activity Assay, Knockdown, Activation Assay, RNA Sequencing, Control

Journal: Cell Death Discovery

Article Title: The Nrf2-HMOX1 pathway as a therapeutic target for reversing cisplatin resistance in non-small cell lung cancer via inhibiting ferroptosis

doi: 10.1038/s41420-025-02564-z

Figure Lengend Snippet: 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.

Article Snippet: The NSCLC cisplatin-resistant cell line A549/DDP (CL-0519) and its parental cell line A549 (CL-0016) were purchased from Procell Life Science & Technology Co., Ltd.

Techniques: Knockdown, Reverse Transcription Polymerase Chain Reaction, Western Blot, Transfection, Negative Control, shRNA, Expressing, Flow Cytometry, GSSG Assay, Cell Counting, Control, Software

Journal: Cell Death Discovery

Article Title: The Nrf2-HMOX1 pathway as a therapeutic target for reversing cisplatin resistance in non-small cell lung cancer via inhibiting ferroptosis

doi: 10.1038/s41420-025-02564-z

Figure Lengend Snippet: A , B Both A549 and A549/DDP cells were treated with the Nrf2 inhibitor ML385 (5 μM) for 48 h. Western blot analysis was conducted to assess the expression levels of Nrf2, HMOX1, ACSL4, GPX4, and SLC7A11 in both cell lines. The WB experiment was 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. C , D Both A549 and A549/DDP cells were treated with cisplatin (20 μM) alone or in combination with ML385 (5 μM). The intracellular ROS level was assessed via DCFH-DA (10 μM) and analyzed via flow cytometry. E , F Intracellular Lip ROS levels were assessed via C11-BODIPY581/591 (2 μM) and analyzed via flow cytometry. G Intracellular GSH levels were measured via GSH and GSSG assay kits. H A LIP assay was performed to quantify labile iron levels. I Cell viability was assessed via a Cell Counting Kit. Statistical analysis was performed via unpaired t- tests, with significance levels denoted as ** p < 0.01, *** p < 0.001, and **** p < 0.0001 relative to the control or differently treated groups.

Article Snippet: The NSCLC cisplatin-resistant cell line A549/DDP (CL-0519) and its parental cell line A549 (CL-0016) were purchased from Procell Life Science & Technology Co., Ltd.

Techniques: Western Blot, Expressing, Control, Software, Flow Cytometry, GSSG Assay, Cell Counting