Cell line and culture

Calu-3, a human submucosal gland cell line derived from bronchial adenocarcinoma, was purchased from Elabscience Biotechnology Inc. (USA). Calu-3 cells were cultured at 37 °C with 5% CO2 in MEM (Nacalai Tesque, Inc., Kyoto, Japan) with 10% fetal bovine serum (Hyclone, Logan, UT, USA), 100 IU/mL penicillin, and 100 μg/mL streptomycin (Nacalai Tesque, Inc.). The cells were stimulated with different D-glucose concentrations as follows: 100 mg/dL D-glucose (Nacalai Tesque, Inc.) in MEM (NG) and 1000 mg/dL D-glucose in MEM (HG). The medium was changed every alternate day. BAY-876 (Medchemexpress, Monmouth Junction, NJ, USA) was used to inhibit GLUT1.

Total RNA extraction and cDNA synthesis

Calu-3 cells were seeded at a density of 4 × 105 cells/well in 24-well plates. After 24 h, the medium was replaced with medium containing different D-glucose concentrations (100 and 1000 mg/dL) and cultured at 37 °C with 5% CO2 for 24, 48, and 72 h. Total RNA was extracted with 180 μL Tripure (Roche, Basel, Switzerland) from each well following manufacturer’s instruction. Total RNA was reverse transcribed to cDNA with ReverTra Ace® qPCR RT Master Mix (Toyobo Life Science, Osaka, Japan).

Real-time qPCR

ACE2 gene expression in Calu-3 cells was examined by real-time qPCR. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control. Real-time qPCR was performed on a Roche LightCycler 480 system (Roche Diagnostics) using THUNDERBIRD® SYBR® qPCR Mix (Toyobo Life Science). Reactions were performed in a final volume of 20 μL containing 1x THUNDERBIRD® SYBR® qPCR Mix, 0.3 μM of forward and 0.1 μM of reverse primers, 7.8 μL of distilled H2O, and 1 μL of the cDNA template. The primers used in this study were as follows: For human ACE2, forward, 5′-CCACTGCTCAACTACTTTGAGCC-3′ and reverse, 5′-CTTATCCTCACTTTGATGCT-3′; for GAPDH, forward, 5′-GAAGGTGAAGGTCGGAGTC-3′ and reverse, 5′-GAAGATGGTGATGGGATTTC-3′; for GLUT1, forward, 5′-AAGGTGATCGAGGAGTTCTACA-3′ and reverse, 5′- ATGCCCCCAACAGAAAAGATG-3′. The amplification conditions were as follows: denaturation at 95 °C for 5 min; 45 cycles of amplification (95 °C for 10 s, 60 °C for 10 s, and 72 °C for 10 s); and cooling at 50 °C for 30 s. Relative quantification of target gene expression was determined using LightCycler 480 software according to ΔΔCT-method.

Western blotting

Lysate samples were isolated from Calu-3 cells using a radioimmunoprecipitation assay lysis buffer (Wako Pure Chemical Industries, Osaka, Japan). Total protein was fractionated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membrane. After blocking with 5% bovine serum albumin in Tris-buffered saline/0.1% Tween-20 (TBST) at room temperature for 1 h, the membrane was incubated overnight at 4 °C with antibodies against ACE2 (1:1000; R&D Systems, Inc. Minneapolis, MN, USA) and GAPDH (1:2000, Santa Cruz Biotechnology, CA, USA). The membrane was washed three times with TBST and incubated with horseradish peroxidase anti-goat or anti-mouse secondary antibody (DakoCytomation, CA, USA) at 20 °C for 1 h. After washing three times with TBST, immunoreactive bands were detected using a ChemiDoc XRS+ Image System (Bio-Rad Laboratories, CA, USA). Protein bands were quantified using Image Lab software (Bio-Rad Laboratories).

Immunofluorescence staining

Calu-3 cells were cultured on cover-glass slides for 72 h. The glass slides were washed three times with phosphate buffered saline (PBS), fixed with 4% paraformaldehyde (Nacalai Tesque, Inc.) for 15 min at room temperature, and rinsed with PBS. After blocking with PBS containing 5% bovine serum albumin for 30 min, fixed Calu-3 cells were incubated overnight at 4 °C with primary antibody mouse anti-human ACE2 (1:50; Santa Cruz Biotechnology). After rinsing with PBS, the slides were incubated with anti-mouse immunoglobulin G H&L (Alexa Fluor® 488; Abcam, MA, USA) in PBS. After rinsing with PBS, slides were stained with DAPI (Tokyo Chemical Industry, Tokyo, Japan) for 15 min at room temperature. The cover slides were mounted using Fluoromount (Diagnostic BioSystems, CA, USA), scanned, and photographed using a light microscope (Keyence BZ-X800, Keyence Corporation of America, NJ, USA).

LDH activity assay

LDH activity was measured to determine the effect of D-glucose concentration on cell viability. Calu-3 cells were plated in 96-well plates (1 × 105 cells/well) and incubated in MEM with different D-glucose concentrations ranging from 100 to 5000 mg/dL, and with BAY-876. After 72 h, cell viability was evaluated using the Cytotoxicity Detection Kit PLUS (Roche Diagnostics), according to the manufacturer’s protocol. Cells cultured in MEM were used as controls. The absorbance was measured at 490 nm, and cell viability was calculated.

The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay

Cell proliferation and viability were assessed using an MTT assay (Nacalai Tesque, Inc.). Calu-3 cells were plated in 96-well plates (1 × 105 cells/well) and incubated in MEM with different D-glucose concentrations ranging from 100 to 5000 mg/dL, and inhibitors (BAY-876) for 72 h. Following instruction, 10 μl MTT solution was added, and the cells were cultured at 37 °C with 5% CO2 for 4 h. 100 μl solubilization solution was added, and the cells were cultured at 37 °C with 5% CO2 for 15 h. The absorbance was measured at 570 nm.

RNA interference (siRNA)

The pre-designed Silencer™ Select Validated siRNA or Silencer™ Select siRNA negative control (Thermo Fisher Scientific, MA, USA) were transfected into Calu-3 cells using 10 nM of Lipofectamine™ RNAiMAX (Thermo Fisher Scientific) manufacturer’s instructions. 24 h after transfection, cells were treated with NG or HG for 72 h.

Statistical analysis

Significant differences among samples were determined by one-way analysis of variance with post-hoc Tukey’s honest significant difference test or Student’s t-test with p < 0.05 indicating statistical significance. A minimum of three samples were tested in three independent experiments. All results are presented as mean ± standard error of the mean. Data analysis was performed using the SPSS software version 24.0 (IBM, Armonk, NY, USA).