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

Flowcytometry analysis
5
HepG2 cells were seeded in 6-well plates in concentration of 2 × 10 cells per well, 25 µL serum of infected
patient with HCV genotype 4 that contains 1 × 10 virus (MOI = 0.5) was added to each well, and incubated
5
for 3 days at CO incubator. The old media was removed and fresh warm media (45 ºC) was added for infected
2
cells for 5 min. Then the media has been removed and cells were washed using PBS, and trypsinized by using
trypsin. Finally the cells were collected in PBS and centrifuged at room temperature at 5000 rpm for 5 min.
The supernatant was removed and the pellet was resuspended in PBS that contains Triton-X-100 (0.01%) for
permeabilization, then cells were centrifuged as previously described. The supernatant was removed and
the pellet was resuspended in PBS that contains 1% BSA and 1:1000 diluted rabbit monoclonal antibodies
for either NS5A or RPL22 protein (Promega, USA) followed by 1 h incubation at room temperature. The
cells were centrifuged as previously described and were washed using PBS for three times. The cells were
then incubated for 1 h in the dark with secondary antibody goat anti-rabbit (Promega, USA) in dilution of
1:100. Finally, the stained cells were centrifuged and washed by PBS and were collected in 500 µL PBS for
flowcytometry (Becton Dickinson Facscalibur).

Prediction tools
To investigate the possible interaction and potential binding site between Alu-repeated sequence and RPL22
sequences, IntaRNA software has been used [29-31] .

Statistical analysis
A student’s two-tailed test was used to determine significance values of relative gene expression in treated
and non-treated cells. SDS 2-2.2 software was used to analyze the Ct values of the q RT-PCR and to drive and
calculate the relative gene expression using ∆∆Ct equations .
[32]

RESULTS
Heat shock has no cytotoxic effect on cell viability rate
HS is the consequences for subjecting the cells to higher temperature than the optimal temperature range for
biological functions. The influence of HS on cells’ viability rate was monitored dependent on cell imaging,
number of living cells and TC following incubation. Additionally, lactate dehydrogenase (LDH) production
50
from treated cells was measured as an indicator for systemic toxic effect of HS. HepG2 cells were seeded
in either 6-well plates or 96-well plates and were incubated overnight. Next, the cells were infected with
HCV by adding the patient-derived serum to fresh media (MOI = 0.5) followed by 3 days of incubation.
The infected cells were subjected to warm media (45 ºC) for the indicated time points. Cell viability rate
dependent HS-time course was detected by using WST-1 assay which revealed that the TC is greater than
50
10 min [Figure 1A]. This result indicates that the cytotoxic effect of HS is initiated upon 10 min of treatment.
Furthermore, cells imaging and living cells upon 5 min of HS treatment showed no detrimental influence on
treated cells in comparison with cells that were left without treatment (NT) [Figure 1B and C]. The relative
LDH production showed negligible differences between 5 min-HS-treated cells, non-treated cells (NT) and
mock in comparison with cells that were treated with Triton-X 100 as detergent agent [Figure 1D]. These data
reveal that treatment with HS (45 ºC) for 5 min has no cytotoxic effect in HepG2 cells.

HS treatment disturbs HCV replication via regulation of viral NS5A gene
In order to investigate whether the HS treatment has an influence on HCV replication, the relative gene
expression of viral NS5A and its corresponding protein level have been detected in HepG2 cells following
HS treatment. NS5A is a zinc-binding and proline-rich hydrophilic nonstructural protein that plays a crucial
role in HCV-RNA replication. NS5A has the ability to modify NS5B polymerase activity and modulate
multiple aspects in cellular immune response . Thus, the expression of NS5A reveals the capability of viral
[33]
replication in infected cells. Here, the expression of HCV-NS5A has been detected at both RNA and protein