Page 134 - Read Online

Ding et al. Hepatoma Res 2018;4:12 I http://dx.doi.org/10.20517/2394-5079.2018.07 Page 3 of 8

Enzyme-linked immunosorbent assay analysis
Enzyme-linked immunosorbent assay (ELISA) detection of targets of interest was performed according to
the manufacturers’ instructions. β2GPI was measured in groups A, B, D, and E; HBsAg in groups B, D and
F; and tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and alpha fetal protein (AFP) in all groups.
[6]
Once β2GPI reached the highest expression level, as determined from previous studies , cell supernatants
from each group were collected for ELISA analysis. Triplicates of standards, samples and blank groups were
prepared. The optical density (OD) value of each well was measured at 450 nm. Data were presented as
means ± SD.

Non-radioactive NF-κB EMSA and NF-κB relative quantification
Assays were performed only with nuclear extracts according to the manufacturer’s instructions. Nuclear
extracts (5 μg) were used for each reaction with 400 fmol bio-labeled (hot) oligonucleotide NF-κB probe
(5’-AGT TGA GGG GAC TTT CCC AGGC-3’) and unlabeled (cold)-NF-κB probe (5’-AGT TGA GGG
GAC TTT CCC AGGC-3’). Poly(dI-dC): poly(dI-dC) was used as a nonspecific competitor. A 25-fold
molar excess of unlabeled homologous oligonucleotide was used as a specific competitor. Non-homologous
oligonucleotide sequences were also used to validate the specificity of the binding of each transcription factor
in the competition assays. Binding reaction resolved by 6.5% acrylamide/bis (30:1 ratio) electrophoresis
in 0.25× TBE on ice. The gel was transferred to nitrocellulose membranes in 0.5× TBE. The membrane
was then UV crosslinked for 10 min, blocked with 1× blocking buffer for 30 min, and then incubated with
streptavidin-HRP in blocking buffer (1:750) at room temperature for 30 min. The membrane was washed
four times with 1× washing solution and was equilibrated with 1× equilibration solution for 5 min with
shaking. Finally, the membranes were incubated with chemiluminescence substrate buffer, and the bands
were visualized using Viagene CoolImager (Viagene Biotech Co., China). NF-κB relative quantification was
based on relative activity of the combination of NF-κB and DNA. The last result was represented by ΔФ (gray
value). The gray values of the image were measured after film exposure by the imaging system CoolImger.
Data were presented as means ± SD.

Statistical analysis
SPSS 22.0 software was used for data processing and statistical analysis. Cell assay data were presented as
means ± SDs and the variance was analyzed. Comparison between groups was measured using Fisher’s least
significant difference (LSD) test. Differences were significant at P < 0.05.

RESULTS
Expression of β2GPI and HBsAg in transfected cells

We used ELISA to measure expression of β2GPI and HBsAg 24h after transfection of recombinant plasmids
in cell supernatants. β2GPI protein expression was found in group B, D, and E, significantly different from
non-transfected, non-treated group A (P < 0.001). There were no differences in expression levels of β2GPI
in groups B, D, and E (P > 0.05) suggesting similar transfection efficiency. HBsAg protein expression was
found in groups B, D, and F. Expression was determined using a cutoff value (COV) that equal to the average
absorbance value of the negative control (0.532). The absorbance of specimen ≥ COV indicated positive
expression of HBsAg.

Activation of NF-κB in β2GPI- and/rHBsAg-transfected cells following LPS stimulation
A representative image of non-radioactive NF-κB EMSA in the six groups is shown in Figure 1, and NF-κB
relative quantification was represented by gray value is shown in Figure 2. Groups B, C, D, E, and F induced
differential levels of activation of NF-κB, with the highest relative activity of NF-κB observed in group D
(1404.5 ± 11.28); this was significantly different compared with the other five groups (P < 0.05). The relative
activity of NF-κB in group B was 914.57 ± 12.51, significantly higher than levels in groups A, C, E, and F
(P < 0.05). The levels in group E (867.76 ± 6.27) and F (882.52 ± 7.92) were much higher than those of group