The human adenocarcinoma A549 and lung fibroblast WI-38 cell lines were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA) and maintained in Gibco™ Dulbecco's modified Eagle's medium (DMEM; Thermo Fisher Scientific, Inc., Paisley, UK) supplemented with 10% fetal bovine serum (FBS), 100 U/ml of penicillin and 0.1 mg/ml of streptomycin (all from Thermo Fisher Scientific, Inc.). For different analyzing purposes, A549 cells was treated with 3.12 µM erastin (Sigma-Aldrich, St. Louis, MO, USA) for 24 h, NAC (Sigma-Aldrich) 5 mM for 4 h or KU-55933 (Selleck Chemicals, Shanghai, China) 15 nM for 24 h. Then cells were harvested as described below for further analysis.

Cells were harvested and pelleted by centrifugation at 1,000 × g and 4°C for 10 min and washed with ice-cold PBS twice. Lysis buffer (100 µl) containing 50 mM Tris-HCl, 150 mM NaCl, 0.02% NaN₃, 100 µg/ml phenylmethanesulfonyl fluoride (PMSF), 1 µg/ml aprotinin, 1 µg/ml pepstatin A, and 1% Triton X-100 was each added into 1×10⁶ cells for obtaining the cell lysate. After centrifugation at 12,000 × g for 10 min at 4°C, the supernatant was collected and assessment of protein concentration was carried out using a bicinchoninic acid (BCA) protein assay kit (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). Protein (50 µg) was resolved by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to a nitrocellulose membrane. After transferring, the membrane was blocked using 5% bovine serum albumin (BSA) in PBS for 30 min at room temperature. The primary antibodies against p53 (1:5,000 dilution; cat. no. ab28), phosphorylated p53 (1:2,000 dilution; cat. no. ab1431), p21 (1:1,000 dilution; cat. no. ab109520), Bax (1:2,000 dilution; cat. no. ab32503), SLC7A11 (1:1,000 dilution; cat. no. ab37185) and β-actin (1:5,000 dilution; cat. no. ab8226) were purchased from Abcam (Cambridge, UK) and incubated with the blocked membrane separately for 2 h at room temperature. After being washed 3 times for 5 min each with PBS-T, the membrane was incubated for 1 h with peroxidase-coupled secondary antibodies (1:5,000 dilution; cat. nos. ab6785 or ab6721), and detected with the ECL Plus Western Blotting Detection reagents (Pierce Biotechnology, Rockford, IL, USA) and imaged using X-ray film.

A549 cells were washed in phosphate-buffered saline (PBS) twice and 100 µl of CFSE fluorescent dye (50 µmol/l; Thermo Fisher Scientific, Inc., Waltham, MA, USA) was added, followed by incubation at 37°C for 30 min. Cells were washed in PBS twice and incubated in medium supplemented with 10% FBS for another 24-h incubation. After two washes with PBS, PI (20 µg/ml; Sigma-Aldrich) was added for incubation at room temperature for 10 min. After two washes with PBS, cells were imaged under a X71 (U-RFL-T) fluorescence microscope (Olympus, Melville, NY, USA).

Cells were plated in 12-well plate and allowed to attach overnight. In addition, 5-ethyny-2′-deoxyuridine (EdU) (Cell-Light EdU Cell Proliferation Detection kit; Guangzhou RiboBio Co., Ltd., Guangzhou, China) was used as a marker of cell proliferation. EdU was added at a final concentration of 50 µmol/l into the medium and the cells were cultured for an additional 120 min. Cells were washed twice with PBS and fixed with 4% paraformaldehyde at room temperature for 10 min, washed with glycine (2 mg/ml) for 5 min in a shaker, treated with 0.2% Triton X-100 for 10 min and washed with PBS twice. Click-iT^® Cell Reaction Buffer kit (Thermo Fisher Scientific, Inc.) was added for further incubation for 30 min. Then the cells were washed with 0.5% Triton X-100 for three times, stained with DAPI for 10 min at room temperature, washed with 0.5% Triton X-100 for three times, immersed in 150 µl of PBS and examined under a X71 (U-RFL-T) fluorescence microscope (Olympus).

A549 cells were suspended and adjusted to 1×10⁶ cells/ml and 5,000 cells/well were plated into a 96-well plate and incubated overnight. Various concentrations of erastin (0.1, 1, 2, 4, 6, 8 and 10 µM) were added into each well. Twenty-four hours later, the Cell Counting Kit-8 (CCK-8, Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) prepared solution was added for a 4-h co-incubation at 37°C in the dark. Absorbance at wavelength 450 nm (A450) was detected by a microplate reader (Synergy 2 Multi-Mode Microplate Reader; BioTek, Winooski, VT, USA) to determine the cell viability.

Cells were co-incubated with [5-(and 6)-carboxy-2′,7′-dichlorodihydrofluorescein diacetate] (carboxy-H₂DCFDA; Thermo Fisher Scientific, Inc.) at a final concentration of 5 µmol/l for 30 min. Subsequently the medium was removed and washed twice with PBS. The green fluorescence was imaged using a X71 (U-RFL-T) fluorescence microscope (Olympus). For quantitative measurement, stained cells were suspended using 0.25% trypsin (Gibco; Thermo Fisher Scientific, Inc.) and the green fluorescence was assessed using a 3-laser Navios flow cytometer (Beckman Coulter Inc., Brea, CA, USA).

Invitrogen™ Oligofectamine transfection reagent (Thermo Fisher Scientific, Inc.) was used for transfecting 50 nM p53 siRNA (p53si), SLC7A11 siRNA (SLC7A11si) or negative control siRNA (CTLsi) purchased from Ambion (Life Technologies; Thermo Fischer Scientific, Inc.) into A549 cells according to the manufacturer's instructions and then after 24 h, the cells were used for subsequent experiments.

Cells were washed with ice-cold PBS for 5 min, 3 times and fixed with ice-cold alcohol for 15 min at 4°C. Subsequently, the cells were blocked with 1% BSA, 0.1% Triton X-100 in PBS for 1 h at room temperature. Then the cells were incubated with anti-γ H2AX antibody (cat. no. ab26350; Abcam) at a 1:200 dilution for 2 h at room temperature. After 3 washes with ice-cold PBS, the cells were incubated with Alexa Fluor^® 647-conjugated donkey anti-mouse IgG secondary antibody (cat. no. ab150107; Abcam) at a 1:2,000 dilution for 1 h at room temperature. After 3 washes with ice-cold PBS, the cells were incubated with 5 µg/ml DAPI for 10 min. The slices were analyzed under a X71 (U-RFL-T) fluorescence microscope (Olympus) at a magnification of ×200.

Cells (1×10⁶) were collected and fixed in 70% ice-cold ethanol at −20°C overnight. Then the cells were collected and resuspended in PBS supplemented with 100 ng/ml RNase A and 50 ng/ml propidium iodide (PI) for 30 min. After staining, samples were analyzed for cell cycle distribution with a 3-laser Navios flow cytometer (Beckman Coulter). Experiments with duplicates were performed independently thrice.

A caspase-3 colorimetric assay kit (R&D Systems, Minneapolis, MN, USA) was used to detect the enzyme activity of caspase-3. The total cell lysate was qualified by the BCA protein assay kit (Sigma-Aldrich; Merck KGaA). Total protein (50 µg) for each sample was moved to a 96-well microplate and quantified using a microplate reader (Synergy 2 Multi-Mode Microplate Reader; BioTek).

Cells (5×10⁵) were collected and co-incubated with 5 µl Annexin V-fluorescein isothiocyanate (Annexin V-FITC) and 10 µl PI supplied by an Annexin V/PI apoptosis kit (BioVision, San Francisco, CA, USA) for 10 min in the dark. After staining, the ells were subjected to flow cytometric analysis (3-laser Navios flow cytometer; Beckman Coulter).

All data are presented as the mean ± SD. Statistical differences among different groups were analyzed by one way analysis of variance (ANOVA) with Dunnett's post hoc test using Prism 6 (GraphPad Software, Inc., San Diego, CA, USA). A value of P<0.05 or P<0.01 was considered to indicate a statistically significant difference.

To ascertain whether erastin exerts a cytotoxic effect and increases ROS in A549 cells and the non-tumour cell line WI-38, both cell types were treated with increasing concentrations of erastin (0.1–10 µM) and were subjected to the CCK-8 assay 24 h later to assess cell viability. As displayed in Fig. 1A, exposure to 0–10 µM erastin for 24 h significantly decreased A549 cell viability in the A549 cells, but not that of WI-38 cells. Erastin has been reported to play a critical role in inducing ferroptosis via ROS accumulation (3). We quantified fluorescence in erastin-treated (IC₃₀ concentration for A549 cells, 3.12 µM) A549 or WI-38 cells stained with a redox-sensitive probe. Consistent with previous literature (6), erastin treatment increased the ROS level in A549 cells but not that in WI-38 cells (Fig. 1B), indicating that NSCLC A549 cells are much more sensitive than non-tumour WI-38 lung cells. Thus, subsequent experiments were focused on the effects of erastin treatment on A549 cells. To determine the role of ROS induced by erastin exposure on p53, the expression levels of p53 protein and its transcriptional targets Bax and p21 were detected. As displayed in Fig. 1C, the upregulation and activation of p53 were evidenced by upregulated p53, Bax and p21. Following pretreatment with N-acetyl-1-cysteine (NAC), an ROS scavenger, erastin exposure failed to significantly affect p53 activation, indicating that activation was dependent on the presence of ROS induced by erastin exposure (Fig. 1C, right panel). According to the literature, activated p53 transcriptionally suppresses SLC7A11, a key component of the cysteine/glutamate antiporter, thus promoting ferroptosis induced by erastin exposure (12). This prompted us to detect the expression of SLC7A11 in A549 cells with or without p53 knockdown after erastin exposure. In the CTLsi group, with the upregulation of p53 induced by erastin treatment, SLC7A11 was obviously downregulated (Fig. 1D). While there was no obvious effect of erastin treatment on the expression of SLC7A11 in A549 cells with p53 knockdown, progressive suppression of the SLC7A11 protein level was observed in A549 cells exposed to erastin (Fig. 1D). These results indicated that ROS induced by erastin potentially contributed to ferroptosis induction via activating p53 transcriptional activity.

By considering the effect of accumulated ROS on the DNA damage response (DDR) (13,14), we then tested whether erastin-induced ROS caused DDR in A549 cells. As expected, erastin exposure produced some typical γ-H2AX positive-stained cells. Conversely, co-exposure of erastin with NAC only produced few γ-H2AX positive-stained cells (Fig. 2A). Without disturbing p53 mRNA levels (data not shown), the activation of p53 by erastin was abolished by NAC co-incubation (Fig. 2B). DDR is responsible for inducing p53 post-transcriptionally, which is phosphorylated by activated ataxia telangiectasia mutated (ATM) kinase (15), and prompted us to determine whether ROS induction of p53 is dependent on DDR and subsequent activation of phosphorylation. Thus, KU-55933, a specific ATM inhibitor, was employed to inhibit the phosphorylation of p53 at S15 by DDR-activated ATM, and then the effects of accumulated ROS on p53 were detected. As displayed in Fig. 2C and D, NAC treatment, but not KU-55933, induced DDR, and both NAC and KU-55933 treatment clearly inhibited p53 phosphorylation following erastin exposure, indicating that the activating effects of erastin-induced ROS on p53 were exerted via ATM kinetic activity after causing DDR (Fig. 2E).

To determine the role of activated p53 on ROS generation induced by erastin, we assessed the ROS level in A549 cells with or without p53 knockdown in A549 cells after erastin exposure. As illustrated in Fig. 3A, while erastin exposure induced accumulating ROS in both A549-p53si and A549-CTLsi cells, the ROS level in A549-p53si cells was lower than that in A549-CTLsi cells. According to research, activated p53 suppresses post-transcriptionally the expression of SLC7A11, a key component of the cysteine/glutamate antiporter, thus inducing ROS accumulation (12). This prompted us to ascertain whether ROS induction by activated p53 after erastin exposure was dependent on the suppression of SLC7A11. We employed SCL7A11si to efficiently knock down the expression of SCL7A11 without disturbing the expression of p53 (Fig. 3B). After erastin exposure, both A549-SLC7A11si and A549-SLC7A11si/p53si cells presented decreased ROS levels. By comparison with the SCL7A11si-transfected group, co-transfection of SCL7A11si and p53si presented a significantly lower level of ROS, indicating that, potentially, ROS induction by p53 occurred partially by suppressing SCL7A11 (Fig. 3C).

To evaluate the effects of p53 induced by erastin exposure on A549 cells, the CFSE/PI double staining or CCK-8 assay was performed to detect the survival rate or cell viability of A549 cells exposed to the IC₅₀ concentration of erastin for 24 h. As illustrated in Fig. 4A and B, erastin potently inhibited A549 cell survival, as evidenced by CCK-8 optical density (OD) reduction. Knockdown of p53 attenuated erastin-exerted cytotoxicity. According to research, erastin triggers ferroptosis, which is a unique iron-dependent form of non-apoptotic death, prompting us to identify the p53-contributed cytotoxic effect on A549 cells after erastin exposure. Under the erastin-exposed condition, ferrostatin-1 (Fer-1) or Z-VAD-FMK was employed to inhibit ferroptotic or apoptotic death, respectively, and the role of p53 in inducing cytotoxicity was assessed. After confirming the significant decrease in cell viability following erastin exposure compared with untreated cells (data not shown), Fer-1 or Z-VAD-FMK was applied to reveal the effects of p53 on erastin-induced ferroptotic and apoptotic death. By inhibiting apoptotic death confirmed by detecting caspase-3 activity (Fig. 4C), knockdown of p53 inhibited erastin-induced cell death, as detected by CFSE/PI double staining (Fig. 4D). By inhibiting ferroptotic death without disturbing caspase-3 activity (Fig. 4E), knockdown of p53 also exerted an inhibitory effect of apoptotic death induced by erastin exposure (Fig. 4F and G).

To test the effect of p53 induced by erastin on A549 cell proliferation, A549 cells were treated with the IC₃₀ concentration of erastin (3.12 µM) for 24 h. We subjected the treated cells to EdU staining, which is a proliferation marker, and observed that erastin treatment promoted stalled replication, which was greatly rescued by p53 knockdown, as shown by the EdU incorporation (Fig. 5A). This result indicated that erastin treatment inhibited cellular proliferation via the presence of p53. Consistently, analysis of the cell cycle distribution by flow cytometry demonstrated that erastin treatment induced slight accumulation in the G₀/G₁ phase that was reversed by p53 knockdown (Fig. 5B). Since both accumulated ROS and activated p53 mediate the cell accumulation in the G₀/G₁ phase (13), we next examined the related kinetics. While activation of p53 or G₀/G₁ phase accumulation was detectable at 12, 18 or 24 h (Fig. 5C), accumulation of ROS was detected at 3 h after erastin treatment (Fig. 5D). We also determined the effects of erastin-induced ROS on the cell cycle arrest by employing the ROS scavenger N-L-acetylcysteine (NAC) with erastin. In Fig. 5E, scavenging of erastin-induced ROS was eliminated by pretreatment of the cells with NAC, leading to entry into the cell cycle. Collectively, erastin exposure generated ROS, thus activating p53, which demonstrated critical cytotoxic and cytostatic effects in A549 cells.