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Modification of the original vector pcDNA5/FRT

To match the introduced frame shift between the promoter and the FRT site, the original Flp-In vector pcDNA5/FRT (Thermo Fisher Scientific, Darmstadt, Germany) was modified by deletion of one cytosine (C1590del) and termed pcDNA5/FRThygro-fs here. This modification was achieved by site-directed mutagenesis26. For this, 1.0 µL (50 ng) of the pcDNA5/FRT vector was mixed with 5 µL Q-Solution (Qiagen, Hilden, Germany), 2.5 µL 10 × KOD buffer (KOD Hot Start DNA Polymerase Kit; Merck, Darmstadt, Germany), 2.5 µL dNTPs (2 mM each), 1 µL MgSO4 (25 mM), 0.65 µL forward primer SDM_hygro_fwd (5′-GTATAGGAACTTCCTTGGCAAAAAGCCTGAACTCACC-3′), 0.65 µL reverse primer SDM_hygro_rev (5′-GGTGAGTTCAGGCTTTTTGCCAAGGAAGTTCCTATAC-3′), 0.5 µL HotStart KOD Polymerase and 11.2 µL H2O, and PCR was carried out at 95 °C for 3 min, followed by 20 cycles of 95 °C for 30 s, 63 °C for 45 s, and 72 °C for 4 min. The product was digested with DpnI. For this, 25 µL of the PCR product were mixed with 3 µL cut smart buffer (New England Biolabs, Ipswich, USA) and 1.5 µL DpnI (20 units/mL; New England Biolabs) and incubated at 37 °C for 1 h. After this, further 1 µL DpnI was added and the mixture was incubated at 37 °C for one more hour. The product was dialyzed and transformed into E.coli using an Electroporator Gene Pulser II (Bio-Rad Laboratories, Hercules, California, USA) for clonal amplification prior to sequence validation.

Generation of second vector (pcDNA5/FRTpuro)

The second vector was built to include the same promoter as in the Flp-In host cell line (from vector pcDNA5/lacZeo, Thermo Fisher Scientific) including the FRT site, the puromycin resistance gene, and the backbone of the regular pcDNA5/FRT vector. To distinguish the two vectors by PCR, we also introduced a unique sequence, at which primers could bind for the eventual validation. For cloning, the three different fragments were generated by PCR using hybrid primers to generate overlapping amplicons (Fig. S1). The fragments of the puromycin resistance gene and the SV40 promoter/FRT region were then fused by overlap-extension PCR27 and recombined with the pcDNA5 backbone by sequence and ligation independent cloning28,29,30.

To amplify the pcDNA5 backbone, 10 µL Q-solution (Qiagen, Hilden, Germany), 5 µL 10 × KOD buffer (KOD Hot Start DNA Polymerase Kit; Merck, Darmstadt, Germany), 5 µL dNTPs (2 mM each), 3 µL MgSO4 (25 mM), 1.5 µL forward primer PuroR_p5_fwd (5′-cacgaccccatgGGCTGGATGATCCTCCAGCG-3′), 1.5 µL reverse primer SV40/FRT_p5_rev (5′-gacacgtacgtacgtGGCGAACGTGGCGAGAAAGG-3′), 1 µL HotStart KOD polymerase, 1.5 µL pcDNA5/FRT DNA (100 ng) and 21.5 µL H2O were mixed and PCR was carried out at 95 °C for 2 min, followed by 35 cycles of 95 °C for 30 s, 68.1 °C for 30 s, 72 °C for 5 min and completed at 72 °C for 10 min. The PCR product was DpnI digested to remove template DNA interfering with subsequent cloning steps as described above. The PCR for the amplification of the SV40 promoter region and the FRT site was composed of 10 µL Q-solution, 5 µL 10 × KOD buffer, 5  µL dNTPs (2 mM each), 3  µL MgSO4 (25 mM), 1.5 µL forward primer p5_SV40/FRT_fwd (5′-gccacgtacgtacgtGTCAGTTAGGGTGTGGAAAG-3′), 1.5 µL reverse primer PuroR_SV40/FRT_rev (5′-tcggtggccaagGAAGTTCCTATACTTTCTAGAG-3′), 1 µL HotStart KOD polymerase, 1.5 µL pcDNA5/lacZeo DNA (100 ng) and 21.5 µL H2O and carried out at 95 °C for 2 min, followed by 35 cycles of 95 °C for 30 s, 64.9 °C for 30 s, 72 °C for 1 min and completed at 72 °C for 10 min. To amplify the puromycin resistance gene, 10 µL Q-solution, 5 µL 10 × KOD buffer, 5 µL dNTPs (2 mM each), 3 µL MgSO4 (25 mM), 1.5 µL forward primer SV40/FRT_PuroR_fwd (5′-ttccttggccACCGAGTACAAGCCCACGG-3′) , 1.5 µL reverse primer p5_PuroR_rev (5′-tcatccagccCATGGGGTCGTGCGCTCC-3′) , 1 µL HotStart KOD polymerase, 1.5 µL of a puromycin resistance gene-containing vector (100 ng) and 21.5 µL H2O were mixed and PCR was carried out at 95 °C for 2 min, followed by 35 cycles of 95 °C for 30 s, 68.1 °C for 30 s, 72 °C for 2 min and completed at 72 °C for 10 min.

For the recombination of the ‘SV40 promoter-FRT’ region and the puromycin resistance gene, overlap-extension PCR composed of 10 µL Q-Solution, 5 µL 10 × KOD buffer, 5 µL dNTPs (2 mM each), 3 µL MgSO4 (25 mM), 5 µL purified PCR product of SV40/FRT PCR (60 ng/µL), 5 µL purified PCR product of puromycin resistance gene (109 ng/µL; equimolar ratio), 1 µL HotStart KOD polymerase and 17 µL H2O was performed at 95 °C for 2 min, followed by 35 cycles of 95 °C for 30 s, 50 °C for 45 s, 72 °C for 2 min and completed at 72 °C for 10 min. Subsequently, 2 µL of the PCR product were mixed with 10 µL Q-Solution, 5 µL 10 × KOD buffer, 5 µL dNTPs (2 mM each), 3 µL MgSO4 (25 mM), 1.3 µL forward primer p5_SV40/FRT_fwd (5′-gccacgtacgtacgtGTCAGTTAGGGTGTGGAAAG-3′), 1.3 µL reverse primer p5_PuroR_rev (5′-tcatccagccCATGGGGTCGTGCGCTCC-3′), 1 µL HotStart KOD polymerase and 22.4 µL H2O and incubated at 95 °C for 2 min, followed by 35 cycles of 95 °C for 30 s, 50 °C for 45 s, 72 °C for 3 min and completed at 72 °C for 10 min.

The final vector was recombined by sequence and ligation independent cloning28,29,30. For this, 6 µL of the purified pcDNA5/FRT backbone (60 ng) were mixed with 1.44 µL purified product of the overlap-extension PCR (1:3 vector to insert molar ratio), 1 µL 10 × BSA (New England Biolabs, Ipswich, USA), 1 µL 10 × NEB Buffer 2.1 (New England Biolabs), 0.56 µL H2O and 0.5 µL T4 DNA polymerase (3 units/µL; New England Biolabs), incubated at 50 °C for 40 s and subsequently kept on ice for 10 min. After this, dialysis was performed and the dialyzed product was electroporated into One Shot TOP10 Electrocomp E. coli. Obtained bacterial clones were screened via colony PCR and correct assembly of the plasmid was confirmed by sequencing.

The created plasmid pcDNA5/FRTpuro was modified by site-directed mutagenesis to correct the FRT site, since the FRT site present in the provided pcDNA5/lacZeo differs in one nucleotide from the one present in the original pcDNA5/FRT vector, and to introduce a frame shift (C1757ins) matching the reading frame of the pcDNA5/FRThygro-fs vector. For this, 0.6 µL (50 ng) of the pcDNA5/FRTpuro vector were mixed with 5 µL Q-Solution, 2.5 µL 10 × KOD buffer, 2.5 µL dNTPs (2 mM each), 1 µL MgSO4 (25 mM), 0.65 µL forward primer SDM_puro_fwd (5′-CATGGCAGAAGTTCCTATTCCGAAGTTCC-3′), 0.65 µL reverse primer SDM_puro_rev (5′-AATAGGAACTTCTGCCATGGTAGCCTCC-3′), 0.5 µL HotStart KOD Polymerase and 11.2 µL H2O and PCR was carried out at 95 °C for 3 min, followed by 20 cycles of 95 °C for 30 s, 58.1 °C for 45 s, 72 °C for 3 min and completed at 72 °C for 10 min. The product was DpnI digested as described above and transformed into E.coli prior to sequence validation. The obtained vector was termed pcDNA5/FRTpuro-fs hereafter.

Cloning CYP2C19 from human liver RNA

Human liver total RNA (TaKaRa Bio, Kusatsu, Japan) was used to clone CYP2C19. For this, 1 µL RNA (1 µg) was diluted by adding 16.75 µL RNase-free water, mixed with 1 µL gene-specific primers (5′-GAGGAAAGAGAGCTGCAGGG-3′), incubated at 72 °C for 10 min and subsequently cooled down to room temperature. After this, 6 µL 5 × RT buffer (SuperScript II Reverse Transcriptase Kit; Thermo Fisher Scientific, Darmstadt, Germany), 3.5 µL DTT (0.1 M), 1 µL dNTPs (2 mM each), 0.5 µL RNase inhibitor (40 U/µL) and 0.25 µL SuperScript II Reverse Transcriptase (200 U/µL) were added and reverse transcription was carried out at 42 °C for 1 h before temperature was raised to 75 °C for 15 min. Synthesized cDNA was amplified with primers containing restriction sites for eventual cloning with HindIII and EcoRV (see Table 1). The PCR for the amplification was composed of 10 µL Q-solution, 5 µL 10 × KOD buffer, 5 µL dNTPs (2 mM each), 2 µL MgSO4 (25 mM), 1.3 µL forward primer CYP2C19_HindIII_fwd (5′-AAGAGGAGaagcttACCATGGATCCTTTTGTGGTCCTTG-3′), 1.3 µL reverse primer CYP2C19_EcoRV_rev (5′-CATCTGTgatatcTCAGACAGGAATGAAGCACAGC-3′), 1 µL HotStart KOD polymerase, 2.0 µL cDNA template and 22.4 µL H2O and carried out at 95 °C for 3 min, followed by 35 cycles of 95 °C for 30 s, 61 °C for 30 s, 72 °C for 2:30 min and completed at 72 °C for 10 min. After amplification, the CYP2C19 was subcloned by TOPO cloning using the TOPO XL PCR Cloning Kit (Thermo Fisher Scientific, Darmstadt, Germany) following the instructions of the manufacturer. Sequencing revealed two single nucleotide polymorphisms: c.99C>T (rs17885098; synonymous) and c.991A>G (rs3758581, p.ile331val). Both polymorphisms define the *1B variant of CYP2C19, which is not associated with changes in its in vitro catalytic activity31 and additionally the most abundant variant in the global population32,33,34. It was therefore used for further cloning into the expression vectors and for performing transport and metabolism experiments.

Table 1 Primers used for PCR.

Transfection protocol

Generally, cell lines used in this study were cultivated in DMEM culture medium (Thermo Fisher Scientific, Darmstadt, Germany) which was supplemented with 10% fetal calf serum (FCS; Thermo Fisher Scientific, Darmstadt, Germany) and penicillin/streptomycin (100 units/mL, 100 µg/mL; Thermo Fisher Scientific, Darmstadt, Germany) unless explicitly described differently. Cells were cultured at 37 °C in a humidified atmosphere (5% CO2, 95% relative humidity). All experiments were carried in HEK293 T-REx cells (Thermo Fisher Scientific, Darmstadt, Germany).

For transfection of HEK293 T-REx cells, one million cells were plated for each transfection in a 6-well plate 24 h in advance. On the day of transfection, for double transfections, 200 ng of each expression plasmid in midi-prep quality were mixed with 3.6 µg pOG44 encoding the Flp recombinase in 100 µL DMEM. For single transfections, 400 ng of expression plasmid was mixed with 3.6 µg pOG44 encoding the Flp recombinase in 100 µL DMEM. Additionally, 12 µL FuGene 6 transfection reagent (Promega Corporation, Walldorf, Germany), were added to 100 µL DMEM. After an incubation period of 5 min at room temperature, both solutions were mixed by repetitive pipetting and incubated for another 15 min at room temperature. In the meantime, the cells were washed once with 2 mL DMEM with 10% FCS and 1.8 mL fresh DMEM with 10% FCS were added to the cells. After incubation, the 200 µL DNA-FuGene 6 was pipetted dropwise onto the cells. After 24 h, the cell culture medium was replaced by DMEM with 10% FCS and penicillin–streptomycin. On the next day (48 h post transfection), cells were transferred to a 100 mm petri dish. After 24 h, the selection was performed by adding hygromycin B (final concentration 200 µg/mL; Thermo Fisher Scientific, Darmstadt, Germany) and puromycin (final concentration 0.25 µg/mL; Thermo Fisher Scientific, Darmstadt, Germany). Five days later, the supernatant was replaced by fresh cell culture medium containing hygromycin and puromycin. Around ten days after selection started, single colonies were picked. For this, cells were washed once with culture medium to remove dead cells and finally the medium was completely removed. Single colonies were resuspended in 2 µL medium and transferred to a 24-well-plate, where 1 mL culture medium with a reduced concentration of the selection antibiotics (hygromycin: 50 µg/mL, puromycin: 0.025 µg/mL) had been placed in advance. After reaching a confluence of 70–80%, cells were transferred to a 6 well plate and later to a T25 culture flask. When cells were passaged the first time, 40% of cells were used to prepare cell pellets for DNA and RNA extraction each, to allow validation of generated cell lines on genomic as well as on transcriptional level. Transfections were usually carried out in duplicates to ensure a sufficient number of clones to analyze.

The same protocol was independently performed in the Institute of Pharmacology and Toxicology at the Justus Liebig University in Giessen to stably transfect two versions of the sodium/bile acid transporter (NTCP) tagged with yellow or cyan fluorescent protein (NTCP-YFP, NTCP-CFP).

Validation of correct integration by PCR

The stable, genomic integration of both plasmids was validated by PCR. For this, the genomic DNA of 2 × 106 cells was isolated using the QIAGEN DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany) in a QIACube robot (Qiagen) following the manufacturer’s protocol. The isolated DNA was analyzed by multiplex PCR using the QIAGen Multiplex PCR Kit (Qiagen). This reaction covered the Integration PCR 2 as well as the Multiple Integration PCRs A-C (for detailed primer information see Table 1). Multiplex PCR was composed of 5 µL 2 × QIAGEN Multiplex PCR Master mix, 2 µL Q-Solution, 1 µL 10 × primer mix (2 µM each), 1 µL genomic DNA (100 ng) and 1 µL H2O. PCR was carried out at 95 °C for 15 min, followed by 35 cycles of 95 °C for 30 s, 62.7 °C for 90 s, 72 °C for 90 s and completed at 72 °C for 10 min. As a positive control, the genomic DNA of a cell clone validated for all types of multiple integrations was used. Cell clones passing the multiplex PCR screening were validated again by single PCRs (Integration PCRs 1 and 2, Multiple Integration PCRs A-C) composed of 5 µL 2 × QIAGEN Multiplex PCR Master mix, 2 µL Q-Solution, 0.25 µL forward primer (10 µM), 0.25 µL reverse primer (10 µM), 1 µL genomic DNA (100 ng) and 1.5 µL ddH2O using the same thermocycler conditions.

Sanger sequencing was performed on the products including the FRT sites. For the third FRT site, the primers PFRT_f and PLacZ (5′-CCTTCCTGTAGCCAGCTTTCATCAA-3′) under the same conditions as mentioned for single PCRs above. The resulting amplicons were separated on a 0.8% agarose gel, cut out and extracted using the QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany). For sanger sequencing, 100 ng DNA were premixed with 30 pmol of one of the primers used for amplification and sent to external sequencing by Microsynth Seqlab, Göttingen.

Tracking of eGFP/tdTomato double-transfected cells by flow cytometry

The stability of stable transfection was tracked by analyzing fluorescence signals of eGFP/tdTomato double-transfected cell lines over 30 passages. Two independently generated cell lines were cultured in parallel in DMEM cell culture medium with or without the culturing concentrations of hygromycin (50 µg/mL) and puromycin (0.025 µg/mL). Every second passage, cells were analyzed using a LSR II (BD Bioscience, Heidelberg, Germany) flow cytometer and the software BD FACSDiva (Version 6.1.3, BD Bioscience). Fluorescence intensities of green channel (laser excitation wavelength 488 nm) and red channel (laser excitation wavelength 561 nm) were plotted to determine double-positive cells. Thresholds for classifying cells as positive were set by comparing the fluorophore expressing cells to a mock-transfected control cell line.

Expression analyses

The RNeasy Plus Mini Kit (Qiagen, Hilden, Germany) was used according to the manufacturer’s instructions for total RNA isolation. Briefly, 1 to 2 × 106 cells were harvested by centrifugation at 500×g for 5 min at RT. The pellet was dissolved in 350 µL of RLT buffer supplemented with 1% β-mercaptoethanol (v/v). The automatic isolation was performed using a QIAcube (Qiagen, Hilden, Germany), in which the genomic DNA eliminator spin column removed the genomic DNA and total RNA was eluted in 50 µL of RNAse free ddH2O.

The cDNA synthesis from isolated RNA was performed using the SuperScript II Reverse Transcriptase Kit (Thermo Fisher Scientific, Darmstadt, Germany). Three µg RNA was diluted in 17.75 µL of RNAse free ddH2O. Primer annealing was initiated with the addition of 1 µL anchored-dT primer (10 µM) and incubation at 70 °C for 10 min. To initiate cDNA synthesis, 11.25 µL of a reverse transcription reaction mix [6 µl 5 × Superscript RT buffer, 3.5 µL DTT (0.1 M), 1 µL dNTPs (10 mM), 0.5 µL RNase Inhibitor P/N (40 U/µL), 0.25 µL SuperScript II Reverse Transcriptase (200 U/µL)] were added and incubated at 42 °C for one hour. Afterward, enzyme denaturation was done by increasing the temperature to 75 °C for 15 min. To this 30 µL synthesized cDNA, 70 µL of RNAse free ddH2O were added and concentration was further adjusted to 3 ng/µL cDNA by 1:10 dilution.

HOT FIREPol EvaGreen qPCR Mix Plus (ROX) kit (Solis BioDyne, Tartu, Estonia) was used to perform the real-time qPCR. Briefly, the reaction mixture constituted 2 µL 5 × EvaGreen qPCR Mix, 5.6 µL ddH2O, 0.4 µL primer mix (10 µM each; HPRT1: forward (5′-TGACACTGGCAAAACAATGCA-3′), reverse (5′-GGTCCTTTTCACCAGCAAGCT-3′); OCT1: forward (5′-TGTCACCGAAAAGCTGAGCC-3′), reverse (5′-TCCGTGAACCACAGGTACATC-3′), CYP2C19: forward (5′-CCTGATCAAAATGGAGAAGGAAAAG-3′), reverse (5′-TCTGTCCCAGCTCCAAGTAAG-3′)), 2 µL cDNA (6 ng total). Standard curve analysis for each primer pair was performed to check the primer efficiency and amplification of a single specific amplicon. To do so, five concentrations of a cDNA pool in a 1:5 dilution series were distributed in a 384 well-plate and amplification was performed in TaqMan 7900HT (Applied Biosystems, Darmstadt, Germany) machine. SDS 1.2 software (Applied Biosystems, Darmstadt, Germany) was used to identify the Cycle threshold (Ct) value. The Primer efficiency was well within the accepted range, namely 107% (HPRT1), 101% (OCT1), and 99% (CYP2C19)35. Subsequently, expression levels of OCT1 and CYP2C19 genes, along with the housekeeping gene HPRT1, were measured in technical and biological triplicate manner. The ΔΔCt method was used for expression analysis36. Relative expression against single transfected OCT1 and CYP2C19 cell lines were calculated based on this equation:

$${text{Relative}};{text{ expression }} = { 2}^{{ – left[ {({text{Ct}};{text{ experimental }}{-}{text{ Ct }};{text{housekeeping }};{text{experimental}}) , {-} , ({text{Ct }};;{text{control }}{-}{text{ Ct }};{text{housekeeping }};{text{control}})} right]}} = { 2}^{{ – [Delta Delta {text{Ct}}]}}$$

Transport experiments

Functional validation of the stably integrated genes was performed via transport and metabolism of proguanil. Two days ahead of the transport experiment, 600,000 cells were plated in poly-d-lysine pre-coated 12-well-plates. On the day of experiment, cells were washed once with 2 mL 37 °C pre-warmed Hanks buffered saline solution (Thermo Fisher Scientific, Darmstadt, Germany) containing 10 mM HEPES (Sigma-Aldrich, Taufkirchen, Germany; from here on named HBSS+) followed by incubation with HBSS+ containing 1 μM Proguanil (Sigma-Aldrich, Taufkirchen, Germany). After 2, 5, 15, 30 and 60 min, the incubation was stopped by collecting the supernatant and cells were immediately washed twice with 1 mL ice-cold HBSS+ and cells were lysed by adding 500 μL lysis buffer [acetonitrile:water 4:1 (v/v)] containing 10 ng/μL proguanil-d6 (Toronto Research Chemicals, North York, Canada) and 10 ng/μL desvenlafaxine (Sigma-Aldrich, Taufkirchen, Germany) as an internal standard for mass spectrometry analysis. For sample preparation, cell lysates were centrifuged in a desktop centrifuge at maximum speed for 15 min, 400 μL were transferred into a collection plate, evaporated at 40 °C under nitrogen flow and the dry residues were dissolved in 250 μL 0.1% formic acid. For the processing of cell supernatant, samples were centrifuged at 400×g for 5 min. After this, 400 μL was transferred to a new reaction tube and 800 μL of precipitation reagent [acetonitrile:methanol 10:1 (v/v) containing the same internal standards as described above] were added and the mixture was incubated for 15 min on a rotation shaker. Precipitated protein was pelleted by centrifugation at full-speed in a desktop centrifuge for 15 min, 800 μL of the supernatant was used for evaporation and dry residues were dissolved in 250 μL 0.1% formic acid for analysis as well.

LC–MS/MS determination of proguanil transport and metabolism

Concentrations of proguanil and cycloguanil were quantified by high-performance liquid chromatography coupled to mass spectrometry. For sample separation, we used a Shimadzu Nexera 2 UHPLC system containing an auto-sampler SIL-30AC, a communications bus module CBM-20A, a liquid chromatograph LC-30AD, a column oven CTO-20AC and a Brownlee SPP RP-Amide column (4.6 × 100 mm inner dimension with 2.7 μm particle size) with a C18 pre-column. For chromatography, an aqueous mobile phase containing 20% organic additive (acetonitrile:methanol 6:1 (v/v)) was used with a flow gradient starting with 0.3 mL/min for the first 4.5 min, increased to 0.7 mL/min at 4.6 min and back to 0.3 mL/min from 9.0 to 9.1 min, which was left for another two minutes to reconstitute the original conditions for the next measurement. The HPLC system was coupled with an API 4000 tandem mass spectrometer (AB SCIEX, Darmstadt, Germany), which enabled the detection of substrates via specific LC retention times and mass transitions in MRM mode with parameters given in Table S1. The quantification was performed by integration of the peak areas using the Analyst software (Version 1.6.2, AB SCIEX, Darmstadt, Germany). Concentrations of proguanil and cycloguanil (Santa Cruz Biotechnology, Heidelberg, Germany) were determined by simultaneous measurement of a standard curve with known concentrations. To calculate the net uptake of proguanil, the measurement of an empty vector-transfected cell line was subtracted from the other cell lines to take passive diffusion and endogenous transporters into account.

Total protein quantification

Results of cellular transport experiments were normalized to the total amount of protein per well to compensate differences in cell density. For this, the total protein of one well per cell line in each transport experiment was quantified using a BCA assay. Cells were lysed by incubation with 500 μL RIPA buffer for 10 min. Five microliter of each sample were incubated after adding 200 μL bicinchoninic acid with 0.008% copper sulfate at 37 °C for 30 min. Subsequently, the absorbance at a wavelength of 570 nm was measured using a Tecan Ultra microplate reader (Tecan Group, Männedorf, Switzerland). The protein concentration was quantified by comparison to a standard curve using bovine serum albumin.

Tracking of double-transfected cells by microscopy

HEK293-Flp-In cells stably transfected by our Double-Flp-In Method with NTCP-CFP and NTCP-YFP, a classical Foerster Resonance Energy Transfer (FRET) pair, were seeded onto IBIDI chamber-slides to reach confluency at the day of microscopy. For comparison HEK293-MSR cells were seeded like above and were transiently transfected with Lipofectamine 2000 (Thermo Fisher Scientific, Darmstadt, Germany) with an equimolar number of premixed plasmids of pcDNA5 vectors coding for NTCP-CFP or NTCP-YFP both under the control of the identical CMV-promoter, which is also applied by the Double-Flp-In Method. After 48 h of standard incubation, slides were washed twice with PBS and transferred to microscopy at room temperature covered in PBS. Images were taken with a Leica DMI6000 B inverted fluorescent microscope at 40× objective magnification. For qualitative analysis and comparison of expression levels and patterns of the fluorescent proteins CFP and YFP channels were adjusted to yield similar signal intensities. Phase contrast channel was applied to demonstrate the confluency of the cell layer and transfection rates. Staining of the cell nuclei and the fixation of the cells was deliberately avoided to not interfere with CFP and YFP signals.

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