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Farghaly et al. Hepatoma Res 2018;4:41 I http://dx.doi.org/10.20517/2394-5079.2018.30 Page 3 of 10

HCV genotype 4. The potential targeted gene by activated Alu molecule has been detected using qRT-PCR
and flowcytometry. HCV replication in treated cells has been monitored to figure out the inhibitory effects
of HS on viral replication.

METHODS
HepG2 cell line
HepG2 cells were obtained from VACSERA, Giza, Egypt and were propagated in order to obtain increasing
numbers of cells for further investigations. Propagation was done using RPMI media which supplemented with
1% L-glutamine, 10% bovine serum albumin (BSA) and 1% penicillin/streptomycin at 37 ºC at CO incubator.
2

HCV infection
Blood sample from a patient with HCV genotype 4 was identified and provided from Ain Shames Specialized
Hospital, Egypt. For infection, HepG2 cells were incubated for three days with the serum of derived sample
in multiplicity of infection (MOI) of 0.5 .
[25]
HS treatment and virus infection
5
To figure out the effect of HS on HCV replication, HepG2 cells were seeded in 6-well plates (2 × 10 cells per
well). Cells were infected for 3 days with HCV (MOI = 0.5), other cells were incubated without infection. All
cells were then stimulated by HS using warm media (45 ºC for 5 min). The infectious media was collected
and stored at -80 ºC for LDH detection as an indicator for cytotoxic effect of heat shock.

Cytotoxic effect and metabolic activity of host cells
To determine the time cytotoxic 50% (TC ) of HS, HepG2 cells were seeded in 96-well plates in a density
50
of 5 × 10 cells per well. The cells were then treated for different time point (0-10 min) with warm media (45 ºC).
4
After each incubation period, the cytotoxic effect was monitored by using water-soluble tetrazoluim salt
(Cell proliferation reagent WST-1, Sigma, USA) according to the manufacture protocol. The number of living
cells was calculated and cell survival was investigated by using inverted microscope and detection of lactate
dehydrogenase (LDH) level using LDH detection kit (Abcam, ab102526). According to the manufacture
procedures, equal amounts of infections medium and LDH buffer (40 µL) were incubated with LDH substrate
for 1 h then the relative LDH production was calculated according to the standard curve. Cells that were
treated with 50 and 100 µL of Triton x-100 served as a positive control .
[26]

RNA isolation and quantitative real time-PCR
Total RNAs were isolated from treated and untreated cells using TriZol (Invitrogen, USA) and chloroform
methods. Isolated RNA was dissolved in RNase free water and the concentration of all samples was adjusted to
final concentration of 100 ng/µL. Then 10 µL from each isolated and purified total RNA was used to generate
cDNA using cDNA synthesis kit (Qiagen, USA). According to the manufacturer protocol, total RNA was
incubated with reverse transcriptase and poly (dTs) primers at 45 °C for 1 h followed by 5 min incubation
at 95 °C. The cDNA was then incubated at -20 °C until used [27,28] . q RT- PCR was used to detect the relative
expression of viral NS5A, non-coding Alu gene and L22 ribosomal gene in infected HepG2 cells upon heat
shock treatment compared to control, the qRT-PCR was performed by using SYBR green and the following
oligonucleotides specific for NS5A, Alu and L22 genes; NS5A-For-5’-ATTCGTTCGTAGTGGGATCCA-3’,
NS5A-Rev-5’-AAGAGTCCAGTATTATCACCTT-3’, Alu-for-5’-AAAACGGTGAAACCCCGT-3’, Alu-
rev5’-TATGTGCCAGGCACTTTT-3’ and L22-for-5’-GAATTCGCACCGACTCGTAC-3’ and L22-rev-
3’-GGTGTTCGCAAAGGTGCTGTCCC-5’. Levels of GAPDH, as internal control, were amplified
using specific oligonucleotides GAPDH-for 5’-TGGCATTGTGGAAGGGCTCA-3’ and GAPDH-rev-5’-
TGGATGCAGGGATGATGTTCT-5’. The following parameters have been used in qRT-PCR program, 94 ºC
for 3 min, 40 cycles (94 ºC for 15 s, 60 ºC for 30 s and 72 ºC for 30 s) and finally 72 ºC for 10 min [19,26] .