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Fig. 1. ABL, β-actin and GUS mRNA levels in HeLa cells after transfection with HPV siRNAs.
HeLa cells were incubated with 30 nM siRNA for 48 h before harvesting the cells and extracting
the RNA. The expression levels of ABL, β-actin, GUS and EF1α were then determined with
quantitative real-time PCR. ABL, β-actin and GUS values were normalized against EF1α values.
Bars represent mean values from triplicate samples, error bars are ± SD. Normalized ABL, β-actin
and GUS values from siRNA-treated samples were compared to same values from their respective
control samples by performing a two-tailed paired t-test for each pair. The values from treated
samples do not significantly differ from control values, with the exception of GUS mRNA after
transfection with 18E7-694 siRNA; *, P < 0.05.
Fig. 2. Effect of siRNA transfection reagent (Oligofectamine) and the control siRNA 16E6-191 on
p53 reporter activity and cell viability. HeLa p53 reporter cells were either treated with
Oligofectamine alone or with Oligofectamine + 30 nM 16E6-191 siRNA. Control cells were treated
with plain medium. At 24, 48, 72 and 96 hours after transfection the amount of viable cells in each
well was determined with colorimetric WST-1 assay, and the luciferase activities were quantitated
and normalized against the WST-1 values. A, kinetic changes in p53 reporter activity. The data
points represent mean values from two independent experiments, both performed in triplicate. Error
bars show 95 % confidence interval limits. B, amount of viable cells at 96 h. Bars represent mean
values from two independent experiments performed in triplicate, ± 95 % CI.
Fig. 3. The combined cytotoxic effect of 18E6-340 siRNA and chemotherapy drugs in HeLa cells.
HeLa cells were incubated with 100 nM 18E6-340 siRNA (inactive reference siRNA) for 24 h
before adding chemotherapy drugs to the medium. Six hours after the drug addition the cells were
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trypsinized, suspended in fresh medium and plated in 6-well plates. After 7 days, the resulting
colonies were fixed, stained and scanned, and the survival fractions were obtained by comparing
pixel counts from wells treated medium only to counts from wells treated with siRNA and/or
chemotherapy. Then, a theoretical zero-interaction curve (exactly additive) was calculated for each
siRNA + drug combination by applying Bliss independence criterion, and the siRNA + drug dose
response curves were statistically modelled as described in Materials and Methods to obtain 95 %
confidence intervals for each curve.
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, siRNA + drug dose-response curve.
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, 95 %
confidence interval limits for the siRNA + drug dose-response curve. ----------, Bliss curve showing
the theoretical situation where the combined cytotoxic effect of siRNA + drug is exactly additive. If
the Bliss curve is within the 95 % confidence interval limits, the combined cytotoxic effect of
siRNA + chemotherapy is considered to be additive. Here, the combined effect of each 18E6-340
siRNA + drug combination is additive with the exception of the slightly subadditive effect seen
after treatment with siRNA + mitomycin.