The genes of human wild-type CRY 2, variant 2 (CRY2-2; NM_001127457.2) and MagR (NM_030940.3) that are necessity for the formation of magnetosensor complexes16, were obtained from MCF-7 cell line. In brief, total RNA samples were isolated from cells using a RiboEx reagent (GeneAll, Korea). For cDNA synthesis, reverse transcription PCR was performed using Prime Script II reverse transcriptase (Takara, Japan) and two specific oligonucleotide primers were designed by oligoanalyzer software, and were synthesized by Pishgam Biotech Co. (Tehran, Iran). The complete open reading frame of CRY2-2 and MagR were amplified using forward (Fwd) and reverse (Rev) primers, which contained a restriction site (underlined) as followed:
CRY2-2-Fwd, 5′-CTAGCTAGCATGCCAGCTCCACCCGGGCG-3′, Nhe1 restriction site;
CRY2-2-Rev, 5′-AAAGAGCTCTCAGGCATCCTTGCTCGGCA-3′, Sac1 restriction site;
MagR-Fwd, 5′-CTAGCTAGCATGTCGGCTTCCTTAGTCCGG-3′, Nhe1 restriction site;
MagR-Rev, 5′-AAAGAGCTCTCAAATATTAAAGCTTTCTCC-3′, Sac1 restriction site.
We designed primers for PCR and resequencing the first exon of CRY2-2 that was incomplete in genome assembly, to achieve the full DNA coding sequence.
Construction of CRY and MagR expressing plasmids
The CRY2-2 and MagR cDNA were cloned into pDB2 (Invitrogen) as a high copy-number plasmid with kanamycin resistance cassette. The pDB2 vector was extracted using Qiagen Midi kit (Qiagen, Hilden, Germany). The DNA sequences and plasmid were double digested with Nhe1 and Sac1 restriction enzymes at 37 °C overnight, and thus gel purified. After that, the purified PCR fragment was ligated to the pDB2 vector by T4 DNA ligase at 14 °C overnight. In order to transform the recombinant DNA into E. coli DH5α competent cells, which were freshly prepared by calcium chloride (CaCl2) treatment. Next, resulting recombinant DNA was transformed to E. coli DH5α according to the protocol. Cells were first grown overnight at 37 °C in LB media, and this starter culture was subsequently diluted 1:100 into LB media. The native pDB2 plasmid containing green fluorescent protein (GFP) followed an identical culturing approach. Clones were confirmed for correct insertion by restriction double digestion of the isolated plasmids (with Nhe1 and Sac1) and colony PCR. All plasmids were constructed by standard molecular biology procedures and subsequently verified using an automatic sequencer (MWG) by cytomegalovirus (CMV) promoter and SV40_PA_terminator universal primers.
The MCF-7 human breast adenocarcinoma and human embryonic kidney 293T (HEK 293T) cell lines were used, originally obtained from National Cell Bank of Iran (NCBI) at Pasteur Institute of Iran (IPI). The cells were grown in Dulbecco's modified eagle's medium (DMEM) with neutral pH (7.2–7.4), containing 10% (v/v) heat-inactivated fetal bovine serum (FBS), L-glutamine (2 mM), 1% penicillin/streptomycin (all from Gibco). Cultures were maintained in a humidified incubator at 37 °C and 5% CO2 atmosphere. The cell media was changed every 2–3 days, and when cells were confluent about 70–80%, they were trypsinized with 0.025% trypsin-EDTA (Bioidea, Iran). After attaching overnight, the cultivated cells were prepared to transfection.
HEK 293T cells were stably transfected with vectors of pDB2_CRY2-2, pDB2_MagR and native GFP-expressing pDB2 plasmid as previously described. Transfection was achieved using 25 kDa branched polyethylenimine (bPEI25, Sigma-Aldrich) at a concentration of 3.57 mg PEI per mg plasmid DNA (pDNA)84. Briefly, 200 × 103 cells were seeded in six-well cell culture plates (SPL Life Sciences Co., Ltd. Korea) and incubated in a humidified atmosphere at 37 °C under 5% CO2, overnight. Before transfection, polymeric nanoparticles (containing PEI and prepared-pDNA) were prepared and diluted in FBS-free DMEM in a total volume of 100 µl. At transfection time, the culture media was completely aspirated from the wells and replaced drop-wise with suspension of nanoparticles. After that, the supplemented DMEM with 10% FBS and 1% penicillin and streptomycin was added at 4 h later and cell culture plates were returned to the 37 °C incubator for 48 h to allow gene expression. As seen in Supplementary Fig. S2, the overexpression of these genes don’t show significant toxic effects on the cells. The transfection efficiency was measured in cells, which were generated in the same way to express enhanced-GFP using a luciferase assay. Luciferase activity was measured by luminometer (Berthold detection systems, GmbH, Germany) at different times, and presented as relative light units (RLU/sec) per number of cells. As shown in Supplementary Fig. S3, maximum transfection efficiency was at 48 h post-transfection.
Characterization of nanoparticles
Gel retardation assay
The stability of DNA-binding polymeric complexes (PEI/pDNA) were examined by gel retardation assay as shown in Supplementary Fig. S4. The formation of electrostatic binding between the amine group of PEI with positive charge and phosphate group of DNA neutralize the negative charge of pDNA. Briefly, the complexes were prepared at defined concentration of 3.57 µg PEI and 1 µg pDNA. The complexes and free pDNA were loaded onto agarose gels (1%) and electrophoresed at 90 V for 50 min. The retardation of plasmid mobility was visualized by ethidium bromide (0.1 mg/ml) and ultraviolet imaging system.
Particle size and charge analysis
The particle size and zeta potential of each sample were measured by a Malvern Nano ZS instrument and DTS software (Malvern Instruments, UK) at room temperature. The samples were prepared at concentration of 3.57 µg PEI and 1 µg pDNA in cold PBS up to 1 ml. The results were analyzed by Zetasizer software (V6.12) as shown in Supplementary Table S1.
The prepared-HEK 293T cells were treated by 0.1 µM DOX (Accord Healthcare) in exposure of 15 mT SMF and 50 Hz ELF-PEMF. In brief, cells were seeded into cell culture plates (SPL Life Sciences Co., Ltd. Korea) containing supplemented DMEM with 10% (v/v) FBS, 2 mM L-glutamine, 100 units/mL of penicillin and 100 mg/mL of streptomycin at 37 °C and 5% CO2, respectively. Cells were initially allowed to attach overnight and then prepared for transfection by clonal plasmids containing either CRY and MagR or GFP. Afterward, the media was removed and treated by new supplemented DMEM in the DOX, sham- and SMF- or ELF-PEMF-exposed conditions for 24 h post-transfection. Generally, cells were characterized at the endpoint of transfection processes and 24 h after that.
Magnetic field exposure system
Exposure to SMF (15 mT) and ELF-PEMF (50 Hz) were used in this study that prepared by a locally designed homogeneous MF-generator. This platform consisted of two coils (1800 loops of 3.0 mm diameter coated copper wire), which were resistant to heat up to 200 °C through passing either direct current (DC) switching power supply or alternating current (AC) (Supplementary Fig. S5a). Wirelength in each coil was about 1 km and each coil weighed approximately 40 kg. The coils received DC voltage up to 50 V and AC voltage 220 V and 50 Hz frequency, current up to 16 A, and the maximum provided-power was equal to 800 W. Coils had a total resistance and inductance of 3 Ω and 2 Henry, respectively. The electric power was provided using a 220 V/AC power supply equipped with a variable transformer as well as a single-phase full-wave rectifier. These two coils guided either SMF or ELF-PEMF through two iron blades (with 1-meter height and a cross-section of 10 × 10 cm2).
A removable cell culture incubator (a plexiglass container with 35 cm length, 23 cm width and 52 cm height) as exposure unit was included between both iron blades (with 1 cm gap) as shown in Supplementary Fig. S5b, which was stabled by plastic bases upon wooden insulation (with 1 cm thickness), and exposure unit is above the coils with 15 cm distance. In the exposure unit, humidity, temperature (37 °C) and CO2 atmosphere (5%) were controlled using three different sensors. To cool off the system, a gas chiller with optimum control on temperature was used. This automatic cooling system included an engine, an evaporator, a condenser, and a refrigerant gas (Freon-12). The engine is far enough from the exposure unit to avoid any significant MFs interference. The evaporator covered the outer surface of the coils, which enabled it to cool the system down effectively.
Overall cells were exposed to either sham or MFs conditions. Cells in sham-exposed conditions were incubated inside the not-energized coils in absence of MFs exposure. The effects of current-induced heating were not observed in the exposure unit (Supplementary Fig. S6). The temperature was measured inside and outside of the exposure unit, and external of the coils by thermometer at sham- and MFs-exposed conditions at different currents of DC and AC.
The exposure system was designed to generate continuously homogeneous either SMF in the range of 0.5 mT to 90 mT or ELF-PEMF at a frequency of 50 Hz in the stable conditions. An electronic board was used to stabilize the exposure system, therefore a SMF or ELF-PEMF were obtained inside of the exposure unit. A Teslameter (13610.93, PHYWE, Gottingen, Germany) with a probe-type of Hall Effect was used to measure MFs, calibrate the system and uniformity of MFs. A Hall Effect sensor is a transducer with the varying output voltage in response to MFs. Presence of any pulsation in the different currents were tested by an oscilloscope (40 MHz, model 8040, Leader Electronics Co., Yokohama, Japan), which resulted from rectifier into MFs exposure system. This pulsation frequency may be related to the shortcoming of single-phase full-wave rectifier used in the experiment, which provided a ripple voltage ~ 5%. However, the ripple voltage is not zero, it is small enough to confirm that generated MFs is highly homogeneous.
As seen in Supplementary Fig. S5c, the homogeneity of generated MFs was theoretically estimated by Complete Technology for 3D Simulation (CST STUDIO 2011 software) to select the best site for our samples within the exposure unit (http://www.CST.com). The profile of field emission in coils is demonstrated with surfaces of the same color. The local value of GMF in our lab was 47 ± 5 µT, according to measurement was performed by Tehran Geomagnetic Observatory, Institute of Geophysics, University of Tehran.
Detection of intracellular ROS generation
Intracellular ROS contents of HEK 293T cells were quantified using 2′,7′-dichlorofluorescein diacetate (DCFDA) assay kit (ab113851, Abcam). For ROS assay, the 10 × 103 cells were seeded per well into 96-well plates by 100 µL supplemented DMEM and prepared for transfection and treatments, respectively. After that, cells were prepared immediately as manufacturer's recommendations. Briefly, the media was removed and cells were washed with PBS. Then, working DCFDA solution (20 µM DCFDA into 1X buffer) was added to each well upon the cells and incubated for 45 min at 37 ºC in the dark. The working solution was freshly made before each trial and did not store. After that, the buffer was completely removed, and rewashed and replaced with PBS. The measurement of ROS levels were monitored immediately by Cytation™ v3.0 Cell Imaging Multi-Mode Reader (BioTek, USA) at excitation / emission wavelengths of 485 / 535 nm.
Cell cycle analysis
Cell cycle distribution of HEK 293T cells was characterized using propidium iodide (PI) staining (Sigma-Aldrich). Briefly, the 150 × 103 cells were seeded per well into 6-well plates by 2 mL supplemented DMEM and prepared for transfection and treatments, respectively. After that for the assay, the medium was initially collected and cells were washed with PBS and trypsinized. Then, cells were rewashed twice with ice-cold PBS and centrifuged at 1200 rpm for 5 min. Supernatant was carefully removed, and cells were fixed by 70% (v/v) cold ethanol (Merck), then incubated at 4 °C overnight. Next, ethanol was removed and cells were rewashed with cold PBS twice, carefully. Then, cells were resuspended in 50 µg/ml RNase-supplemented cold PBS and incubated at 37 °C for 1 h. After that, cell suspension was stained with a 50 µg/mL PI solution in PBS supplemented with 0.1% (v/v) Triton X-100 at room temperature for 1 h in the dark. Finally, cell cycle analysis was quantified immediately by FACSCalibur flow cytometer (Becton-Dickinson, Franklin Lakes, NJ). Data were collected from at least 104 cells and analyzed using FlowJo™ v10.6.2 software (FlowJo LLC, USA).
Cell viability experiments
The cell viability of HEK 293T cells was determined using tetrazolium salt ((3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide [MTT]) (Sigma-Aldrich). For MTT assay, the 10 × 103 cells were seeded per well into 96-well plates by 100 µL supplemented DMEM and prepared for transfection and treatments, respectively. After that, the media was removed and added 100 µL dye solution (0.5 mg/mL in FBS-free DMEM) to each well, and incubated at 37 °C for extra 4 h in the dark. The mitochondrial succinate dehydrogenases are able reduce the MTT to formazan as an indicator of cellular viability. Finally, the supernatants were removed and 100 µL Dimethyl sulfoxide (DMSO) (Daejung Chem., Korea) was added to each well and incubated for dissolve of formazan crystals. Then, the optical density (OD) of each well was quantified by a microplate reader (uQuant MQX200, BioTek, USA) at 570 nm. The percentage of cell viability was calculated based on the intensity of formazan compared to untreated cells (either control or sham) using standard formula as shown in Eq. (1).
Quantification of gene expression
Total cellular RNA was isolated from HEK 293T cells using a RiboEx reagent (GeneAll, Korea) according to the manufacturer’s protocol. Briefly, the 150 × 103 cells were seeded per well into 6-well plates by 2 mL supplemented DMEM and prepared for transfection and treatments, respectively. After that, total RNA was extracted and quantified at 260/230 nm and 260/280 nm ratios indicated the yield of extracted RNA by a UV spectrophotometer (NanoDrop 2000c, Thermo Scientific, USA). RNA integrity was confirmed using an agarose gel electrophoresis. The cDNA synthesis was performed according to the following protocol: (1) DNase-1 treatment at 37 °C for 30 min to avoid genomic DNA contamination then followed by inactivation at 72 °C, (2) For mRNA reverse transcription enhancement, random hexamer and oligo dT mix primers were used, (3) cDNA synthesis was performed using the Prime Script II reverse transcriptase (Takara, Japan) at 42 °C for 70 min followed by RT inactivation at 72 °C for 12 min. Then, quantitative real-time PCR (qRT-PCR) was performed using high ROX SYBR® Green master mix assay kit (Biofact biofactory, Korea) by a StepOne™ Thermocycler (Applied Biosystems, USA). The primers were designed by oligoanalyzer software and, were synthesized by Pishgam Biotech Co. (Tehran, Iran). The quality of the qRT–PCR reactions was confirmed by melting curve analyses. The qRT–PCR data were calculated using the comparative Ct method. The sequences of qRT-PCR forward (Fwd) and reverse (Rev) primers to amplify the fragments of OGG1 (173 bp), MTH1 (180 bp) and ITPA (167 bp) genes were respectively:
OGG1-Rev, 5′-CCGCTCCACCATGCCAGTGATG − 3′;
MTH1-Fwd, 5′-TGGGCCAGATCGTGTTTGAGTTCGT − 3′;
Expression data were normalized to the housekeeping gene of GAPDH as reference gene, which generated a 115 bp fragment to control the variability in expression levels. Reference gene was approximately equal in all experiments. The fold-change values were calculated for each time point.
GraphPad Prism v6.07 (GraphPad Software Inc., San Diego, CA) was used for statistical analysis and data graphing. We were applied one-way, (3 × 2) and (3 × 2 × 2) factorial ANalysis Of VAriance (ANOVA) followed by post-hoc analysis using Newman–Keuls multiple comparison test to compare between independent variables (e.g., cell type, DOX, SMF and EMF) in the ROS production, cell cycle distribution, cell viabilities, and gene expression. The error bars represent the mean ± standard deviation (SD) of at least three independent experiments (n = 3) for all biological-measured parameters in graphs. For all experiments: *p < 0.05; **p < 0.001; ***p < 0.0001, and α,p < 0.05; β,p < 0.01; χ,p < 0.001 were considered as a statistical significance difference.