The data presented in this article are related to the research article entitled “The effects of deoxyelephantopin on the cardiac delayed rectifier potassium channel current (IKr) and human ether-a-go-go-related gene (hERG) expression” (Y.F. Teah, M.A. Abduraman, A. Amanah, M.I. Adenan, S.F. Sulaiman, M.L. Tan) [1], which the possible hERG blocking properties of deoxyelephantopin were investigated. This article describes the construction of human embryonic kidney 293 (HEK293) cells overexpressing HERG potassium channel and verification of the presence of hERG mRNA and protein expression in this recombinant cell line

Abduraman, Muhammad Asyraf

Adenan, Mohd Ilham

Amanah, Azimah

Mei, Lan Tan

Sulaiman, Shaida Fariza

Yi, Fan Teah

Data in Brief

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Contents lists available at ScienceDirectData in BriefData in Brief 14 (2017) 584–591http://d2352-34(http://cDOIn CorrBertam,E-mjournal homepage: www.elsevier.com/locate/dibData ArticleData on the construction of a recombinantHEK293 cell line overexpressing hERG potassiumchannel and examining the presence of hERGmRNA and protein expressionYi Fan Teah a, Muhammad Asyraf Abduraman b,Azimah Amanah a, Mohd Ilham Adenan c,Shaida Fariza Sulaiman d, Mei Lan Tan a,b,na Malaysian Institute of Pharmaceuticals & Nutraceuticals, National Institutes of BiotechnologyMalaysia (NIBM), Ministry of Science, Technology and Innovation Malaysia, Pulau Pinang, Malaysiab Advanced Medical & Dental Institute, Universiti Sains Malaysia, Pulau Pinang, Malaysiac Atta-ur-Rahman Institute for Natural Product Discovery, Universiti Teknologi MARA (UiTM),Selangor Darul Ehsan, Malaysiad School of Biological Sciences, Universiti Sains Malaysia, Pulau Pinang, Malaysiaa r t i c l e i n f oArticle history:Received 7 July 2017Received in revised form31 July 2017Accepted 3 August 2017Available online 12 August 2017Keywords:hERGRecombinant cell linehERG mRNA expressionhERG protein expressionx.doi.org/10.1016/j.dib.2017.08.00809/& 2017 The Authors. Published by Elsevreativecommons.org/licenses/by/4.0/).of original article: http://dx.doi.org/10.1016espondence to: Lifestyle Science Cluster,Kepala Batas, Pulau Pinang, Malaysia. Tel :ail address: tanml@usm.my (M.L. Tan).a b s t r a c tThe data presented in this article are related to the research articleentitled “The effects of deoxyelephantopin on the cardiac delayedrectiﬁer potassium channel current (IKr) and human ether-a-go-go-related gene (hERG) expression” (Y.F. Teah, M.A. Abduraman, A.Amanah, M.I. Adenan, S.F. Sulaiman, M.L. Tan) [1], which the pos-sible hERG blocking properties of deoxyelephantopin were inves-tigated. This article describes the construction of human embryo-nic kidney 293 (HEK293) cells overexpressing HERG potassiumchannel and veriﬁcation of the presence of hERG mRNA and pro-tein expression in this recombinant cell line.& 2017 The Authors. Published by Elsevier Inc. This is an openaccess article under the CC BY license(http://creativecommons.org/licenses/by/4.0/).ier Inc. This is an open access article under the CC BY license/j.fct.2017.07.011Advanced Medical and Dental Institute, Universiti Sains Malaysia, 13200604-5622309, Fax : 604-5622349.Y.F. Teah et al. / Data in Brief 14 (2017) 584–591 585Speciﬁcations TableSMTHDEEDubject area Pharmacology and Toxicologyore speciﬁcsubject areaStable transfection, RT-qPCR and Western Blot analysisype of data Figures, text ﬁleow data wasacquiredCFX96™ Real-Time PCR Detection System (Bio-Rad Laboratories, USA),ChemiDoc™ XRS Imaging System (Bio-Rad Laboratories, USA)ata format AnalyzedxperimentalfactorsTransfected HEK293 cells with pCMV6-Neo-hERG plasmid and non-transfectedHEK293 cells were analyzed using RT-qPCR and Western Blot analysisxperimentalfeaturesThe presence of hERG mRNA and protein expression in HEK 293-hERG cell linewere determinedThe absence of hERG mRNA and protein expression in non-transfected HEK293cells were observedata sourcelocationUniversiti Sains Malaysia, Pulau Pinang, MalaysiaMalaysian Institute of Pharmaceuticals and Nutraceutical, NIBM, Pulau Pinang,Malaysiaata accessibility Data is accessible with this articleDValue of the data The data is beneﬁcial to researchers who are interested in the pharmacology properties of deox-yelephantopin and Elephantopus scaber Linn This data set is beneﬁcial to researchers who want to construct a heterologous mammalian systemexpressing hERG potassium channel The data is helpful to determine the mRNA and protein expression of hERG in cell lines after stabletransfection The data is helpful to ensure that the recombinant cell line (HEK293-hERG) is expressing hERG atboth transcriptional and translational level.1. DataFig. 1 shows the plasmid map of pCMV6-Neo-hERG (Origene, USA). Fig. 2 shows the restrictionenzyme digestion products of the plasmid. Fig. 3 shows the sequence alignment and comparisonbetween the sequences of the cDNA insert against the hERG sequence [NM 000238.3 homo sapienspotassium voltage-gated channel, subfamily H (eag-related), member 2 (KCNH2), transcript variant 1,mRNA]. Fig. 4 shows the melt curves displaying the melting temperature (Tm) as single peaks. Fig. 5shows the standard curve plots for ampliﬁcation efﬁciency for hERG and β-actin. Fig. 6 shows sig-niﬁcant hERG mRNA expressions in different batches of HEK293-hERG cells. Fig. 7 shows a repre-sentative image of the hERG channel protein expression in hERG-transfected and non-transfectedHEK293 cells [1].2. Experimental design, materials and methods2.1. Cell culture and plasmid ampliﬁcationHEK293 cell line was purchased from American Type Culture Collection (ATCC, USA). Cells weremaintained in Dulbecco's Modiﬁed Eagle medium (DMEM) supplemented with 10% (v/v) fetal bovineserum (FBS), 100 U/ml penicillin, 100 µg/ml of streptomycin, 1% (v/v) sodium pyruvate, 1% (v/v)Fig. 1. Plasmid map of pCMV6-Neo-hERG (adapted from Origene Technologies, USA).Fig. 2. Gel electrophoresis of restriction enzyme digestion products of the plasmid map. Lane 1:1 kb DNA ladder; Lane 2: NotIrestricted plasmid sample where 6 kb refers to the fragment of plasmid and 4 kb band refers to the hERG cDNA insert.Y.F. Teah et al. / Data in Brief 14 (2017) 584–591586sodium bicarbonate and 1% (v/v) Minimum Essential Medium (MEM) non-essential amino acid.Cultures were incubated at 37 °C in a humidiﬁed incubator supplemented with 5% (v/v) CO2. Mam-malian expression vector carrying hERG cDNA (pCMV6-Neo-hERG) was purchased from OriGeneTechnologies (USA). The recombinant plasmid map is illustrated in Fig. 1. Ampliﬁcation of thisplasmid DNA was carried out by transformation into the DH5α strain of Escherichia coli bacteria(Invitrogen, USA) using standard laboratory protocol. Extraction of plasmid DNAwas carried out usingPlasmid Midi kit (Qiagen, USA). Digestion of the plasmid was carried out using restriction enzymeFig. 3. Sequence alignment and comparison between the sequences of the cDNA insert against hERG sequence [NM 000238.3homo sapiens potassium voltage-gated channel, subfamily H (eag-related), member 2 (KCNH2), transcript variant 1, mRNA].Y.F. Teah et al. / Data in Brief 14 (2017) 584–591 587NotI (New England Biolabs, USA), where the restriction sites were indicated as illustrated in Fig. 1. Gelelectrophoresis was performed with 0.7% (w/v) agarose gel to conﬁrm the size of plasmid fragmentsafter restriction enzyme digestion. The recombinant plasmid, pCMV6-Neo-hERG was sent for DNAsequencing at Macrogen (Seoul, Korea). The obtained sequence was then analyzed using NucleotideBasic Local Alignment Searching Tool (BLAST) from National Center for Biotechnology Information(NCBI) (http://blast.ncbi.nlm.nih.gov/).2.2. Stable transfectionHEK293 cells were suspended in an appropriate volume of ice-cold Opti-MEM® (Gibco, USA)electroporation buffer. Cell suspension containing approximately 2.5 × 105 cells were aliquoted intopre-chilled electroporation cuvette. An aliquot containing 2.5 µg of pCMV6-Neo-hERG clone (OriGeneTechnologies, USA) was added into the cuvette. Sterile deionized water added into cell suspensionwas used as blank electroporation control. The cuvette was then inserted into the Gene Pulser Xcell™electroporation system (Bio-Rad Laboratories, USA) and electroporation was carried out using singlepulse at 240 V and for 25 ms. The cells were then transferred into a T25 ﬂask containing 5 mL ofcomplete medium and incubated at 37 °C for 48 h. After 48-h maintenance in complete mediumwithout selection antibiotic, both sets of cells (with cDNA clone and blank control) were supple-mented with complete medium containing Geneticin® in increasing concentration over severalweeks. Surviving cells cultured in complete medium containing 500 µg/mL of G418 sulfate wereselected manually using a pipette tip and transferred into 24-well plates. These cells were grownseparately and propagated to ensure monoclonal populations of recombinant cells were obtained.Fig. 4. Melt curves displaying the melting temperature (Tm) as peaks. The Tm for hERG expression was 80.5 °C and the Tm for β-actin expression was 82.0 °C.Y.F. Teah et al. / Data in Brief 14 (2017) 584–5915882.3. Veriﬁcation of hERG mRNA expressionThe mRNA expression of hERG gene in selected stably transfected cells (HEK293-hERG) and thenon-transfected HEK293 cells were determined using RT-qPCR. Total RNA was isolated usingQIAshredder™ and RNeasy® Mini Kit according to the manufacturer's instructions. Quantiﬁcation andpurity of RNA samples was determined using Ultrospec™ 3100 pro UV/Visible spectrophotometer(Amersham Biosciences, U.K.). Primers were designed using Beacon Designer software, Version 7.7(Premier Biosoft International, USA) based on sequence data obtained for hERG (NM_000238.3) andβ-actin (NM_001101.3) from the National Centre of Biotechnology Information (NCBI) database(http://www.ncbi.nlm.nih.gov/) (Table 1). RT-qPCR was carried out using Bio-Rad® CFX™ real timePCR system and iScript™ One-Step RT-PCR kit with SYBR® Green (Bio-Rad Laboratories, USA). Brieﬂy,the RT-qPCR ampliﬁcation was carried out in ﬁnal volume of 25 µL according to manufacturer'sprotocol and program as shown in Table 1. The hERG mRNA expression was determined by analyzingthe threshold cycle (Ct) value for each RNA sample extracted from both transfected HEK293-HERGcells and non-transfected HEK293 cells (control). The data were presented as fold change in geneexpression normalized to an endogenous reference β-actin gene and relative to the non-transfectedcontrol.Fig. 5. Standard curve plots for ampliﬁcation efﬁciency of hERG and β-actin. PCR efﬁciency (E) was determined using CT slopemethod with data points covering a log dilution range. Data was calculated automatically by CFX Manager software version 3.1(Bio-Rad Laboratories, USA). The calculated ampliﬁcation efﬁciency for hERG was 102.3% and that of β-actin was 104.7%.Y.F. Teah et al. / Data in Brief 14 (2017) 584–591 5892.4. Veriﬁcation of hERG protein expressionTotal protein was isolated using Mem-PER™ Plus membrane protein extraction kit (Thermo-Scientiﬁc, USA) and was puriﬁed using Pierce® SDS-PAGE Sample Prep Kit (ThermoScientiﬁc, USA)according to the manufacturer's protocol. Protein concentration was measured using Biorad DC Pro-tein Assay and Protein Standard II BSA (Bio-Rad Laboratories, USA). Total membrane protein wasseparated using SDS-PAGE with 4% (v/v) stacking gel and 10% (v/v) resolving gel. Brieﬂy, fractionatedproteins were ﬁrst transferred to Immobilon-P PVDF membrane (Milipore, USA) and blocked with 3%(w/v) skim milk for 1 h and incubated with optimized primary antibodies against hERG [1:250; Santa-Cruz Biotechnology (USA)] and β-actin [1:1000; Cell Signaling Technology (USA)] overnight at 4°C.The membrane was then incubated in horseradish peroxidase conjugated secondary antibodies [CellSignaling Technology (USA)] for 2.5 h followed by ECL™ Western Blotting Detection Reagents (GEHealthcare, UK). Chemiluminescence signal was analyzed using ChemiDoc™ XRS Imaging System(BioRad Laboratories, USA).Fig. 7. A representative image showing the Western blot analysis of the hERG protein expression in hERG-transfected and non-transfected HEK293 cells.Fig. 6. Graphical representation of the hERG mRNA expressions in HEK293 and HEK293-hERG cell line. Data are represented asthe means 7 SD from 2 independent experiments (n¼6).Table 1Primer sequence, RT-PCR composition and thermal cycler program.PrimersGene accession no. Sequence Amplicon size (bp) Optimized annealingtemperature (°C)hERG (NM_000238.3) F 5′-AAATCACCCTCAACTTTG-3′ 96 53R 5′-TTCGCTCCTTTATCTTAG-3′β-actin (NM_001101.3) F 5′- ATCACCATTGGCAATGAG-3′ 105R 5′-GATGGAGTTGAAGGTAGTT-3′Reaction MixtureReagent Final concentration Volume (μl)2 X SYBR Green RT-PCR Reaction Mix 1 X 12.510 µM forward primer 300 nM 0.7510 µM reverse primer 300 nM 0.75Nuclease-free water - 9.5iScript™ Reverse Transcriptase - 0.5RNA template 100 ng 1Total volume 25ProgramRT-PCR cycle Steps Temperature and durationReverse transcription cDNA synthesis 50.0 °C for 10 minInactivation of reverse transcriptase 95.0 °C for 5 minPCR cycling and detection (40 cycles) Denaturation 95.0 °C for 10 sAnnealing & extension Gradient for 30 sMelt curve analysis (60 cycles) 65 °C to 95 °C:Increment 0.5 °C for 5 sHold 10.0 °C ∞Y.F. Teah et al. / Data in Brief 14 (2017) 584–591590Y.F. Teah et al. / Data in Brief 14 (2017) 584–591 591AcknowledgementsThis study was supported by the Sciencefund Grant (02–05-23-SF0002) from the Ministry ofScience, Technology and Innovation Malaysia (MOSTI).Transparency document. Supporting informationTransparency data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.dib.2017.08.008.Reference[1] Y.F. Teah, M.A. Abduraman, A. Amanah, M.I. Adenan, S.F. Sulaiman, M.L. Tan, The effects of deoxyelephantopin on the cardiacdelayed rectiﬁer potassium channel current (IKr) and human ether-a-go-go-related gene (hERG) expression, Food Chem.Toxicol. 107 (2017) 293–301.

Data on the construction of a recombinant HEK293 cell line overexpressing hERG potassium channel and examining the presence of hERG mRNA and protein expression

Data on the construction of a recombinant HEK293 cell line overexpressing hERG potassium channel and examining the presence of hERG mRNA and protein expression

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