NSCLC cell lines (A549, H2122, H292, H1299 and H460) and normal human lung epithelial cells (BEAS-2B) were purchased from The Cell Bank of Type Culture Collection of the Chinese Academy of Sciences. All cells were maintained in RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin and 100 µg/ml streptomycin in an incubator at 37°C with a 5% CO₂ atmosphere. Cell lines in the logarithmic growth phase were selected for subsequent experiments.

Cell transfections were performed using Lipofectamine^® 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. Untransfected cells were used as a blank control. Briefly, 4×10⁵ H2122 or A549 cells were plated into a six-well plate and cultured to 90–95% confluence. The overexpression of GSTO1 in NSCLC cells was induced through transfection with a pcDNA3.1 plasmid (4 µg; Invitrogen; Thermo Fisher Scientific, Inc.) carrying the GSTO1 cDNA insert, with an empty vector (4 µg) as the negative control (pcDNA3.1-NC). The knockdown of GSTO1 in NSCLC cells was induced using 2 µg short hairpin RNA (shRNA; Thermo Fisher Scientific, Inc.) against GSTO1, using stable non-specific shRNA (2 µg) as the NC (shRNA-NC). The shRNA-NC sequence was 5′-CCGGCCTAAGGTTAAGTCGCCCTCGCTCGAGCGAGGGCGACTTAACCTTAGGTTTTTC-3′ and the shRNA-GSTO1 sequence was 5′-GCTGGAAGCAATGAAGTTA-3′. Cells were transfected for 24 h at 37°C in an atmosphere containing 5% CO₂ to obtain stably transfected cells for future use. At 48 h post-transfection, the overexpression and knockdown of GSTO1 was confirmed using reverse transcription-quantitative PCR (RT-qPCR) and western blotting.

Total RNA was extracted from cells using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). Total RNA was reverse transcribed into cDNA using a RevertAid First Strand cDNA Synthesis kit (Thermo Fisher Scientific, Inc.), which was conducted according to the manufacturer's protocol. qPCR analysis was subsequently performed using a SYBR Green RT-PCR kit (Takara Bio, Inc.) on an ABI 7500 Fast Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.). The following thermocycling conditions were used for qPCR: Initial denaturation for 10 min at 95°C; followed by 40 cycles of denaturation at 95°C for 15 sec and annealing/extension at 55°C for 45 sec. The following primer sequences were used for the qPCR: GSTO1 forward, 5′-AGCATACCCAGGGAAGAAG-3′ and reverse, 5′-CTGCCATCCACAGTTTCAG-3′; and β-actin forward, 5′-CATCGTCCACCGCAAATGCTTC-3′ and reverse, 5′-AACCGACTGCTGTCACCTTCAC-3′. Expression levels were quantified using the 2^−∆∆Cq method (18). β-actin was used as the endogenous control and expression levels were normalized to β-actin.

Total protein was extracted from cells using RIPA lysis buffer (Beyotime Institute of Biotechnology). Total protein was quantified using a Bradford assay and 25 µg total protein/lane was separated via SDS-PAGE on 10% gels. The separated proteins were subsequently transferred onto PVDF membranes (EMD Millipore) and blocked with 5% non-fat milk in TBS with 0.05% Tween-20 for 30 min at room temperature. The membranes were then incubated with the following primary antibodies at 4°C overnight: Anti-GSTO1 (1:500; cat. no. ab129106; Abcam), anti-Bax (1:500; cat. no. ab182733; Abcam), anti-caspase 3 (1:500; cat. no. ab44976; Abcam), anti-JAK (1:500; cat. no. ab47435; Abcam), anti-phosphorylated (p)-JAK (1:500; cat. no. ab138005; Abcam), anti-STAT3 (1:500; cat. no. ab119352; Abcam), anti-p-STAT3 (1:500; cat. no. ab30647; Abcam) and anti-β-actin (1:500; cat. no. ab8227; Abcam). Following the primary antibody incubation, the membranes were incubated with anti-rabbit HRP-conjugated secondary IgG antibody (1:50,000; cat. no. ab205718; Abcam) or anti-mouse HRP-conjugated secondary IgG antibody (1:5,000; cat. no. ab205719; Abcam) for 1 h at room temperature. Protein bands were visualized using enhanced chemiluminescence (Pierce; Thermo Fisher Scientific, Inc.). Several X-ray films were analyzed to verify the linear range of the chemiluminescence signals and densitometric analysis was performed using ImageJ software (version 1.41; National Institutes of Health).

Cell proliferation was analyzed using CCK-8 solution (Beyotime Institute of Biotechnology), according to the manufacturer's protocol. Briefly, cells (1×10⁴−10⁵ cells/well) were seeded into a 96-well plate with the culture medium and incubated in a CO₂ incubator at 37°C for 24 h. Subsequently, 10 µl CCK-8 solution was added to each well and incubated for another 2 h at 37°C. Finally, the absorbance was measured at 450 nm using a microplate reader.

Cell migration and invasion were analyzed using Transwell plates. For the migration assay, 1.0×10⁵ cells in 200 µl serum-free RPMI-1640 medium were plated into the upper chambers of 8-µm Transwell plates. For the invasion assays, 1.0×10⁵ cells were plated into the upper chamber of Transwell plates precoated at 37°C for 4–5 h with Matrigel. The lower chambers for both assays were filled with RPMI-1640 medium supplemented with 20% FBS. After 24 h of incubation at 37°C, non-invasive or non-migratory cells were removed from the upper chambers, and invasive or migratory cells in the lower chamber were fixed with 100% methanol for 30 min at room temperature. Finally, cells were stained with 0.1% crystal violet for 30 min at room temperature, and then visualized and counted using an inverted fluorescence microscope (Olympus Corporation; magnification, ×100) with ImageJ software (version 1.8; National Institutes of Health).

Cell apoptosis was analyzed using an Annexin V-FITC/propidium iodide (PI) apoptosis kit (BD Biosciences). Briefly, 1×10⁵ cells were stained with Annexin V-FITC and PI, according to the manufacturer's protocol. Apoptotic cells were subsequently analyzed with a BD FACSAria™ Fusion flow cytometer (BD Biosciences) using ModFit software version 3.2 (BD Biosciences). The apoptotic rate was calculated as the percentage of early and late apoptotic cells.

All statistical analyses were performed using SPSS version 22.0 software (IBM Corp.). Data from each experiment are presented as the mean ± SD of three independent experiments. Statistical differences between ≥3 groups were determined by one-way ANOVA followed by a Tukey's post hoc test for multiple comparisons. P<0.05 was considered to indicate a statistically significant difference.

To determine the function of GSTO1 in NSCLC cell lines, GSTO1 expression levels in several NSCLC cell lines were analyzed (Fig. 1A). As the expression levels of GSTO1 were the lowest in the H2122 cell line compared with BEAS-2B cells (P<0.01), these cells were selected to construct GSTO1-overexpressing cells. GSTO1 expression levels were knocked down using shRNA in the A549 cell line, as this cell line expressed the highest levels of GSTO1 compared with BEAS-2B cells (P<0.01). RT-qPCR was subsequently used to confirm the transfection efficiency through analyzing GSTO1 expression levels in A549 and H2122 cells following the transfections. As shown in Fig. 1B, GSTO1 expression levels were significantly downregulated in A549 cells transfected with shRNA-GSTO1 compared with those in the shRNA-NC and control groups (P<0.01), whereas GSTO1 expression levels were significantly upregulated in H2122 cells transfected with pcDNA3.1-GSTO1 compared with pcDNA3.1-empty and H2122 control cells (P<0.01).

As shown in Fig. 1C, the proliferative ability of A549 cells transfected with shRNA-GSTO1 was significantly inhibited compared with cells transfected with shRNA-NC (P<0.01). Conversely, the proliferative ability was significantly increased in cells transfected with pcDNA3.1-GSTO1 compared with pcDNA3.1-empty- transfected cells (P<0.01). These results indicated that GSTO1 may promote the proliferation of NSCLC cells.

Transwell assays were used to analyze the effect of GSTO1 on the migratory and invasive abilities of NSCLC cells. The knockdown of GSTO1 in A549 cells by shRNA significantly decreased the cell migratory and invasive abilities compared with the shRNA-NC and control groups (P<0.01), whereas overexpression of GSTO1 with pcDNA3.1-GSTO1 significantly increased the migratory and invasive abilities of H2122 cells compared with the pcDNA3.1-empty-transfected and control cells (P<0.01) (Fig. 2A and B). These results indicated that GSTO1 may promote the migration and invasiveness of NSCLC cells.

To further confirm the role of GSTO1 in NSCLC cells, the effects of GSTO1 knockdown or overexpression on apoptosis were analyzed using flow cytometry. The knockdown of GSTO1 with shRNA significantly promoted the apoptosis of A549 cells compared with the shRNA-NC and control groups (P<0.01; Fig. 3A). Following successful transfection with pcDNA3.1-GSTO1, the overexpression of GSTO1 was discovered to significantly inhibit the apoptosis of H2122 cells compared with the pcDNA3.1-empty and control groups (P<0.01). Meanwhile, the genetic knockdown of GSTO1 significantly upregulated the expression levels of Bax and caspase 3 compared with the shRNA-NC and control groups (P<0.01), whereas the overexpression of GSTO1 significantly downregulated the expression levels of Bax and caspase 3 compared with the pcDNA3.1-empty and control groups (P<0.01) (Fig. 3B). These results indicated that GSTO1 may inhibit the apoptosis of NSCLC cells.

To determine the possible mechanisms underlying the GSTO1-mediated increases in the aggressive phenotypes observed in NSCLC cells, the effects of GSTO1 on JAK and STAT3 expression levels were analyzed. The results revealed that the genetic knockdown of GSTO1 in A549 cells significantly decreased the phosphorylation levels of JAK and STAT3 compared with the shRNA-NC and control groups (P<0.01), whereas pcDNA3.1-GSTO1-transfected H2122 cells had significantly increased phosphorylation levels of JAK and STAT3 compared with pcDNA3.1-empty-transfected and control cells (P<0.01) (Fig. 3B). These results indicated that GSTO1 may promote aggressive phenotypes in NSCLC cells via activation of the JAK/STAT3 signaling pathway.