SUPPPLEMENTAL MATERIAL
ADDITIONAL DISCUSSION
To further verify whether inhibition of IR-induced ERK1/2 is essential for the inhibition of
IR-induced G2/M checkpoint activation, MCF-7 cells were treated with IR first and then
incubated with U0126. Under this condition, inhibition of ERK1/2 by U0126 after irradiation had
no effect on IR-induced G2/M arrest (data not shown). This result indicates that once the
signaling cascade leading to G2/M arrest is activated following IR, continued activation of
ERK1/2 is not necessary for the maintenance of the G2/M checkpoint activation in MCF-7 cells
following IR treatment.
Although JNK kinase is also activated in MCF-7 cells following IR treatment (data not
shown), time course experiments indicate that, while ERK1/2 activation occurs within 15min
following IR exposure, JNK activation is not observed until 2-3hr post IR (data not shown).
Furthermore, JNK inhibition using JNK specific inhibitor SP600125 had little, if any, effect on IRinduced G2/M arrest in MCF-7 cells (data not shown). Furthermore, previous studies have
shown that ATM-dependent activation of p38 occurs following irradiation of HeLa and U2OS
cells and that this signaling pathway may contribute to IR-induced G2/M arrest in these cells
(Wang et al., 2000). However, we observed no increase in p38 phosphorylation following IR
exposure of MCF-7 cells (data not shown). Thus, these results indicate that both JNK and p38
are not involved in the regulation of IR-induced G2/M checkpoint activation in MCF-7 cells.
Previous studies in H1299 lung cancer cells and HeLa cervical cancer cells have shown
that IR exposure induces Cdc25A phosphorylation and that this results in decreased stability of
Cdc25A (Xiao et al., 2003; Zhao et al., 2002). While IR treatment of MCF-7 cells was not
associated with any concomitant change in the steady state levels of Cdc25A protein (see
1
Figure 1E), ERK1/2 inhibition in MCF-7 cells resulted in increased Cdc25A protein levels even
in the absence of IR treatment (see Figure 7C). These results suggest a specific effect of
ERK1/2 signaling on the regulation of Cdc25A protein expression and that this effect is
independent of ATM/ATR activities, since treatment with caffeine had no effect on Cdc25A
protein expression.
SUPPLEMENTAL MATERIALS AND METHODS
Cell culture and drug treatment
For the studies involving drug treatment, log-phase growing cells were incubated in
medium containing caffeine (Sigma-Aldrich Corporation), PD98059 (LC Laboratories) or U0126
(LC Laboratories). PD98059 and U0126 were dissolved in DMSO and caffeine dissolved in
DMEM. Control cells were incubated in medium containing the same amounts of vehicle alone.
For experiments involving IR exposure, exponentially growing cells were treated with IR at the
indicated doses and then incubated at 37oC for the indicated times prior to analysis. For
experiments involving both drug treatment and IR exposure, cells were pre-incubated with drug
for 1hr or 2hr prior to IR exposure.
Antibodies and recombinant proteins
All antibodies were obtained from Santa Cruz Biotechnology unless indicated elsewhere.
These include mouse monoclonal antibodies specific for Cdc2 (17), Cdc25C (H-6), Chk1 (G-4),
and Chk2 (B-4); rabbit polyclonal antibodies specific for ATM (Ab-3) (EMD Biosciences),
Cdc25A (Cell Signaling Technology), Cdc25C (C-20), Chk1 (FL-476), Chk2 (H-300) and Wee1
(C-20) and goat polyclonal antibody for ATR (N-19). Antibody against phospho-ERK1/2 (E-4) is
a mouse monoclonal IgG that binds to a short peptide sequence of ERK1 and ERK2 containing
phosphorylated Tyr-204. Antibody C-14-G is a goat polyclonal IgG which recognizes full-length
2
ERK2 and to a lesser extent ERK1. Antibody p-Cdc2 (Tyr15) is a goat polyclonal IgG that
recognizes a peptide sequence containing phosphorylated Tyr15 of Cdc2. Anti-phospho-Histone
H3 is affinity-purified rabbit polyclonal IgG that recognizes specifically Histone H3
phosphorylated at Ser10 (Upstate Biotechnology). Anti-phospho-p53 antibody (16G8) is a
mouse monoclonal IgG1 that detects p53 phosphorylated at Ser-15 (Cell Signaling Technology).
As control for protein loading, affinity-purified anti-Actin goat IgG (I-19) was used for quantitating
Actin protein levels on all immunoblots.
Recombinant Cdc2 protein was purified as a glutathione S-transferase fusion protein
containing full-length human Cdc2 (Santa Cruz Biotechnology); Cdc25C protein, substrate for
Chk1/Chk2 kinase assay, was purified as a glutathione S-transferase fusion protein containing
residues 200-256 of human Cdc25C [kindly provided by Dr. Helen Piwnica-Worms (Washington
University School of Medicine)]; p53 full-length protein was purified as a glutathione Stransferase fusion protein (Santa Cruz Biotechnology); Chk1 protein was purified as a
glutathione S-transferase fusion protein containing full-length human Chk1 [kindly provided by
Dr. Junjie Chen (Mayo Clinic and Foundation)]; glutathione S-transferase protein was used as a
negative control substrate in all kinase assays and was prepared according to the standard
procedure (Pharmacia); Histone H1 protein was used as a substrate for Cdc2 kinase assay
(Upstate biotechnology).
Immunoblotting, Immunoprecipitation and kinase assays
Cell lysates preparation, immunoblotting and immunoprecipitation assays were
performed as described previously (Yan and Mumby, 1999; Yan et al., 2005). For ATM and ATR
kinase assays, ATM and ATR proteins were immunoprecipitated from 500g cell lysate using
AB-3 (EMD Biosciences) and N-19 (Santa Cruz Biotechnology) respectively. ATM kinase
activity was assayed using p53 recombinant protein (Canman et al., 1998; Gatei et al., 2000)
and ATR kinase activity assayed using Chk1 recombinant protein (Hall-Jackson et al., 1999;
3
Sarkaria et al., 1999). Both ATM and ATR kinase assays were performed at 30oC for 50min. For
Cdc2 kinase assay, Cdc2 protein was immunoprecipitated from 200 g cell lysate using an antiCdc2 antibody (17) (Santa Cruz Biotechnology), and the immunoprecipitates assayed with a
Cdc2 kinase assay kit (Upstate Biotechnology) using histone-H1 recombinant protein as
substrate. Chk1 and Chk2 were immunoprecipitated from 250g of cell extract, using anti-Chk1
(G4) and anti-Chk2 (B-4) antibody respectively, and kinase assays performed as described
previously using Cdc25C recombinant protein as substrate (McGowan and Russell, 1993;
McGowan and Russell, 1995; Ward et al., 2001; Yarden et al., 2002; Yu et al., 2002). Chk1 and
Chk2 kinase reactions were incubated at 30oC for 20min (Yarden et al., 2002). Wee1 kinase
was immunoprecipitated from 500g of cell extract using C-20 anti-Wee1 antibody (Santa Cruz
Biotechnology) and Wee1 activity was assayed using Cdc2 recombinant protein as substrate
(Santa Cruz Biotechnology) as described previously (Yan et al., 2005). Kinase assays were
terminated by addition of 20l of 6 X Laemmli SDS sample buffer and protein substrate
phosphorylation analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and
autoradiography (Yan and Mumby, 1999). Substrate phosphorylation of Cdc2 Kinase assay was
quantitated either by using a scintillation counter according to manufacture’s instruction (Upstate
Biotechnology) or by SDS-PAGE/autoradiography as described previously (Yan and Mumby,
1999).
To quantitate protein levels on Western blot, specific protein signals were visualized by
chemiluminescence exposed to X-ray film and then scanned using EPSON Perfection
1200XPHOTO scanner. The intensity of individual signals was analyzed using ImageQuant 5.2
analytical program (Amersham Biosciences).
siRNA transfection
4
Negative control siRNA, siCONTROL Non-Targeting siRNA (Control-siRNA), contains at
least four mismatches to any human, mouse, or rat gene, as previously determined by the
manufacture
using
Microarray.
The
sequence
for
the
is
Control-siRNA
5’-
UAAGGCUAUGAAGAGAUAC-3’. SMARTpool siRNAs targeting ERK1/2 consists of eight
siRNAs targeting multiple sites on ERK1/2 (ERK1/2-siRNAs). The sequences for ERK1-siRNAs
are
5’-PUAAAGGUUAACAUCCGGUCUU-3’,
PAGAGACUGUAGGUAGUUUCUU-3’,
sequences
for
ERK2-siRNAs
PAGCUUGUAAAGAUCUGUUUUU-3’,
5’-PAACUUGUACAGGUCAGUCUUU-3’,
5’-PUACUGCAACUGCGUGUAGCUU-3’;
are
and
5’-PAAUAAGUCCAGAGCUUUGGUU-3’,
5’-PUUCUACUUCAAUCCUCUUGUU-3’,
5’the
5’5’-
PAAUUUCUGGAGCCCUGUACUU-3’. SMARTpool siRNAs targeting ATR consists of four
siRNAs targeting multiple sites on ATR (ATR-siRNAs). The sequences for ATR-siRNAs are 5’PCAAACCAGCAGUGUUGUUCUU-3’,
5’-PUAACAGUAGACAGCUGACCUU-3’,
5’-
PUAUCUGUUAGGCGAGUUGCUU-3’ and 5’-PUAAAUAAUCAGCCAUCAGUUU-3’.
Cells were transfected with siRNAs at 100nM using DhamaFECT1 siRNA transfection
reagent (Dharmacon Research Inc.) according to the manufacture’s instruction. Transfected
cells were incubated for additional indicated times and analyzed for protein expression of the
relative targeted protein using immunoblotting with specific antibodies. For experiment involving
both siRNA transfection and IR exposure, the transfected cells were incubated for the indicated
times and then treated with IR. The irradiated cells were incubated for additional indicated times
and analyzed for either protein expression or DNA-content as described above.
5
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