We acquired the original MEC-1 cell line (#ACC 497, DSMZ) as a kind gift from Prof. Marek Mraz, M.D., Ph.D. (Laboratory of Microenvironment of Immune Cells, Central European Institute of Technology (CEITEC MU) and University Hospital Brno, Czech Republic). We modified the original MEC-1 cells using CRISPR/Cas9. A CRISPR/Cas9 plasmid (U6gRNA-Cas9-2A-GFP MiR155) was introduced into MEC-1 cells by nucleofection by Amaxa Nucleofector II (Lonza) with use of the B-cell nucleofection kit (#VPA1001, Lonza) and U-015 program. The CRISPR/Cas9 plasmid we ordered from Sigma-Aldrich, was designed at our laboratory and targeted the mature hsa-miR-155-5p sequence. More information about the creation and validation of miR-155 deficient MEC-1 clone #48 are in the Supplemental Fig. 1. MEC-1 cell line was cultured in IMDM medium (#LM-I1090, Biosera) supplemented with 10% of fetal bovine serum (#FB-1090, Biosera), 1% of P/S (#P4333, Sigma Aldrich) at 37 °C in 5% CO2 incubator. We followed the recommended protocol for cell culturing from DSMZ for MEC-1 cells (#ACC-497). The hypoxic MEC-1 cell line was incubated in hypoxic glove box (Coy O2 Controlled InVitro Glove-Box–Hypoxia Chamber, Genetica) in IMDM medium supplemented with 10% of fetal bovine serum, 1% of P/S at 37 °C, 1% O2 (for optimizing experiments we used 0.2%, 1% and 5% O2) and 5% CO2 for 24 h, and 48 h (for optimizing experiments also 72 h, 96 h and 120 h). Cells were collected for RNA isolation, proliferation (WST-1 assay), cell viability (AnnexinV/PI staining), cell cycle kinetics (BrdU staining) and metabolic assays.
Chemically induced hypoxiaChemically induced hypoxia was made in vitro through the addition of deferoxamine mesylate salt—DFO (#D9533, Sigma-Aldrich) and dimethyloxalylglycine, N-(Methoxyoxoacetyl)-glycine methyl ester – DMOG (#D3695, Sigma-Aldrich) to a well containing 2 × 106 cells. Cells were cultured in IMDM medium (LM-I1090, Biosera) supplemented with 10% of fetal bovine serum (#FB-1090, Biosera) and 1% of P/S (#P4333, Sigma Aldrich). Cells were then incubated for 3 h, 6 h, and 24 h at 37 °C in 5% CO2 with DFO (final concentration 200 µM) and DMOG (final concentration 1 mM). After each time interval of incubation with DFO or DMOG, cells were collected, and total RNA was extracted for further gene expression detection. In parallel, cells were collected for flow cytometry measurement of apoptosis (AnnexinV/PI staining).
Proliferation and cell viability testsThe WST-1 assay was used for the cell proliferation and viability measurement, followed by manufacturer’s protocol (ROCHE, #11 644 807 001). MEC-1 cells were seeded 5000 cells per well (used 96 well plate, flat bottom) in culture medium and incubated with 10 µL of WST-1 solution for 3 h [in normoxia and hypoxia conditions (1% O2)]. After 3 h, the substrate reaction absorbance was measured at 440 nm wavelength (blank 600 nm) by using Spark® multimode microplate reader (TECAN) spectrophotometer (Tecan i-control, 1.10.4.0, infinite 200Pro). The absorbance of blank was subtracted from measured samples wavelength. Data are from three independent experiments. Data were analyzed using t-test, two-tailed, paired.
Cell growth curve was created by cell count determined by hemocytometer (counting-chamber). Cells were counted daily for one-week (in parallel normoxia vs hypoxia (1% O2) conditions). Seeding density of cells at day 0 was 10,000. Data are from three independent experiments (samples were done in quadruplicate). Data were analyzed using t-test, two-tailed, paired.
Annexin V/PI staining was performed for evaluation of apoptotic and dead cells. MEC-1 cells (1 × 106 cells) were washed with 1 × PBS, resuspended in 1 × Annexin V binding buffer, stained by 5 µL of Annexin V (15 min at RT) (FITC, # BMS500FI-300, Invitrogen). Cells were washed with 1 × PBS and kept on ice until measurement on flow cytometer. Shortly before measurement 5 µL of PI (# BMS500FI-300, Invitrogen) was added. Data were evaluated by Diva software, the software FlowJo was used for data visualization, and the measurement was performed with the use of the FACS Canto II BD flow cytometer (30 000 events).
Cell cycle measurement by BrdU flow kitFor BrdU staining, 2 × 106 cells/well was used. A volume of 30 uL of BrdU were added to the MEC-1 cell culture (final concentration 1 µM) and stained for 20 min at 37 °C in 5% CO2 and 21% O2 under normoxic and under hypoxic (1% O2) conditions as followed by the manufacturer’s protocol (# 552598, BrdU flow kit, BD). Before flow cytometry measurement, cells were stained with 7AAD (provided by BrdU kit, 20 µL/tube) for 10 min on ice. Measurement was performed on cytometer FACS Canto BD (50 000 events). The software FlowJo was used for data visualization. Data are from three independent experiments. Data were analyzed using t-test, two-tailed, paired.
RNA isolation and qRT-PCRTotal RNA including microRNA was extracted from MEC-1 cells (2 × 106) by TRI reagent (#TR118, MRC) with slight modification as over night precipitation with isopropanol at − 20 °C and followed by manufacturer’s protocol. An amount of 100 ng of total RNA including microRNA was reverse-transcribed by High-Capacity cDNA Reverse Transcription Kit with RNase Inhibitor (#4,374,966, ThermoFisherScientific). TaqMan-based PCR with specific probes (Universal Probe Library, ROCHE and ThermoFisherScientific primers designed with probes) was performed on QS7 Pro instrument (ThermoFisherScientific). As reference genes, GAPDH (for mRNA) and RNU44 (for miRNA) were used. The miRNA/mRNA expression was calculated by 2 delta-delta CT algorithm from target and reference CT values [specific (s) and control (c) amplicons calculated by 2 − (CTc-CTs) equation] [26]. Data were acquired using t-test, two-tailed, paired (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001).
Western blotCells were lysed (2 × 106) in RIPA buffer and sonicated (1 cycle, 10” at 40% power). Four micrograms of proteins were separated by 1-D polyacrylamide gel electrophoresis (Mini-PROTEAN® TGX™ Precast Gels, Bio-Rad). Proteins were transferred to Immuno-Blot® PVDF Membrane (#1,620,174, Bio-Rad) using Trans-Blot® Turbo™ Transfer System (Bio-Rad) and probed with primary antibody overnight at 4 °C (anti-HIF-1α, sc-10790; anti-GAPDH sc-51907, Santa Cruz Biotechnology). The following day, the membrane was washed with 1 × TBS and probed with secondary anti-rabbit antibody (#A0545, Sigma-Aldrich), conjugated with horseradish peroxidase and detected using detection kit (#1,705,060, Clarity™ Western ECL Substrate, Bio-Rad). Signalling was detected using ChemiDoc MP Imaging System (Bio-Rad).
Metabolic assaysMitochondria isolation was followed by the referred protocol in [27]. Mitochondria protein content was measured by the BCA kit (#BCA1-1KT, Sigma-Aldrich) on Biotek Synergy HT Microplate Reader at 562 nm wavelength. The concentration of protein was calculated using a standard curve prepared using the BSA protein standards.
Electron transport chain complex I to complex III was followed protocol in [27]. Mitochondria protein content was measured by the BCA kit (#BCA1-1KT, Sigma-Aldrich). Absorbance (reduction from succinate to cytochrome C) was read at 550 nm wavelength for 6 min (1 read/15 s) on Biotek Synergy HT Microplate Reader. Results were calculated according to Lambert–Beer equation.
The Glucose Uptake-GloTM Assay (#J1341, Promega) was used according to the manufacturer´s manual. The MEC-1 cells were cultured in normoxia and hypoxia (1% O2, 24 h, and 48 h), then counted and 10,000 cells were transferred to a non-translucent 96-well cell culture plate. The reaction solution from the kit was added and the luminescence signal was acquired at 0.5 s on the Spark® multimode microplate reader (Tecan i-control, 1.10.4.0, infinite 200Pro) and is shown as counts/s.
2-NBDG (2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)Amino)-2-Deoxyglucose)(2-DG) (#N13195, ThermoFisherScientific) was used for the measurement of glucose uptake in MEC-1 cells (ctrl and miR-155 deficient) as followed by the manufacturer´s recommendations. The MEC-1 cells were counted and 200,000 cells were seeded on 6-well plate (in triplicates) and then cultured in normoxia and hypoxia (1% O2) for 24 h and 48 h. Cells were washed with 1 × PBS, resuspended in 1 mL of Seahorse XF DMEM medium (#103,575–100, Agilent), and then incubated 10 min with 2-DG in final concentration 10 µM (in normoxia and in hypoxia). Next, cells were washed with 1xPBS and the cell pellet was resuspended in 200 µL of 1xPBS, followed by flow cytometry. Shortly before measurement, 5 µL of PI (# BMS500FI-300, Invitrogen) were added. As 2-DG has an excitation/emission maximum of ∼465/540 nm, we used channel FITC 530 in this case. Data were evaluated by Diva software and the measurement was performed with the use of the FACS Canto II BD flow cytometer (50 000 events).
The Lactate Assay (#MAK064, Sigma-Aldrich) was used according to the manufacturer´s manual. The MEC-1 cells were cultured in normoxia and hypoxia (1% O2, 24 h, and 48 h), were then centrifuged (350´ g, RT, 5 min), and 10 µL of supernatant were aspirated for the assay and transferred to 96-well plate. Each sample was brought to a final volume of 50 µL/well with Lactate Assay Buffer. The reaction solution from the kit was mixed and 50 µL of Master Reaction Mix were added to each sample. Cells in the 96-well plates were incubated for 30 min at RT protected from light. Colorimetric absorbance was measured on Biotek Synergy HT Microplate Reader at 570 nm wavelength. Concentration of lactate was calculated using standard curve.
mtATP level was obtained by the Adenosine 5′-triphosphate (ATP) Bioluminescent Assay Kit (#FLAA, Sigma Aldrich). The MEC-1 control group and the MEC-1 miR-155 deficient cells (2 × 105 cells) were cultivated 24 h and 48 h under the normoxia and hypoxia (1% O2) conditions. Cells were cultured under standard cell culture conditions. Mitochondria were isolated and further mtATP measurement was carried out according to the manufacturer´s protocol. Lumenescence was measured by the Biotek Synergy HT Microplate Reader.
ATP level was determined by the CellTiter-Glo® 2.0 Cell Viability Assay (#G9242 Promega). The MEC-1 ctrl and the MEC-1 miR-155 deficient cells were cultivated 24 h and 48 h under the normoxia and hypoxia (1% O2) conditions. Cells were cultured under standard cell culture conditions and 5,000 cells were used for measurement. The assay was performed according to the manufacturer's manual. The luminescence signal was acquired at 0.5 s on the Spark® multimode microplate reader (Tecan i-control, 1.10.4.0, infinite 200Pro) and is shown as counts/s.
LDH level was acquired by the LDH-Glo™ Cytotoxicity Assay (#J2380 Promega). The MEC-1 ctrl and the MEC-1 miR-155 deficient cells were cultivated 24 h and 48 h under the normoxia and hypoxia (1% O2) conditions. Cells were cultured under standard cell culture conditions and 2 µL of the cultivation media out of 1 ml was used for the measurement. The assay was performed according to the manufacturer´s manual. The luminescence signal was acquired at 0.6 s on the Spark® multimode microplate reader (Tecan i-control, 1.10.4.0, infinite 200Pro).
Pyruvate concentration was determined by the Pyruvate Assay Kit (#MAK332 Sigma-Aldrich). The MEC-1 control group and the MEC-1 miR-155 deficient cells were cultivated 24 h and 48 h under the normoxia and hypoxia (1% O2) conditions. Cells were cultured under standard cell culture conditions and 1 × 106 were homogenized mechanically in 1 ml of cold PBS; 10 µl were used for the measurement. The assay was performed according to the manufacturer´s manual. The fluorescence signal was acquired, excited by 530/20 nm and emission was read at 585/20 nm on the Spark® multimode microplate reader (Tecan i-control, 1.10.4.0, infinite 200Pro).
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