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Fig. S1 Epirubicin inhibited cell viability and induced TIGAR expression in A549
cells. A549 cells were treated with different concentrations of epirubicin for 12 h, 24
h and 36 h, respectively. Cell growth inhibitory was detected with MTT assay. (A)
A549 cell growth after treatment of different concentrations (1.25-40 g/ml) of
epirubicin for different length of time (12-36 h). To detect effect of epirubicin on
TGIAR and TP53 expression, A549 cells were treated with different concentrations of
epirubicin (0, 2.5, 5, 10, 20, 40 μg/ml) for 12 h or 5 μg/ml epirubicin for different
length of time(12 h, 24 h, and 36 h). The protein levels of TIGAR and TP53 were
detected with Western blotting. β-actin protein was used as a loading control.
Quantitative analysis was performed with Image J. (B and D) Protein expression of
p53. (C and E) Protein expression of TIGAR. Values are means ± SD from 3
independent experiments. * p< 0.05, ** p< 0.01, *** p<0.001, ns p>0.05 versus
control group.
Fig. S2 TIGAR expression was regulated by TP53 in HepG2 cells. HepG2 cells
were treated with 25 μM or 50 μM PFT-α for 2, 4 or 6 h, respectively. The protein
levels of TIGAR and Bax were detected with Western blot analysis. β-actin protein
was used as a loading control. Quantitative analysis was performed with Image J. (A)
Protein expression of Bax and TIGAR. (B and C) Quantitative analysis of Bax and
TIGAR protein levels. Values are means ± SD from 3 independent experiments. * p<
0.05, ** p< 0.01, ns p>0.05 versus control group.
Fig. S3 Down-regulation of TIGAR inhibited cell survival and enhanced
epirubicin-induced apoptosis in A549 cells. (A) Transfection efficiency of
FAM-scramble siRNA in HepG2 cells. Cells were trasfected with FAM-scramble
siRNA using lipo2000 for 6 h and Green fluorescence of FAM was detected with a
fluorescence microscopy. (B) Knockdown efficiency of TIGAR in A549 cells. Cells
were transfected with TIGAR siRNAs for 72 h and epirubicin was added 12 h before
the end of experiment. The protein levels of TIGAR were detected with
immunoblotting. β-actin protein was used as a loading control. Quantitative analysis
was performed with Image J. (C) Effects of epirubicin on cell viability after TIGAR
knockdown. A549 cells were treated as described above and cell viability was
assessed with MTT assay. (D) Quantitative analysis of percentage of apoptotic A549
cells after TIGAR knockdown with or without treatment of epirubicin with flow
cytometry. Values are means ± SD from 3 independent experiments. * p< 0.05, ** p<
0.01, *** p<0.001, ns p>0.05 versus corresponding group.
Fig. S4 Knockdown of TIGAR enhanced epirubicin-induced activation of
caspase 3 and apoptosis. (A) The effects of epirubicin on levels of active caspase 3.
HepG2 cells were transfected with scramble siRNA or TIGAR siRNA1 and TIGAR
siRNA2 for 60 h and then were left untreated or treated with 5 μg/ml epirubicin for 12
h. The protein levels of pro-caspase 3 and cleaved caspase 3 were detected with
Western blotting. β-actin protein was used as a loading control. Quantitative analysis
was performed with Image J. (B and C) Cell apoptosis after TIGAR knockdown and
epirubicin treatment in the presence and absence of Z-VAD-FMK. The HepG2 cells
were transfected with scramble siRNA or TIGAR siRNA2 for 60 h and then were left
untreated or treated with 5 μg/ml epirubicin for 12 h in the presence or absence of
Z-VAD-FMK. The cell apoptosis was detected with flow cytometry. Values are means
± SD from 3 independent experiments. * p< 0.05, ** p< 0.01, *** p<0.001, ns p>0.05
versus corresponding group.
Fig. S5 NAC reduced TIGAR knockdown-induced alterations in ROS levels,
GSH/GSSH ratio, NADPH levels and cell survival after epirubicin treatment.
(A) ROS levels after TIGAR knockdown and epirubicin treatment in the presence and
absence of NAC. The HepG2 cells were transfected with scramble siRNA or TIGAR
siRNA2 for 60 h and then were left untreated or treated with 5 μg/ml epirubicin for 12
h in the presence or absence of NAC. The ROS levels were measured with flow
cytometry after treatment of epirubicin for 12 h. (B) Quantitative analysis of ROS
levels after TIGAR knockdown and epirubicin treatment in the presence and absence
of NAC. (C) The effects of NAC supplementation on GSH/GSSG ratio in HepG2
cells after TIGAR knockdown with or without treatment of 5 μg/ml epirubicin. (D)
The effects of NAC supplementation on NADPH levels in HepG2 cells after TIGAR
knockdown with or without treatment of
5 μg/ml epirubicin. (E) Quantitative
analysis of the effects of NAC supplementation on colony numbers of HepG2 cells
after TIGAR knockdown with or without treatment of epirubicin. * p< 0.05, ** p<
0.01, *** p<0.001, ns p>0.05 versus corresponding group.