The A549 human lung adenocarcinoma cell line was obtained from the American Type Culture Collection (ATTC; Rockville, MD, USA). Cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) and supplemented with non-essential amino acids, 50 U/ml penicillin and 50 µg/ml streptomycin at 37°C in a humidified atmosphere with 5% CO₂/95% air.

After cells reached 80% confluence, 2×10⁴ cells/well were cultured into 48-well plates for 24 h. A stock solution of 33 mM 2-ME (Sigma-Aldrich, St. Louis, MO, USA) was prepared in dimethyl sulfoxide (DMSO). Final 2-ME concentrations were prepared by diluting the stock solution with DMEM. Medium was replaced with fresh medium with 10% FBS and with the corresponding 2-ME concentrations (0.001, 0.1, 0.1, 1 and 10 µM final concentrations). A549 cells with 10% FBS medium (non-stimulated cells) were used as growth control. Cells were also cultured with 10% FBS plus 0.03% DMSO (vehicle-control group for all experiments). Cells were cultured for 12, 24, 48, 72 and 96 h in normoxia (5% CO₂ and 95% air) and hypoxia (1% oxygen and 5% CO₂) conditions, at 37°C in a humidified incubator. The culture medium, with and without 2-ME, was not changed during the assay.

Cells were placed into a chamber MIC-101 (Billups-Rothenberg, Del Mar, CA, USA) to expose them to hypoxic conditions. Briefly, a mixture of 95% nitrogen and 5% CO₂ gas that displaces the oxygen into the chamber was injected. The oxygen concentration was measured by an oxygen sensor (Vascular Technology, Nahua, NH, USA) and maintained at 1%. At the end of the incubation period, the media were discarded and the cells were washed with phosphate-buffered solution. Five hundred microliters of 1% glutaraldehyde solution were added to each well and incubated for 20 min at room temperature; subsequently the glutaraldehyde solution was discarded and 0.1% crystal violet (N-hexamethylpararosaniline) (Sigma-Aldrich) solution was added to each well and incubated under constant stirring for 15 min at room temperature. Crystal violet was washed exhaustively, allowed to dry and afterwards 400 µl of 10% acetic acid solution were added to each well. The sample absorbance was measured at 590 nm in 96-well microtiter plates with an ELISA reader (Molecular Devices, Sunnyvale, CA, USA). All experiments were carried out in triplicate. Cell growth was expressed as the relative increase in light absorbance at 590 nm with respect to the value at 0 h.

A549 cells were cultured in 6-well plates (3×10⁵ cells/well) under normoxic or hypoxic conditions for 72 h. Three different media were used: DMEM (control), DMEM with DMSO (without 2-ME) and DMEM with 10 µM 2-ME. Apoptosis was examined by flow cytometry using the Annexin V (Becton-Dickinson Biosciences, Franklin Lakes, NJ, USA) staining kit. Briefly, 1×10⁶ cells in 100 µl Annexin buffer were stained with propidium iodide and FITC-Annexin V staining solutions. Cells were incubated at room temperature in the dark for 15 min, and the data were subsequently acquired through a FACSAria flow cytometer (Becton-Dickinson Biosciences). Data were analyzed using the FlowJo X.0.7 software (Stanford University, Stanford, CA, USA). Results are expressed as percentage; 100% = 5,000 cells.

Cells were plated into 6-well cell culture plates and were grown until they reached 60% confluence. Cells were then exposed to hypoxic conditions and lysed with 0.1% Triton (Sigma-Aldrich) in PBS without calcium to obtain total cell extracts. Protein quantification was performed by the bicinchoninic acid protein assay (BCA protein assay kit; Pierce Biotechnology, Rockford, IL, USA). Western blotting was carried out using 30 µg of cell extract proteins in 8% SDS-polyacrylamide gels (PAGE) under reducing conditions with 5% 2-mercaptoethanol boiled for 10 min. After electrophoresis, the proteins were transferred to PVDF membranes and blocked with 2.5% non-fat dry milk in 100 mM Tris-HCl buffer, pH 7.5 with 150 mM NaCl and 0.1% Tween-20 (TTBS buffer); they were then incubated for 90 min at room temperature with the corresponding antibody: 1:500 anti-HIF-1α (mouse monoclonal antibody, NB100-479) or 1:500 anti-HIF-2α (rabbit polyclonal antibody, NB100-122) (both from Novus Biologicals, Littleton, CO, USA). β-tubulin antibody (1:500) was used as a loading control (sc-53140; Santa Cruz Biotechnology, Santa Cruz, CA, USA). Unbound antibodies were washed with TTBS buffer and bands were detected using the Vectastain^® ABC kit (Vector Laboratories, Burlingame, CA, USA). Western blotting bands were analyzed by densitometry scanning using the Kodak Digital Science ID Image analysis software (Eastman Kodak, Rochester, NY, USA). The results are expressed as densitometry units (DU).

Five hundred thousand cells were cultured in a Nunc Lab-Tek chamber slide system (Thermo Fisher Scientific, Carlsbad, CA, USA) and allowed to grow in normoxia or hypoxia with or without 10 µM 2-ME treatment for 72 h. The cells were fixed with 1% glutaraldehyde (Sigma-Aldrich) and washed thrice with distilled water for 5 min. Antigen retrieval was performed with 1:10 citrate buffer (Sigma-Aldrich). The slides were heated during 5 min in a microwave and they were then allowed to cool for 20 min. Endogenous peroxidase activity was quenched with 2% H₂O₂ solution (Sigma-Aldrich). Antibody blockade and incubation were performed in a humid chamber with 100 µl of blocking solution with serum, followed by the blockade of non-specific sites with 4% (wt/vol) non-fat dry milk in PBS at 4°C overnight. Cells were incubated with 100 µl of the corresponding antibody for 90 min at room temperature: 1:100 anti-HIF-1α (mouse monoclonal antibody, 1NB100-479) or 1:100 anti-HIF-2α (rabbit polyclonal antibody, NB100-122; Novus Biologicals). In another set of experiments, cells were processed with a non-immune IgG instead of the primary antibody as a negative control. Visualization of antibody localization was achieved with a Vectastain^® ABC detection kit. Between each step, the slides were thoroughly washed with distilled water. Finally, the slides were manually counterstained with Harris hematoxylin and mounted with non-aqueous medium. Images were captured with an Evos-FL Auto microscope (Thermo Fisher Scientific).

Cells were incubated under normoxic or hypoxic conditions with or without 10 µM 2-ME for 72 h. After incubation, total RNA and protein were extracted. RNA was extracted using TRIzol reagent (Invitrogen Life Technologies, Thermo Fisher Scientific) and reverse transcribed into cDNA (Advantage RT-for-PCR kit; Clontech, Palo Alto, CA, USA). One microgram of total RNA was reverse transcribed using 2 µg of random primers and Moloney murine leukemia virus reverse transcriptase according to the manufacturer's protocol (Advantage RT-for-PCR kit). Real-time PCR was carried out on a One-Step system (Applied Biosystems, Thermo Fisher Scientific). The expression assay was carried out using pre-designed TaqMan gene expression assay Hs00153153 for HIF-1α labeled with FAM, Hs01026149_m1 for HIF-2α labeled with FAM and normalized with Hs99999901_s1 for 18S ribosomal RNA labeled with VIC (Applied Biosystems). The PCR duplex reactions were performed in a 20-µl reaction volume containing 10 µl of TaqMan Universal PCR Master Mix 2X, 0.5 µl of TaqMan gene expression assay Hs99999901_s1 20X used as an endogenous control (18S rRNA), 1 µl TaqMan gene expression assay of the target gene (Hs00153153 or Hs01026149_m1), 50 ng of cDNA (4 µl) and 4 µl of RNAse-free water.

Relative quantitation method was used to analyze the results of two independent experiments made in triplicate. For each experimental sample, a gene was considered as not expressed if amplification was not detected by threshold cycle Ct = 40. The results are expressed in arbitrary units of ΔCt, where ΔCt = Cttarget - Ct_18S. ΔCt values represent mRNA transcripts.

Cell growth was expressed as a percentage of their relative controls. The mean and standard deviation (SD) were obtained in triplicate. Differences between experimental assays in hypoxia, normoxia (0, 12, 24, 48, 72 and 96 h) and apoptosis assays were analyzed using the Student's t-test. Statistical analysis was conducted using the statistical software SPSS version 20.0 (IBM SPSS). p≤0.05 was considered to indicate a statistically significant result.

Cell growth rates in the 2-ME-stimulated cells were decreased in comparison to the rates in the DMSO-incubated control cells, mainly at 48 and 72 h under normoxic conditions (Fig. 1A). The lowest cell growth rate was observed using a 10 µM concentration of 2-ME in comparison with the DMSO control cells: 179.4±16.6 and 347.7±9.6%, respectively (p<0.0001) at 48 h; 189.0±11.6 and 526.3±7.2%, respectively (p<0.0001) at 72 h, and 179.7±14.2 and 552.9±12.1%, respectively (p<0.0001) at 96 h. In contrast, there were no significant differences among the growth rates between the control cells and those treated with 10 µM 2-ME at 72 and 48 h under hypoxic conditions (Fig. 1B). However, a significant decrease in the growth rate was found in the 10 µM 2-ME-treated cells in comparison with the DMSO-treated cells (66.2±7.2 and 101.2±2.3%, respectively; p=0.04) at 96 h. An exponential cell growth was observed in the DMSO medium without stimulation, as expected.

2-ME at a concentration of 10 µM was used for the apoptosis and HIF-1α and HIF-2α expression assays, due to the significance found for this concentration when cells were incubated under normoxic conditions at 72 h. Longer incubation periods were not used since absence of nutrients could have biased the results. Apoptosis induced by 2-ME in the A549 cells was differentially affected by the oxygenation conditions (Fig. 2A). The presence of 10 µM 2-ME significantly increased the percentage of apoptosis (34.5±2.8%) in comparison with the DMSO control cells (12.5±6.5%) (p=0.006) and non-stimulated cells (NS) (p=4.8×10⁻⁵) in a normoxic condition. There were no significant differences among 10 µM 2-ME-treated and control cells grown under hypoxic conditions. A significant increase in apoptosis was observed in cells treated with 10 µM 2-ME in a normoxic condition in comparison with cells under lower O₂ concentration (5.8±0.2%; p=0.003) (Fig. 2B).

Western blot densitometry analysis showed differences in the protein expression of HIF-1α and HIF-2α under hypoxic conditions (Fig. 3).

HIF-1α was significantly increased in hypoxic cells (44,998.1±1,079.3 DU) in comparison with cells cultured in normoxic conditions (35,200.8±3,726.9 DU; p=0.01). HIF-1α protein expression was not modified when the cells were treated with 2-ME in a normoxic condition. In contrast, there was a decrease in HIF-1α when cells were treated with 2-ME under hypoxic conditions (29,390.1±3,542.9 DU; p=0.001).

HIF-2α protein levels were significantly increased in the cells cultured under hypoxia (65,834.3±1,957.7 DU) in comparison with cells incubated under a normoxic condition (51,537±1,451.3 DU, p=0.001). The synthesis of this protein was not significantly modified by the exposure to 2-ME under a normoxic condition. In contrast, the HIF-2α level was significantly decreased in cells treated with 10 µM 2-ME under hypoxic conditions (25,921±3,5442.9 DU; p=6.8×10⁻⁵).

Significant differences were also found when HIF-1α and HIF-2α levels of cells grown under normoxic (p=0.02) and hypoxic conditions (p=8.6×10⁻³) were compared, but not among the same cell groups treated with 2-ME.

HIF-1α and HIF-2α were detected in the normoxic-grown cell cytoplasm, particularly HIF-2α, using immunoperoxidase staining (Fig. 4). When cells were cultured under hypoxic conditions, HIF-1α was observed with higher intensity in the cell nucleus; however, staining for HIF-2α was low. Neither HIF-1α nor HIF-2α were noted in the cells treated with 2-ME under a normoxic condition. The incubation with 2-ME decreased the nuclear staining for HIF-1α in cells under hypoxia, while no staining for HIF-2α was observed under the same experimental conditions.

Apart from the effects of 2-ME on HIF-1α and HIF-2α protein expression in both experimental conditions, some morphological and physiological effects were also evident. Nuclear lobation (open arrows) and nuclear fragmentation (apoptosis, solid arrows) were present in cells treated with 2-ME under a normoxic condition; these phenomena were more frequent in cells exposed concomitantly to hypoxia and 2-ME.

The gene expression assay revealed that there were significant differences in HIF-1α mRNA expression among normoxic (5.9±0.9 ΔCt values) and hypoxic (9.2±0.8 ΔCt values; p=0.0004) cells (Fig. 5). A significant decrease in HIF-1α mRNA expression was observed when cells were cultured with 2-ME under a normoxic (0.63±0.3 ΔCt values; p=0.03) condition. HIF-1α mRNA was untraceable in cells exposed to 2-ME under hypoxia.

There were no differences in HIF-2α mRNA expression among cells cultured under normoxic and hypoxic conditions. When normoxic-grown cells were exposed to 2-ME, HIF-2α gene expression was significantly decreased (0.3±0.1 ΔCt values) in comparison with the untreated cells (3.9±0.4 ΔCt values; p=0.02). Neither HIF-1α nor HIF-2α mRNA was detected in hypoxic cells exposed to 2-ME.

The mRNA expression of HIF-1α was significantly higher than HIF-2α gene expression under hypoxia (p=0.04); but no differences were noted when mRNA expression for HIF-1α and HIF-2α grown in normoxic conditions was compared.