The Animal Ethics Committee of the Eye, Ear, Nose and Throat Hospital of Fudan University (Shanghai, China) reviewed and approved the study protocol. The Hep2 cell line employed in the present study was from our own laboratory (Laboratory Center, Eye, Ear, Nose and Throat Hospital of Fudan University, Shanghai, China). Cells were maintained in RPMI-1640 (Hyclone; GE Healthcare Life Sciences, Logan, UT, USA) with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and penicillin (50 U/ml)-streptomycin (50 µg/ml) solution (Gibco; Thermo Fisher Scientific, Inc.). The cell line was incubated at 37°C in a humidified atmosphere of 95% air and 5% CO₂. The mixed cancer Hep2 cell line, which was originally considered to be of the laryngeal carcinoma type but was later reported to be contaminated with cervical carcinoma HeLa cells, was used as a cancer cell model in the current study (14–16).

According to the human KEAP1 transcript in GenBank (https://www.ncbi.nlm.nih.gov/nuccore/; NM_203500), three target RNA interference sequences that silence the KEAP1 gene were identified. Lentiviral vectors expressing RNAi specific for the KEAP1 gene and a scrambled sequence encoding a green fluorescent protein (GFP) sequence were designed and constructed by Obio Technology Co., Ltd. (Shanghai, China). The following sequences were used: 5′-GCAAGGACTACCTGGTCAAGA-3′ (shKEAP1), 5′-CGGGAGTACATCTACATGCAT-3′ (shKEAP1-1), 5′-GTGGCGAATGATCACAGCAAT-3′ (shKEAP1-2) and 5′-TTCTCCGAACGTGTCACGT-3′ (scRNA). Pairs of complementary oligonucleotides with these sequences were synthesized, annealed and cloned into the lentiviral plasmid vector [pLKD-CMV-G&PR-U6-short hairpin (sh)RNA] (Obio Technology, Ltd., Shanghai, China) using the AgeI and EcoRI enzymes (Takara Bio, Inc., Otsu, Japan). The recombinant plasmid vectors (32 µg) containing shRNA were co-transfected into 293T cells along with the helper plasmids psPAX2 and pHCMV-VSV-G (Takara Bio, Inc.) using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). Subsequently, the supernatants were collected and purified after 48 h of transfection and then titrated.

The constructed lentiviral vectors contained a green fluorescence gene that emits green fluorescence in response to excitation with blue light. This characteristic may be utilized to optimize the transfection conditions. Following the screening of a series of preliminary experiments using 2 µg/ml puromycin, the optimal transfection efficiency was determined. Briefly, for cell transfection, Hep2 cells in the exponential growth phase were collected, counted and seeded in 24-well tissue culture plates (Corning Incorporated, Corning, NY, USA) at a density of 2×10⁴ cells/well to attain 30% confluence on the day of transfection. The lentivirus containing specific KEAP1 shRNA and the negative control lentivirus were applied to Hep2 cells with the multiplicity of infection of 10 or 20. In addition, polybrene was added into each well with a final concentration of 5 µg/ml. At 12–20 h post-transfection, the medium was completely replaced and the cells were incubated at 37°C in a 5% CO₂ incubator for an additional 72 h. The transfection rate was evaluated by counting GFP-positive cells under an inverted fluorescence microscope; silencing efficacy was verified using real-reverse transcription-quantitative polymerase chain reaction (RT-qPCR), western blotting and an immunofluorescence technique. Hep2 cells that exhibited the highest degree of KEAP1 silencing following transfection with lentivirus containing KEAP1-shRNA were selected for the establishment of a stable cell line in the presence of puromycin at a final concentration of 2 µg/ml. The media containing puromycin were changed every 2–3 days, for 2 weeks. Parent Hep2 and scHep2 cells served as the controls.

Cells were seeded in 6-well plates (Corning Incorporated) at a density of 4×10⁵ cells/well and cultured for 24 h. Subsequently, cells were treated with two different concentrations of H₂O₂ (0.1 and 0.25 mmol/l) for 24 h at 37°C. The floating original medium was collected and the cells were digested with 0.25% trypsin without EDTA (Hyclone; GE Healthcare Life Sciences) at 37°C for 1 min. Cell suspensions were centrifuged for 5 min at 1,000 × g at room temperature. Cells were resuspended and gently washed twice with ice-cold phosphate-buffered saline (PBS; pH 7.2–7.4; Hyclone; GE Healthcare Life Sciences). Finally, the cells were collected in 1.5 ml Eppendorf tube. Cell density was adjusted to 1×10⁶ cells/tube. According to the operating instructions of an Annexin V-allophycocyanin (APC)/7-aminoactinomycin D (7-AAD) double staining apoptosis detection kit (Nanjing KeyGen Biotech Co., Ltd., Nanjing, China), 500 µl binding buffer was added to suspend cells. A total of 5 µl Annexin V-APC and 7-AAD was subsequently added respectively for flow cytometry within 1 h using a Beckman MoFlo XDP flow cytometer with Summit 5.2 (Beckman Coulter, Inc., Brea, CA USA). Cells not treated with any H₂O₂ served as the controls.

shKEAP1 Hep2 and scHep2 cell lines in the logarithmic growth phase were trypsinized, counted, resuspended and plated at a density of 2×10³ cells/well in a volume of 200 µl RPMI-1640 medium per well in a 96-well plate in triplicate. After 6, 24, 48, 72 and 96 h incubation individually (data not shown), cell proliferation was measured using a Cell Counting Kit-8 (CCK-8; Dojindo Moleular Technologies Inc., Kumamoto, Japan) according to the manufacturer's protocols. The absorbance value was recorded at 450 nm using a microplate spectrophotometer. To quantify the ability of H₂O₂ to inhibit the shKEAP1 Hep2 cell line, a cell growth assay was also performed using CCK-8. Briefly, cells were seeded in a 96-well tissue culture plate (2×10⁴/well), incubated overnight at 37°C and subsequently treated with 0, 0.25, 0.5, 0.75 and 1.0 mmol/l of H₂O₂ for 24 h. CCK-8 was subsequently added to each well to detect the inhibitory effect of H₂O₂ on the cells using a microplate spectrophotometer at a wavelength of 450 nm.

Cells in the exponential growth phase were digested with trypsin, resuspended and the cell density was adjusted to 1×10⁶ cells/ml with culture media. Prior to cell density adjustment, one glass coverslip (22×22 mm) was placed into each well of a 6-well dish. A total of 5 µl cell suspensions was seeded onto the glass coverslip in order to grow up to 30–50% confluence 24 h later. H₂O₂ was added into the wells at final concentrations of 0.25 and 0.5 mmol/l for 6 and 9 h. Equal volumes of PBS were added into the wells that were not treated with H₂O₂ for the same aforementioned durations. Following incubation for the specified durations, the media were removed and cells were washed three times with ice-cold PBS. The slides of cells were fixed in fresh 4% formaldehyde for 20 min at room temperature. Subsequently, the cells were washed three times with PBS and incubated in PBS containing 1% (v/v) Triton X-100 and 1% bovine serum albumin (Beyotime Institute of Biotechnology) for 1 h on ice to permeabilize the cells and block non-specific protein-protein interactions. The glass coverslips were removed from the 6-well plates and subsequently incubated with 30 µl NRF2 primary antibody (cat. no. ab31163; Abcam, Cambridge, UK) diluted at 1:100 and placed in a humidified chamber overnight at 4°C. The secondary antibody used was cyanine 3-conjugated goat anti-rabbit IgG (H+L; cat. no. A01516; Beyotime Institute of Biotechnology) used at a 1:1,000 dilution for 1 h. DAPI was used to stain the cell nuclei for 5 min at room temperature (cat. no. C1002; Beyotime Institute of Biotechnology). Finally, the coverslips were sealed with antifade mounting medium (Beyotime Institute of Biotechnology) and observed under fluorescence microscopy (magnification, ×40). The immunofluorescence detection of NRF2 without H₂O₂ and the staining of KEAP1 were performed as described above but using a KEAP1-specific antibody (cat. no. ab139729; Abcam) also diluted at 1:100 and the same secondary antibody (cyanine 3-conjugated goat anti-rabbit IgG; H+L; cat. no. A01516).

Cells were seeded in 6-well plates and treated with 0, 0.5 and 1.0 mmol/l H₂O₂ Total RNA was extracted from cells using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.). RNA quality and quantity was measured using a NanoDrop 2000 spectrophotometer (NanoDrop Technologies; Thermo Fisher Scientific, Inc.). A total of 1 µg RNA was reverse transcribed into cDNA using the PrimeScript RT reagent kit (Perfect Real Time; Takara Bio, Inc.) in a final 20 µl volume reaction, according to the manufacturer's protocol. A volume of 1 µl RT reaction mixture and 9 µl qPCR mixture were mixed and SYBR Green I-based RT-qPCR analyses of human KEAP1, NRF2, NQO1 and HO1 were performed by using the SYBR Premix Ex Taq system (Tli RNaseH Plus; Takara Bio, Inc.), according to the manufacturer's protocol in triplicate on an ABI 7500 system (Applied Biosystems; Thermo Fisher Scientific, Inc.). Negative RT controls were included in each assay. All primers were designed and synthesized by Sangon Biotech Co., Ltd. (Shanghai, China), as presented in Table I. GAPDH was used as a reference gene. Relative quantitative levels of samples were determined by the 2^−∆∆Cq method (17).

Total protein was acquired by lysing cells in a radioimmunoprecicpitation assay buffer containing phenylmethylsulfonyl fluoride with a final concentration of 1 mmol/l (Beijing Cowin Biotech Co., Ltd., Beijing, China). The cytosolic and nuclear fractions of NRF2 were prepared using a NE-PER Nuclear and Cytoplasmic Extraction Reagents kit (Pierce; Thermo Fisher Scientific, Inc.). Protein extracts were obtained via centrifugation at 4°C and 10,000 × g for 10 min; proteins were quantified using a Bicinchoninic Acid Protein assay kit (Beyotime Institute of Biotechnology). A total of 40 µg total protein was electrophoretically separated via 10% SDS-PAGE and transferred to polyvinylidene fluoride membranes. To block the membranes, 5% non-fat milk in Tris-buffered saline containing 0.1% Tween-20 (TBST; pH 7.4–7.5) was applied at room temperature for 1 h. Membranes were subsequently incubated overnight with anti-NRF2 (cat. no. ab31163; Abcam), anti-KEAP1 (cat. no. ab139729; Abcam), anti-NQO1 (cat. no. A180; Cell Signaling Technology, Inc., Danvers, MA, USA), anti-HO1 (cat. no. ab68477; Abcam), and anti-β-actin (cat. no. GB13001-3; Servicebio, Inc.) or lamin B primary antibodies (GB11408; Servicebio, Inc.) in blocking buffer at 4°C at 1:1,000 dilution. Subsequently, membranes were washed three times in TBST followed by incubation for 1 h with HRP-labeled goat anti-rabbit/mouse IgG (H+L) (GB23303/GB23301; Servicebio, Inc.) at a 1:2,500 dilution and washed in TBST again. Bands were visualized using hypersensitive enhanced chemiluminescence kit (cat. no. G2020; Servicebio, Inc. Wuhan China). β-actin was used for cytoplasmic extracts and lamin B used for nuclear extracts.

The significance of intergroup differences among the multiple groups was determined using one-way analysis of variance. The Least Significant Difference test was used for the post hoc test. P<0.05 was considered to indicate a statistically significant difference. The comparisons of the cell viability among the groups were performed by Bonferroni following one-way analysis of variance. All statistical analyses were conducted using the SPSS 17.0 statistical package (SPSS, Inc., Chicago, IL, USA). The experiments were repeated at least three times and data are presented as the mean ± standard deviation.

In order to establish a stable shKEAP1 Hep2 cell line with effective knockdown of the KEAP1 gene, Hep2 cells were transfected with three KEAP1 shRNAs and one scrambled shRNA expression lentiviral plasmids bearing GFP, which was used to assess the efficacy of transfection. RT-qPCR was performed on exponentially growing cells to determine the expression levels of KEAP1 following 72 h transduction. Of the three shKEAP1 sequences, one exhibited the highest knockdown efficiency, which reduced KEAP1 mRNA expression levels by up to 67±1% compared with the Hep2 cells transfected with the other shRNAs (P<0.05; Fig. 1A). Based on this, this sequence was selected to establish a stable shKEAP1 Hep2 cell line with puromycin for 2 weeks. Subsequently, the protein expression levels of KEAP1 in shKEAP1 Hep2 and scHep2 cell lines were measured by western blotting. KEAP1 protein expression levels were markedly reduced following KEAP1 knockdown using the shKEAP1 construct (Fig. 1B). Furthermore, the transfection efficiency of scHep2 cells was demonstrated to be equivalent to that of shHep2, which was ~80% (Fig. 1C). Immunofluorescence staining demonstrated that KEAP1 protein was primarily expressed in the cytoplasm. Weak fluorescence and bands were detected within the shKeap1 group, which were markedly lower compared with in scHep2 and Hep2 cells; however, within the control groups, expression levels were similar (Fig. 1D), indicating that a stable and effective shKEAP1 Hep2 cell line was established.

The effects of KEAP1 knockdown on the mRNA and protein expression of NRF2 and NRF2-associated genes were investigated. The results demonstrated that shRNA-mediated depletion of KEAP1 was associated with significant increases in the mRNA expression levels of NRF2, NQO1 and HO1 by 1.42±0.05-, 1.75±0.10- and 1.59±0.07-fold, respectively, compared with the scHep2 group (Fig. 2A). Notably, immunofluorescence staining demonstrated that the expression levels of nuclear NRF2 were low, with NRF2 primarily located in the cytoplasm within parent Hep2 cells; however, NRF2-reactive fluorescent signals were observed within the nucleus of shKEAP1 cells, indicating that KEAP1 knockdown may cause NRF2 migration into the nuclei (Fig. 2B). Furthermore, western blot analysis demonstrated that delivery of specific shKEAP1 by lentiviral transduction reduced the cytosolic levels of NRF2, which was observed alongside a marked increase in nuclear NRF2 levels (Fig. 2C). Total protein levels of NQO1 and HO1 were markedly elevated in shKEAP1 Hep2 cells compared with the scHep2 cell line and the blank control Hep2 group (Fig. 2D).

To investigate the role of KEAP1 knockdown against oxidative damage in the Hep2 cell line, the expression of this system and the resistance of the cells to oxidative stresses induced by H₂O₂ were analyzed. Immunofluorescence analysis demonstrated that when cultured shKEAP1 Hep2 cells were treated with 0.5 and 1 mmol/l H₂O₂ for 6 h, NRF2 markedly migrated into the cell nucleus compared with in scHep2 cells. In addition, the expression of NRF2 appeared to be markedly higher in response to the concentration of 1 mM/l l H₂O₂ for 6 h compared with 0.5 mmol/l (Fig. 3A-C). As presented in Figs. 3D and E, and 4, the results of RT-qPCR analysis and western blotting demonstrated consistencies with immunofluorescence analysis; the increases in the NRF2, NQO1 and HO1 transcript and protein levels demonstrated time- and dose-dependency within the knockdown cell line. Knockdown of KEAP1 via shRNA in Hep2 cells increased the NRF2, NQO1 and HO1 mRNA expression levels by 1.99, 2.32 and 1.91-fold respectively, compared with scHep2 cells when treated with 0.5 mM/l H₂O₂ for 6 h (Figs. 3D, 4A and B). At the concentration of 1 mM/l H₂O₂ for 9 h, NRF2, NQO1 and HO1 mRNA levels were 2.85, 3.47 and 2.96-fold higher in the KEAP1 knockdown group, respectively, compared with in the scHep2 cells. mRNA expression levels were analyzed upon exposure to 0.5 mmol/l H₂O₂ for 6 and 9 h. NRF2, NQO1 and HO1 were all expressed highly within shKEAP1 cells compared with in scHep2 cells at both concentrations. Western blot analyses revealed that the protein expression levels of nuclear NRF2, and total NQO1 and HO1, were markedly upregulated in shKEAP1 Hep2 cells compared with in scHep2 control cells at the concentration of 0.5 and 1 mmol/l H₂O₂ for 6 h (Figs. 3E and 4C). Supporting the results of immunofluorescence, KEAP1 knockdown led to a reduction in the protein expression levels of NRF2 within the cytoplasm and an increase within the nuclei at 0.5 and 1 mM/l H₂O₂ concentrations, compared with scHep2 cells (Fig. 3E). The expression patterns of NQO1 and HO1 were similar to the nuclear NRF2 in shKEAP1 Hep2 cells upon exposure to H₂O₂ (Fig. 4C).

As the expression levels of NRF2, NQO1 and HO1 were altered within NRF2-activated Hep2 cells, the effects of KEAP1 knockdown on cell function were subsequently investigated. Cell viability decreased in a dose-dependent manner within the shKEAP1 and scHep2 cell lines with increasing doses of H₂O₂ with the range of final concentrations from 0–1 mM/l for 24 h. At concentrations of 0.25, 0.5 and 0.75 mM/l, the mean relative viabilities of shKEAP1 and scHep2 cells were 75 and 51, 33 and 12, and 18 and 6%, respectively. It was observed that transfected shKEAP1 Hep2 cells demonstrated significantly higher cell viabilities following H₂O₂ treatment compared with in the scHep2 group, as demonstrated in Fig. 5. To validate the results obtained from the CCK-8 assay and to investigate whether apoptosis served a role against oxidative stress injury, the percentage of the apoptotic cells was analyzed by flow cytometry. Cells were cultured with varying concentrations of H₂O₂ (0, 0.1 and 0.25 mmol/l) for 24 h. Marked increases were observed in the apoptosis rates of scHep2 cells at 0.1 and 0.25 mmol/l H₂O₂ from 31.8 and 45.3%, while 14.1 and 27.9% in shKEAP1 cells (Fig. 6).