The cell lines used in this study, Huh-7 and Huh-7/E1E2 were kindly provided by Dr. Zafar Nawaz (University of Miami, USA) and Dr. Muhammad Idrees (Incharge Molecular Virology Division, CEMB, university of the Punjab, Lahore, Pakistan respectively. The Huh-7/E1E2 cell line was derived from the parental Huh-7 cell line. The Huh-7/E1E2 cell line was a stable clone of Huh-7 cells transfected with and stably over-expressing HCV Proteins E1E2 (derived from local HCV isolates of genotype 3a) linked to the antibiotic selection marker G418 which was under the control of human cytomegalovirus promoter 28 Huh-7 cell line was cultured in Dulbecco Modified Eagle's Medium (DMEM) supplemented with 100 U/ml of penicillin and 100 ug/ml of streptomycin and 10% heat inactivated fetal bovine serum (complete DMEM medium). Huh-7/E1E2 cell line was maintained in complete medium containing 500 μg/ml G418. The medium was replaced every third day, and cells were passaged every 4-5 days. All cells were maintained at 37°C in a humidified environment containing 5% CO2 in a cell culture incubator.

Total RNA from Huh-7 and Huh-7/E1E2 cells was isolated using Gentra RNA Isolation Kit (Puregene, Minneapolis, MN 55441, USA), following the manufacturer's instructions. Each sample of isolated RNA was further treated with DNase (Fermentas) to remove any residual DNA present that could generate false -positive results. Quantity and quality of extracted RNA was assessed using NanoDrop^® (Spectrophotometer) (ND-1000). Quality of extracted RNA was also checked on agarose gel electrophoresis using 1% agarose gel, stained with ethidium bromide and photographed. To investigate the presence of isoforms of P2X receptors cDNA was synthesized with 1 μg and 5 μg of total isolated RNA using RevertAid™ H minus First Strand cDNA synthesis kit (Fermentas, Cat no.K1632). All the tubes, pipette tips and containers used in the RNA isolation and cDNA synthesis were either manufacturer certified RNAase free or were made RNAase free by treating with 1% DEPC treated water and were autoclaved before use.

Independent sense and antisense primers were designed for P2X6 from known cloned receptor sequences, available in GenBank using primer 3 software, and were synthesized by the core synthesis facility of the centre (CEMB). Previously published primer sequences were used for the remaining receptors, P2X1, P2X2, P2X3, P2X4, P2X5, and P2X6. Primer sequences are shown in Table 1. PCR conditions for amplification of P2X2, P2X3 and P2X5 were 90°C (10 min), 35 cycles [95°C (45 s), 60°C (45 s) and 72°C (45 s)], and 72°C (10 min), PCR conditions for P2X4 were 94°C for 45 s 35 cycles [94°C (1 min), 60°C (45 s) and 72°C (1 min)], and 72°C 10 min. PCR amplifications were repeated using 10 separate preparations of synthesized cDNA, from 10 separate experiments to confirm the consistency of results obtained. Two different concentrations of cDNA (1 μg & 5 μg) were used for optimization of PCR amplification, so that if any isoform has a faint expression in Huh-7 cell, then could also be detected at 5 μg/μl cDNA concentration. Amplification products were separated by gel electrophoresis (2% agarose) and visualized with ethidium bromide staining. PCR products were extracted by using a QIAEX II Gel Extraction Kit (Qiagen), and sequenced by using an automated sequencer to confirm their identities.

Real time qRT-PCR was performed to examine the mRNA expression of identified isoforms of P2X receptors in Huh-7/E1E2 cell line compared to wild type/parental Huh-7 cells as control. Equal numbers of cells (3 × 10⁵/culturing flask) of both cell lines were plated in 25 cm² culturing flasks (6culturing flasks, 1 for each cell line in 3 separate studies) at the same time and kept at 37°C incubator in humid air with 5% CO2. On 4 day, media was removed; cells were washed with sterile 1X PBS (Phosphate Buffer Saline), trypsinized using 0.5% trypsin in EDTA. The RNA extractions and cDNA symthesis were done Gentra RNA Isolation Kit (Puregene, Minneapolis, MN 55441, USA) and RevertAid™ H minus First Strand cDNA synthesis kit (Fermentas, Cat no.K1632) respectively as per manufacturer's instructions.

Quantification of P2X receptors RNAs was performed by Real Time PCR Cepheid smart cycler II (France). The relative levels of P2X2, P2X3, P2X4, P2X5, P2X6 genes were determined using GAPDH mRNA for normalization. The calculation was based on the Δ-Ct (Threshold cycle number difference between control, Huh-7, and Huh-7/E1E2 cells), and the ratios were normalized to the ratios of GAPDH of the corresponding samples. Some isoforms were unresponsive to HCV structural proteins E1E2, while some were responsive. The most responsive isoform was P2X4.

All statistical analysis was performed with GraphPad Prism 5 software Version 5.02. Data are presented as mean ± SEM. Comparisons between parameters were performed using ANOVA followed by Bonferroni or unpaired t test. A p-value of less than or equal to 0.05 was considered statistically significant.

To evaluate whether transcripts for P2X receptors are present in human liver cells, total cellular RNA was extracted from human hepatoma cell line (Huh-7) and was analyzed by RT PCR using primers specific for P2X1 through P2X7 (Table 1). Amplification of GAPDH mRNA served as an internal control. In whole liver, which is composed primarily of hepatocytes but also includes multiple additional cell types, mRNA for different isoforms of P2X receptors, P2X2, P2X3, P2X4, P2X5 and P2X6 were detected by RT- PCR in 10 separate studies. All studies were performed under identical culture conditions using cells from passage 5-15 and capable of forming differentiated monolayers. Figure 1 shows the detection of transcripts of different isoforms of P2X receptors in Huh-7 cell line.

Identification of amplified transcripts was confirmed by sequencing PCR. The purified DNA of P2X2, P2X3, P2X4, P2X5 and P2X6 were used as templates for sequencing PCR in the Big-Dye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems). Samples were analyzed on an automated sequencer (ABI PRISM 3100 genetic analyzer; Applied Biosystems). Products were sequenced from both strands to confirm their identity with reference sequences.

Sequences of the sequenced transcripts of P2X receptors were searched for homology with other sequences in GeneBank using Blast2 (Basic Local Alignment Search Tool), at http://www.ncbi.nlm.nih.gov/BLAST/ Sequences of amplified P2X receptors showed 96-100% homology with reported sequences (GenBank accession numbers, HQ323686, JF807485).

To examine the mRNA expression of identified isoforms of P2X receptors in presence of HCV structural proteins E1E2, Real time qRT-PCR was performed. A significant increase (6.2 fold) was observed in the gene expression of P2X4 receptor in Huh-7/E1E2 cells (stably expressing HCV structural protein E1E2) in comparision with control Huh-7 cells (Figure 2). Whereas expression of P2X5 receptor decreased significantly in Huh-7/E1E2 cells (Figure 2). However, P2X2, P2X3 andP2X6 were unresponsive to E1E2.