Human senescence-related genes were obtained using the Aging Atlas database (ngdc.cncb.ac.cn/aging/index), with 2,916 relevant gene targets screened with a relevance score threshold of >5. Differentially expressed genes (DEGs) induced by chemotherapy were identified using the R package ‘limma’ (version 4.4.1; Posit Software) using the Gene Expression Omnibus (ncbi.nlm.nih.gov/geo/) dataset GSE6410 (15), with thresholds of log₂ fold change >0.3 and P<0.05. KEGG enrichment analysis (https://www.kegg.jp/) of intersection genes was performed using the clusterProfiler package (version 4.4.1), with significant pathways defined as P<0.05.

NSCLC cell lines A549 (cat. no. CL-0016) and PC-9 (cat. no. CL-0668) were maintained in media (cat. nos. CM-0016 and CM-0668; all Procell Life Science and Technology Co., Ltd.) in a humidified atmosphere with 5% CO₂ at 37°C. All cells were authenticated through short tandem repeat analysis and tested for Mycoplasma contamination. Untreated cells were designated as the control group.

To induce senescence, A549 and PC-9 cells were exposed to 100 nM DOX (cat. no. HY-15142A; MedChemExpress) for 4 days at 37°C (16). For generating persistent A549 or PC-9 cell (PAC/PPC), the senescent cells were washed with PBS (cat. no. C0221A; Beyotime Biotechnology) and stimulated with fresh media containing 10% FBS (cat. no. C0252; Beyotime Biotechnology) for 10 days at 37°C.

pGPU6-cloned short hairpin RNA against AREG (pGPU6-shAREG; cat. no. C02001; 5′-CCTCTTTCCAGTGGATCATAA-3′), pEX-2-cloned plasmids overexpressing MAFK (pEX-2-MAFK; cat. no. C05002) and their respective negative controls (NCs) pGPU6-cloned shNC (cat. no. C02001; 5′-CCTAAGGTTAAGTCGCCCTCG-3′) and pEX-2-cloned NC (cat. no. C05002) were procured from Shanghai GenePharma Co., Ltd. A total of 0.5 µg of each plasmid was used for transfection.

A total of 2 days after treatment, A549 and PC-9 cells (~80% confluence during the emergence phase of persistent cells) were transfected (24 h at 37°C) with shAREG, shNC, plasmids overexpressing MAFK or the empty vector NC using Lipofectamine^™ 3000 Transfection Reagent (cat. no. L3000015; Thermo Fisher Scientific, Inc.). For western blotting, cells were cultured in DMEM (cat. no. C0891; Beyotime Biotechnology) containing 10% FBS for 24 h following transfection, and then harvested for lysis 48 h after transfection.

Using a SA-β-gal assay kit (cat. no. 9860; Cell Signaling Technology, Inc.), SA-β-gal activity was measured to assess A549 and PC-9 cell senescence. Briefly, cells were fixed with 4% paraformaldehyde (cat. no. C104190; Shanghai Aladdin Biochemical Technology Co., Ltd.) at room temperature for 15 min and treated with SA-β-gal reaction solution overnight at 37°C. A TE2000 fluorescence microscope (Nikon Corporation) was utilized to detect the positively stained cells in 20 randomly selected fields of view at ×200 magnification. The activity of SA-β-gal was determined by the proportion of the positively stained cells (4).

The proportion of senescent cells (designated as persistent A549/PC-9 senescent cells; PAS/PPS) and proliferating cells (designated as persistent A549/PC-9 dividing cells; PAD/PPD) within the emerging cell population (defined as PACs/PPCs in the preceding methods) was detected through a flow cytometry assay. Trypsinized PAS and PPS cells were washed with PBS, permeabilized using cold 70% ethanol (cat. no. E130059; Shanghai Aladdin Biochemical Technology Co., Ltd.) at 4°C for 30 min and washed twice in Teknova 1X PBS with 0.1% Tween 20 and 1% BSA (cat. no. 11020021; Thermo Fisher Scientific, Inc.). A total of 2×10⁵ cells was incubated with FITC mouse anti-human Ki67 (cat. no. 556026) or FITC mouse IgG1 (cat. no. 550616; both BD Pharmingen^™; BD Biosciences) as control isotype for 30 min at room temperature. The forward-scatter (FSC)/side-scatter (SSC) values and the corresponding Ki67 plots were analyzed using the Attune^™ NxT flow cytometer (cat. no. A24860; Thermo Fisher Scientific, Inc.) and the results were calculated using FlowJo software (version 10.8.1; FlowJo; BD Biosciences) (4). Based on Ki67 expression levels, cells with any detectable Ki67 positivity (Ki67⁺) were defined as the proliferating population (PAD/PPD), while Ki67-negative (Ki67⁻) cells were collected as the senescent population (PAS/PPS), respectively, using a BD FACSAria III cell sorter (BD Biosciences). The sorted cells were collected for subsequent analyses.

Total RNA was extracted from A549 and PC-9 cells using TRI reagent^® (cat. no. 93289; Sigma-Aldrich; Merck KGaA), followed by quantification using a DR6000 UV spectrophotometer (HACH China). Complementary DNA was synthesized using SuperScript^™ IV reverse transcriptase (cat. no. 18090010; Thermo Fisher Scientific, Inc.) at 50°C for 10 min. RT-qPCR analysis was performed with TB Green^® Premix Ex Taq^™ II (cat. no. RR820A; Takara Bio, Inc.) on the CFX Connect^™ Real-Time PCR Detection System (cat. no. 1855201; Bio-Rad Laboratories, Inc.). The RT-qPCR conditions were as follows: Denaturation at 95°C for 5 min, followed by 40 cycles of 95°C for 10 sec, 60°C for 30 sec and 72°C for 30 sec. The relative expression of MAFK and AREG was calculated using the 2^−ΔΔcq method and normalized to that of GAPDH (17). Primers were as follows: MAFK forward, 5′-GAGAGTGGCTCACACGTCG-3′ and reverse, 5′-CTGCTCACCGTCAAATGATGG-3′; AREG forward, 5′-GTGGTGCTGTCGCTCTTGATA-3′ and reverse, 5′-CCCCAGAAAATGGTTCACGCT-3′; IL-6 forward, 5′-CGAGCCCACCGGGAACGAAA-3′ and reverse, 5′-GCTTCGTCAGCAGGCTGGCAT-3′; IL-8 forward, 5′-TTTTGCCAAGGAGTGCTAAAGA-3′ and reverse, 5′-AACCCTCTGCACCCAGTTTTC-3′ and GAPDH forward, 5′-GGAGCGAGATCCCTCCAAAAT-3′ and reverse, 5′-GGCTGTTGTCATACTTCTCATGG-3′.

A549 and PC-9 cells were cross-linked with formaldehyde (cat. no. F111941; Shanghai Aladdin Biochemical Technology Co., Ltd.) for 10 min at room temperature and the reaction was terminated by 5 min incubation with 0.125 M glycine (cat. no. 1686386; Sigma-Aldrich; Merck KGaA). Cells were washed three times in cold PBS, scraped and washed three times again. Pellets were resuspended in 1 ml lysis buffer (cat. no. RABLYSIS1; Sigma-Aldrich; Merck KGaA). Protease and phosphatase inhibitors (cat. no. PPC1010; Sigma-Aldrich; Merck KGaA) were added to all buffers. Samples were incubated for 15 min at 4°C and mixed by vortex for 30 sec every 2 min. Following centrifugation at 7,200 × g for 10 min at 4°C, the supernatant was removed. Pellets were resuspended in 500 µl SDS lysis buffer (cat. no. 20-163; Sigma-Aldrich; Merck KGaA) to obtain DNA fragments. The supernatant was centrifuged at 7,200 × g for 10 min at 4°C and diluted using IP buffer (cat. no. 20-153; Sigma-Aldrich; Merck KGaA). Each 500 µl extract was incubated with 5 µl dithiothreitol (0.1 M; Sigma-Aldrich; Merck KGaA) and 15 µl Protein A/G magnetic beads (cat. no. 88802; Thermo Fisher Scientific, Inc.) pre-conjugated with 2 µg specific antibodies overnight at 4°C. After washing in Tris-EDTA buffer (cat. no. 93283; Sigma-Aldrich; Merck KGaA), 200 µl fresh elution buffer (1% SDS and 0.1 M sodium bicarbonate) was used to elute samples. Following reversal of the DNA-protein cross-links, DNA was purified using a DNA Purification kit (cat. no. D0033; Beyotime Biotechnology) and subjected to PCR amplification as aforementioned targeting the AREG promoter region. Antibodies were as follows: AREG (cat. no. sc-74501; 1:200; Santa Cruz Biotechnology, Inc.), MAFK (cat. no. ab229766; 1:200; Abcam) and control IgG (cat. no. 3900; 1:200; Cell Signaling Technology, Inc.). The regions were amplified using the primer for AREG promoter region from −1,263 to −1,249 relative to the transcription start site (forward, 5′-TTTGGACTACGCACTGTGAT-3′; and reverse, 5′-CAGTGCATCTGTCCTAATTCTGG-3′).

A549 and PC-9 cells were co-transfected with MAFK-overexpressing plasmids/empty vectors (pEX-2 vector; GenePharma Co., Ltd.) and AREG wild-type (WT) or mutant (MUT) by Lipofectamine 3000 Transfection Reagent (cat. no. L3000015; Thermo Fisher Scientific, Inc.) at 37°C. After 48 h of transfection, Firefly and Renilla luciferase activities within cells were determined using the Dual-Luciferase^® Reporter Assay System (cat. no. E1910; Promega Corporation) according to the manufacturer's instructions. Data were measured using a Veritas Microplate Luminometer (Turner BioSystems; Promega Corporation).

Expression of MAFK, AREG and senescence-related protein p21 wild-type p53-activated fragment 1 (p21waf1) in A549 and PC-9 cells was measured through western blotting. RIPA lysis buffer (cat. no. 20-188; Sigma-Aldrich; Merck KGaA) was used for cell lysis. Proteins were quantified using a BCA Protein Assay kit (cat. no. 7780S; Cell Signaling Technology, Inc.). Equal amounts of protein were separated by 12% SDS-PAGE and transferred to PVDF membranes (cat. no. 88518; Thermo Fisher Scientific, Inc.). Following 2 h incubation in 5% non-fat milk at room temperature, the membranes were incubated overnight at 4°C with the following primary antibodies: MAFK (cat. no. ab50322; 17 kDa; 1:4,000; Abcam), AREG (cat. no. ab89119; 28 kDa; 1:1,000; Abcam), p21waf1 (cat. no. 2947; 21 kDa; 1:1,000; Cell Signaling Technology, Inc.) and heat-shock cognate protein 70 (loading control; cat. no. ab51052; 71 kDa; 1:1,000; Abcam). Membranes were washed three times with TBS with 0.1% Tween-20 (Beijing Solarbio Science & Technology Co., Ltd.) and incubated for 1 h with the horseradish peroxidase-conjugated secondary goat anti-mouse (cat. no. A3562) and anti-rabbit IgG (cat. no. AP132; both 1:3,000; both Sigma-Aldrich; Merck KGaA) at room temperature. Visualization was performed using Pierce^™ ECL Plus Western Blotting Substrate (cat. no. 32132X3; Thermo Fisher Scientific, Inc.). The blots were detected using FluorChem M Chemiluminescence Imaging Analysis System (Protein Simple; Bio-Techne) and semi-quantified using ImageJ software (version 1.53; National Institutes of Health).

A cell colony is a cluster of cells formed by the growth of a single ancestor cell in culture; in the present colony formation assay, a colony was defined as containing ≥60 cells. A549 and PC-9 cells subjected to DOX treatment, AREG knockdown or MAFK overexpression were seeded into a 6-well plate (cat. no. 140675; Thermo Fisher Scientific, Inc.) with 1×10³ cells/well and maintained in culture medium at 37°C. After 9 days, the colonies were exposed to 4% formaldehyde for 10 min at room temperature, then stained with 0.1% crystal violet (cat no. G1062; Beijing Solarbio Science & Technology Co., Ltd.) for 30 min at room temperature. Colony images were acquired using a light microscope (IX73, Olympus, Japan) and captured with a digital camera (Canon EOS 857D; Canon, Inc.) was employed to capture the colonies. Colony numbers were manually counted in three random fields per well.

Statistical analyses were performed using GraphPad Prism (version 8.0; Dotmatics). All data are presented as the mean ± SD of three independent experiments. Two-group comparisons were performed using the independent unpaired sample t-test. Multi-group comparisons were performed using one-way ANOVA, followed by Tukey's post hoc test. Normality (Shapiro-Wilk test) and homogeneity of variance (Levene's test) were also assessed. P<0.05 was considered to indicate a statistically significant difference.

A total of 138 senescence-related DEGs were identified by intersecting genes from Aging Atlas and the GSE6410 dataset (Fig. 1). Kyoto Encyclopedia of Genes and Genomes enrichment analysis revealed that these genes were significantly enriched in key signaling pathways including ‘cell cycle’ and ‘MAPK signaling pathway’ (Fig. 2A). Among the DEGs, AREG was markedly involved in four pathways (Fig. 2B and C) and exhibited the most significant expression difference. Based on its role in a number of senescence-related pathways and pronounced differential expression, AREG was selected as the target gene for experimental validation. To explore how DOX influenced NSCLC cell senescence, A549 and PC-9 cells were exposed to 100 nM DOX for 4 days. The expression of senescence-related protein p21waf1 in the cells was identified and western blotting results exhibited DOX upregulated p21waf1 expression in A549 and PC-9 cells (Fig. 3A-C). The proportion of SA-β-gal-positive cells and expression of IL-6 and IL-8 in both A549 and PC-9 cells increased after DOX treatment (Fig. 3D-I). These experimental data revealed DOX induced NSCLC cell senescence. Persistent cells were generated from A549 and PC-9 cells. DOX suppressed A549 and PC-9 cell proliferation but enhanced persistent cell proliferation (Fig. 3J and K). Therefore, DOX induced senescence and suppressed proliferation of NSCLC cells, while enhancing the proliferative capacity in persistent cells.

Proportions of senescent (PAS/PPS cells) and proliferating cells (PAD/PPD cells) in emerging cells (PACs/PPCs) were detected by Ki67 staining (Fig. 4A-C). The dividing PAD/PPD subpopulation within emerging cells can be identified by low FSC and SSC profiles and high Ki67 staining intensity (18,19). Flow cytometry suggested that PAD/PPD subpopulations exhibited a relatively high proliferative activity, yet PAS/PPS subpopulations demonstrated low proliferation (Fig. 4A-C). The percentage of SA-β-gal-positive PAS/PPS cells was also higher compared with that of PAD/PPD cells (Fig. 4D and E), suggesting that the surviving cells were primarily senescent cells but contained a minority of proliferating cells. The RT-qPCR results showed that MAFK and AREG expression levels were significantly higher in PAD/PPD compared with in PAS/PPS subpopulations in A549 and PC-9 cells, implying that MAFK and AREG upregulation was associated with proliferating cells (Fig. 4F-I). DOX downregulated the levels of MAFK and AREG in A549 and PC-9 cells, but the levels were significantly enhanced in PACs/PPCs (Fig. 4J-M). The transfection efficiency of shAREG in emerging A549 and PC-9 cells was demonstrated by downregulated AREG (Fig. 4N and O). Consequently, senescent cells constituted the majority of persistent cells and DOX suppressed MAFK and AREG expression in NSCLC cells, but expression was enhanced in persistent cells.

To evaluate how AREG affected the proliferation of emerging NSCLC cells, A549 and PC-9 cells during emergence were transfected with shAREG or shNC. AREG knockdown suppressed cell proliferation (Fig. 5A and B). Flow cytometry and SA-β-gal assay showed AREG knockdown markedly increased the proportion of senescent PACs/PPCs while decreasing the proportion of proliferating cells (Fig. 5C-H).

ChIP showed that MAFK directly bound to the promoter region of AREG (Fig. 6A and B). The transfection efficiency of plasmids overexpressing MAFK in A549 and PC-9 cells was corroborated by upregulated MAFK in western blotting (Fig. 6C-E). A dual-luciferase reporter assay was used to evaluate the targeted regulation between MAFK and AREG. Luciferase activity was significantly increased by WT AREG and MAFK, but only slightly increased by MUT AREG and MAFK, implying that MAFK promoted luciferase activity in AREG-WT-transfected A549 and PC-9 cells (Fig. 6F and G). MAFK overexpression enhanced the proliferation of emerging A549 and PC-9 cells and reversed the anti-proliferative effect of AREG knockdown (Fig. 6H and I), indicating that the pro-proliferative effect of MAFK was not solely mediated by AREG but may also involve additional target genes or pathways. Collectively, MAFK overexpression reversed the inhibitory effect of AREG knockdown on proliferation of emerging NSCLC cells.