Figure 7. Overexpression of CERKL protects
HeLa cells from apoptosis caused by oxidative stress. A: The
western blot of the PARP apoptosis-dependent cleavage was obtained and
immunodetected 24 h after treatment with several oxidative reagents, as
indicated. The PARP precursor protein size is 116 kDa, whereas the
proteolytic product after caspase-3 cleavage is 85 kDa. Cells were
transfected with either the CERKLa (532aa, encompassing the kinasic
domain) or the empty vector pcDNA3. Immunodetection of tubulin was used
for normalization. This is one image of several similar replicates. The
transfection efficiency was comparable as assessed by western
immunodetection (data not shown). B and C:
Quantification of the PARP-cleaved peptide with respect to total PARP
immunodetected protein (both precursor plus peptide) in empty vector
(red bars) versus CERKLa transfected cells (solid bars) under the
different treatments. Basal apoptosis in empty vector untreated cells
was arbitrarily considered 100%. CERKL protection against apoptosis was
clearly detected after 12 h treatment with 300 μM H2O2
compared with the empty-vector transfected cells, whereas in the other
oxidative conditions this protective effect was not significant (B).
This protective effect of CERKL against apoptosis induced by 300 μM H2O2
was much more pronounced after 24 h treatment (C). D:
The histogram shows the quantification of the PARP-cleaved peptide with
respect to total PARP immunodetected protein (both precursor plus
peptide) in empty vector-transfected cells (red bars) versus cells
transfected with either CERKLa (solid bars), the R257X CERKLa mutant
(white bars) or the R257X CERKLb mutant (blue bars). Cells were grown
under normal conditions or treated with different concentrations of H2O2.
Basal apoptosis in empty vector untreated cells was arbitrarily
considered 100%. The protective effect is clearly detected for the
full-length protein but not for the truncated mutants. NT-untreated
cells; 300-cells treated with 300 μM H2O2;
400-cells treated with 400 μM H2O2; FBS-cells
grown in medium deprived of fetal bovine serum; SNP-cells grown in
medium supplemented with 0.3 mM sodium nitroprusside; R257Xa- cells
transfected with the construct bearing the R257X mutation in the CERKLa
sequence background; R257Xb-cells transfected with the construct
bearing the R257X mutation in the CERKLb sequence background. At least
3 independent experiments were used for replication. Statistical
significance is indicated by an asterisk (Mann–Whitney test, p<0.05).